Emilia Lubecka

Anna Kwiatkowska

Jerzy Ciarkowski

Emilia Sikorska

NMR studies of new arginine vasopressin analogues modified with α-2indanylglycine enantiomers at position 2 bound to sodium dodecyl sulfate micelles

Keywords: α-2-Indanylglycine, vasopressin, SDS micelle, NMR spectroscopy, molecular modeling AVP, arginine vasopressin, GPCR, G-protein coupled receptor, Igl, α-2indanylglycine, Mpa, 3-mercaptopropionic acid, OT, oxytocin, SDS, sodium dodecyl sulfate

In this paper, we use NMR spectroscopy and molecular modeling to examine four new vasopressin analogues modified with α-2-indanylglycine (Igl) at position 2, [L-Igl 2 ]AVP (I), [D-Igl 2 ]AVP (II), [Mpa 1 ,L-Igl 2 ]AVP (III) and [Mpa 1 ,D-Igl 2 ]AVP (IV), embedded in a sodium dodecylsulfate (SDS) micelle. All the analogues display antiuterotonic activity. In addition, the analogues with D-Igl reveal antipressor properties.

Each analogue exhibits the tendency to adopt β-turns at positions 2,3 and/or 3,4, which is characteristic of oxytocin-like peptides. Mutual arrangement of aromatic residues at positions 2 and 3 has been found to be crucial for binding antagonists with the OT and V 1a receptors. The orientation of the Gln 4 side chain seems to be important for the V 1a receptor affinity. In each of the peptides studied, the Gln 4 side chain is folded back over the ring moiety. However, it lies on the opposite face of the tocin moiety in analogues with L and D enantiomers of Igl.

A C C E P T E D

M A N U S C R I P T

INTRODUCTION

Arginine vasopressin (AVP), a neurohypophyseal hormone and a neuromodulator, is a nonapeptide with a disulfide bridge between Cys residues at positions 1 and 6. It plays a very important role in controlling resorption of water by the distal tubules of the kidney and regulating the osmotic pressure of blood [START_REF] Barlow | Vasopressin[END_REF][START_REF] Birnbaumer | Vasopressin receptors[END_REF]. Besides, AVP is responsible for stimulation of the adrenocorticotropine secretion [START_REF] Jard | Vasopressin antagonists allow demonstration of a novel type of vasopressin receptor in the rat adenohypophysis[END_REF] and stability of the body temperature [START_REF] Wang | Voles and vasopressin: a review of molecular, cellular and behavioral studies of pair bonding and paternal behaviors[END_REF]. It exhibits also oxytocic activity [START_REF] Barberis | Structural bases of vasopressin/oxytocin receptor function[END_REF]. Moreover, recent studies have shown that vasopressin, together with oxytocin, might also take part in autism and provide an effective treatment of autism's repetitive and affiliative behaviours [START_REF] Newschaffer | The epidemiology of autism spectrum disrorders[END_REF][START_REF] Insel | Oxytocin, vasopressin, and autism: is there a A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT connection?[END_REF].

The biological effects of vasopressin are mediated by four different receptor subtypes: V 2 (renal), V 1a (vasopressor), V 1b (pituitary) and OT (uterine), being typical members of class A GPCR, which are membrane-spanning proteins [START_REF] Palczewski | Crystal structure of rhodopsin: a G protein-coupled receptor[END_REF]. The current model for peptide hormone interactions with their receptors suggests that the bioactive conformation of the peptide is induced upon association with the cell membrane followed by a two-dimensional diffusion process, whereby the peptide is recognized and then interacts with the receptor [START_REF] Schwayzer | In search of the 'bio-active conformation' -is it induced by the target cell membrane?[END_REF][START_REF] Mierke | Peptide hormone binding to G-protein-coupled receptors: structural characterization via NMR techniques[END_REF]. Therefore, exploring the conformational and dynamic properties of a ligand in a membrane-mimicking environment can contribute to better understanding of the molecular features involved in their interactions with the target receptor.

It is believed that the tyrosine residue at position 2 of AVP plays a part initiating the pressor response of AVP [START_REF] Hlavacek | Important structural modifications. Noncoded amino acid. In Handbook of Neurohypophyseal Hormone Analogs[END_REF]. The substitution of Tyr with its D enantiomer produces an analogue with only partial agonist activity, whereas the deletion or O-alkylation of Tyr 2 hydroxyl group results in antagonistic properties [START_REF] Hruby | Conformational and structural considerations in oxytocin-receptor binding and biological activity[END_REF]. Similarly, replacement of Tyr 2 of AVP with bulky or sterically restricted substituents is generally favorable for generation of effective antagonists [START_REF] Bakos | Synthesis and receptor binding of oxytocin analogs containing conformationally restricted amino acids[END_REF][START_REF] Jójárt | Comparative study of eight oxytocin antagonists by simulated annealing[END_REF].

In this paper, we use NMR spectroscopy and molecular modeling to examine four new vasopressin analogues modified with α-2-indanylglycine (Igl) (Fig.

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dodecylsulfate (SDS) micelle. Although, dodecyphosphocholine (DPC) provides a zwitterionic surface on the micelle that better mimics the biological membranes of the vertebrates, we decided to use the SDS micelle with negatively charged head groups, because the AVP analogues modified with Igl are poorly soluble positively charged peptides, which advises towards incorporation of net negative charges on the SDS micelle surface in order to improve peptide/micelle solubility [START_REF] Sankararamakrishnan | Recognition of GPCRs by peptide ligands and membrane compartments theory: structural studies of endogenous peptide hormones in membrane environment[END_REF].

The α-indanylglycine was earlier successfully applied for the synthesis of potent and totally enzyme-resistant bradykinin antagonists [START_REF] Gera | A new class of bradykinin antagonists containing indanylglycine[END_REF]. In turn, the deamination of cysteine at position 1, usually enhances antidiuretic activity and V 2 /V 1a selectivity [START_REF] Sawyer | Structural changes in the arginine vasopressin molecule that prolong its antidiuretic action[END_REF]. However, most of oxytocin antagonists describe to date contain Mpa (Fig. 1) instead of Cys 1 [START_REF] Grzonka | In vitro degradation of some arginine-vasopressin analogs by homogenates of rat kidney, liver, and serum[END_REF].

All the studied analogues exhibit only negligible antidiuretic activity. The [L-Igl 2 ]AVP (I)

and [Mpa 1 ,L-Igl 2 ]AVP (III) analogues are moderately potent but selective OTR antagonists. In turn, those modified with enantiomer D of Igl have dual activity -they show high antioxytocic potency at low and full oxytocic agonism at high concentrations [START_REF] Kwiatkowska | Novel analogu es of arginine vasopressin containing α-2indanylglycine enantiomers in position 2[END_REF]. This phenomenon is difficult to explain. However, similar effect has been reported for naloxone, an opioid antagonist at low concentration and partial agonist at a high one. The changeover of the activity of naloxone from antagonistic to agonistic is probably the result of a slight desensitization and down regulation of the opioid receptors [START_REF] Cosola | Opioid agonist/antagonist effect of naloxone in modulating rabbit jejunum contractility in vitro[END_REF].

MATERIALS AND METHODS

Peptide synthesis and purification. The peptides were synthesized using the standard 9fluorenylmetoxycarbonyl (Fmoc) methodology (full details including protecting groups, deprotection and cyclication have been reported recently [START_REF] Kwiatkowska | Novel analogu es of arginine vasopressin containing α-2indanylglycine enantiomers in position 2[END_REF]). After HPLC purification, their purity was higher than 98% as determined by analytical HPLC. The MALDI TOF mass spectrometry confirmed that the purified peptides were the desired products.

Sample preparation.

The SDS-d 25 was purchased from Sigma Aldrich. The samples in the SDS

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micelle were prepared at a concentration of about 4 mM of a peptide in 0. resonance assignments were achieved by use of the proton-proton total chemical shift correlation spectroscopy (TOCSY) [START_REF] Bax | Enhanced NMR resolution by restricting the effective sample volume[END_REF], double quantum filtered correlation spectroscopy (DQF-COSY) [START_REF] Rance | Improved spectral resolution in cosy 1 H NMR spectra of proteins via double quantum filtering[END_REF],

the nuclear Overhauser effect spectroscopy (NOESY) [START_REF] Kumar | A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross relaxation networks in biological macromolecules[END_REF], the rotating-frame Overhauser enhancement spectroscopy (ROESY) [START_REF] Bothner-By | Structure determination of a tetrasaccharide: Transient nuclear Overhauser effects in the rotating frame[END_REF][START_REF] Bax | Practical aspects of two-dimensional transverse NOE spectroscopy[END_REF], as well as the gradient heteronuclear single quantum coherence ( 1 H- 13 C gHSQC) [START_REF] Palmer | Sensitivity improvement in proton-detected two-dimensional heteronuclear correlation spectroscopy[END_REF][START_REF] Kay | Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity[END_REF][START_REF] Schleucher | A general enhancement scheme in heteronuclear multidimensional NMR employing pulsed field gradients[END_REF]. All the spectra were measured with a water signal presaturation pulse typically of 2 dB and 1.5 s. In the case of the 1D NMR spectra, 16 K data points were collected and a spectral width of 8 kHz was used. The homonuclear TOCSY (80 ms), NOESY (200 ms), ROESY (200 and 300 ms) and DQF-COSY were recorded in the range of 0.5-9.5 ppm, with 32-64 scans per t 1 , a spectral width of 4.5 kHz in both dimensions, and 512 x 2K data sets, which were then zero-filled to 1K x 2K after Fourier transform. Data size for 1 H- 13 C HSQC spectrum was 256 (t 1 ) -1024 (t 2 ) and spectral widths were 18 kHz in the 13 C dimension and 4.5 kHz in the 1 H dimension. For this experiment, a total number of 128 transients were used.

Vicinal coupling constants, 3 J HNHα , were assigned using DQF-COSY and 1D NMR spectra.

The DQF-COSY spectra were processed to enhance the resolution to 1.2 Hz per point in F2. For

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Gly residues, the two 3 J HNHα coupling constants are equal within the limits of error.

The proton chemical shifts were referenced to the H 2 O frequency measured with respect to external 2,2-dimethyl-2-silapentanesulfonic acid (DSS). The 13 C chemical shifts were referenced to DSS according to the following relationship: 13 C/ 1 H = 0.251449530 [29]. The data were processed using VNMR 6.1B (Varian Inc., Palo Alto, CA) and the spectra were analyzed using XEASY [START_REF] Bartles | The program XEASY for the computer-supported NMR spectral analysis of biological macromolecules[END_REF].

Molecular modeling. The calculations consisted of the following three steps: (i) search of the conformational space by employing molecular dynamics simulations, (ii) simulation of the NOE spectrum and vicinal coupling constants for each conformation, and (iii) determination of the statistical weights of the conformations to obtain the best fit of the averaged NOE intensities and coupling constants to the experimental quantities [START_REF] Groth | Maximum entropy approach to the determination of solution conformation of flexible polypeptides by global conformational analysis and NMR spectroscopy -Application to DNS 1 -c-[D-A 2 bu 2 , Trp 4 ,Leu 5 ]-enkephalin and DNS 1 -c-[d-A 2 bu 2 , Trp 4 , D-Leu 5 ]enkephalin[END_REF].

Molecular dynamics simulations were carried out with the AMBER force field [START_REF] Case | AMBER 9[END_REF]. The valence geometries of the residues not specified in the standard AMBER database, were parameterized as recommended by the AMBER 9.0 manual [START_REF] Case | AMBER 9[END_REF]. Specifically, these residues were modeled using bond lengths, the valence and torsion angles of appropriate residues and compatible molecular segments taken from the CSDS [START_REF] Allen | Applications of the Cambridge Structural Database in organic chemistry and crystal chemistry[END_REF] database. The point charges were optimized by fitting them to the ab initio molecular electrostatic potential (6-31G* basis set, GAMESS'04 [START_REF] Schmidt | General atomic and molecular electronic structure system[END_REF] ab initio molecular electronic structure program) for two different conformations of every nonstandard residue, followed by consecutive averaging the charges over all conformations, as recommended by the RESP protocol [START_REF] Bayly | A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model[END_REF].

The preparation of the micelle was initiated by construction of a single molecule of sodium dodecyl sulfate (SDS) based on the literature parameters [START_REF] Schweighofer | Simulation of sodium dodecyl sulfate at the water-vapor and water-carbon tetrachloride interfaces at low surface coverage[END_REF][START_REF] Bruce | Molecular dynamics simulation of sodium dodecyl sulfate micelle in water: micellar structural characteristics and counterion distribution[END_REF]. The entire procedure has been described by us previously [START_REF] Rodziewicz-Motowidło | Conformational studies of vasopressin and mesotocin using NMR spectroscopy and molecular modeling methods. Part II: Studies in the SDS micelle[END_REF]. Initial structures of the peptides were generated in random conformation. The peptide groups were kept fixed to trans geometry with force constant f = 50 kcal/ (mol x rad 2 ) during the entire simulations. The calculations were performed only for major conformations. Each peptide was placed in a simulation box with its centre of mass coinciding with that of the micelle. Owing to the spherical symmetry of the micelle, the orientation of the peptide

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was unimportant. The side chain of Arg 8 and the N-terminus were protonated. Thus, the [Igl 2 ]AVP (I) and [D-Igl 2 ]AVP (II) had a total charge of +2, whilst the total charge of [Mpa 1 ,Igl 2 ]AVP (III)

and [Mpa 1 ,D-Igl 2 ]AVP (IV) was equal to +1. To neutralize the entire system, chloride ions were added.

To eliminate initial bad contacts between a peptide and the micelle core, and to prevent penetration of water during equilibration, the peptide and the bulk water were kept under weak harmonic constraints with force constants of 10 and 5 kcal/mol⋅Å, respectively. These constraints were removed in 20,000 steps of minimization (steepest descent method). The entire system was then minimized for 20,000 steps. Thereafter, the system was equilibrated under a constant pressure at 303 K for 2 ns. Afterwards, the LES (locally enhanced sampling) [START_REF] Cheng | Improved conformational sampling through an efficient combination of mean-field simulation approaches[END_REF] system was used. This technique allows selective application of additional computational effort to a portion of the system, thus increasing the sampling of the region of interest. The enhanced sampling is achieved by replacing the region of interest with multiple copies. During the simulation, the copies are free to move apart and explore different regions of conformational space, thereby increasing the statistical sampling. A total of three copies of each peptide were generated by ADDLES module of the AMBER 9.0 program. During MD simulation, a 9 Å cutoff radius was chosen. The MD simulations were carried out at 303 K in a periodic box of constant volume, with the Particle-mesh Ewald (PME) procedure. The time step was 2 fs. The total duration of the LES run was 4 ns. The coordinates were collected every 2000 th step. The conformations obtained during the last 800 ps of simulation were considered in further analysis. As a result, the set of 600 conformations for each peptide was obtained.

Next, the theoretical NOE spectrum and 3 J HNHα vicinal coupling constants were calculated using the MORASS [START_REF] Meadows | MORASS 2.1 Program[END_REF] program for each conformation. This program solves the system of Bloch differential equations for the cross-relaxation of a system of interacting proton spins. The vicinal couplings, 3 J HNHα , were calculated from the empirical Bystrov-Karplus relationship [START_REF] Bystrov | Spin-spin coupling and the conformational states of peptide system[END_REF]. The NOE effects were generated using a correlation time of 0.45 ns [START_REF] Yu | Combined use of NMR, distance geometry, and retrained energy minimization for the conformational analysis of 8-lysinevasopressin[END_REF], mixing time of 200 ms and a cut-off

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value of 6 Å. The weight of the coupling-constant term was 0.2. The populations of the conformations, described by statistical weights, were determined by fitting a linear combination of the generated spectra and coupling constants to the experimental data with an entropy factor, α=0.2 [START_REF] Groth | Maximum entropy approach to the determination of solution conformation of flexible polypeptides by global conformational analysis and NMR spectroscopy -Application to DNS 1 -c-[D-A 2 bu 2 , Trp 4 ,Leu 5 ]-enkephalin and DNS 1 -c-[d-A 2 bu 2 , Trp 4 , D-Leu 5 ]enkephalin[END_REF]. To describe the structural preference of each peptide we used the structures constituting about 60% of the ensemble obtained from calculations. As a result 194, 111, 24 and 42 conformations of analogues I, II, III and IV, respectively, were considered.

The results were analyzed using the Carnal and Ptraj programs from the AMBER 9.0 package [START_REF] Case | AMBER 9[END_REF]. To find the peptide-micelle interactions, the radial distribution functions (RDFs)

between the side chains and negatively charged groups of the SDS micelle and also between the side chains and the micelle core atoms were calculated. The data were averaged over the conformations with the sum of their statistical weights amounting to 60%. To estimate the interactions between the peptides and aqueous environment, hydration numbers were also calculated, as a method of quantifying the interactions between the peptides and water. The hydration number is an integral radial distribution function showing how many water molecules are located near the residue. Two sets of the hydration numbers were calculated for each system, one for the side chains heavy atoms, and the other for the carbonyl oxygen for each residue for a hydration radius of 3.8 Å.

The molecular structures were drawn and analyzed with the MOLMOL [START_REF] Koradi | MOLMOL: a program for display and analysis of macromolecular structures[END_REF] graphic program.

RESULTS AND DISCUSSION

Analysis of the NMR spectra. The NMR spectra of analogue II and IV indicate that the Phe 3 , Gln 4 , Asn 5 , and Arg 8 residues are at equilibrium between two conformational states. In analogue IV, two distinct sets of residual proton resonances were also found for Gly 9 . The intensity ratio of both conformers is equal approximately to 5.6:1 for each peptide. On the other hand, only one set of the chemical shifts was found for the remaining residues. The appearance of two conformers is likely to

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be due to either the cis/trans isomerization of the Cys 6 -Pro 7 peptide bond or different interactions of the peptides with the SDS micelle. The former hypothesis could be verified by an exchange transHα(Pro)-cisHα(Pro) cross peak and/or cisHα(Cys)-cisHα(Pro) connectivity. However, they were not found in the ROESY and/or NOESY spectra. In turn, taking into account the latter hypothesis, strong electrostatic interactions between the positively charged Arg 8 side chain and the negatively charged sulfate groups at the micelle surface could be expected. Hence, the Arg 8 should be strongly bound to the micelle surface and a smaller conformational space should be accessible than for the remaining residues. Consequently, only one set of proton resonance for Arg is likely to exist. Therefore, we cannot validate the origin of the two conformers unambiguously. The proton chemical shifts of the peptides are summarized in Tables 1S and2S (Supplementary Materials).

It is known that the sequential d NN (i,i+1) NOE connectivities are seen only in those regions of peptides which preferentially adopt folded conformations [START_REF] Dyson | Folding of immunogenic peptide fragments of proteins in water solution. I Sequence requirements for the formation of a reverse turn[END_REF]. Therefore, the scrutiny of the NOE patterns (Fig. 2) suggests that all the analogues adopt primarily reverse structures in their cyclic part. In addition, existence of the d αN [START_REF] Birnbaumer | Vasopressin receptors[END_REF][START_REF] Wang | Voles and vasopressin: a review of molecular, cellular and behavioral studies of pair bonding and paternal behaviors[END_REF] It is known that proline φ dihedral angle is relatively fixed at about -60°. The proline ring has also a significant effect on ψ dihedral angle resulting in two minima, -40° and 150° [START_REF] Wiliamson | The structure and function of proline-rich regions in proteins[END_REF]. In tun, the

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high value of 3 J HNHα coupling constant for Arg 8 of analogue I (10.2 Hz) corresponds to dihedral angle φ in the range of -140°÷-100° [START_REF] Pardi | Calibration of the angular-dependence of the amide proton-C-alpha proton coupling-constants, 3 J HNHα , in a globular protein -use of 3 J HNHα  for identification of helical secondary structure[END_REF][START_REF] Eberstadt | Scalar Coupling Constants -Their Analysis and Their Application for the Elucidation of Structures[END_REF]. Bearing all these facts in mind, we can expect one of the three types of β-turns, βI: φ i+1 =-60, ψ i+1 =-30, φ i+2 =-90, ψ i+2 =0, βII: φ i+1 =-60, ψ i+1 =120, φ i+2 =80, ψ i+2 =0 βVIII-turn (φ i+1 =-60, ψ i+1 =-30, φ i+2 =-120, ψ i+2 =120) [START_REF] Lewis | Chain reversals in proteins[END_REF] in the fragment Cys 6 -Pro 7 -Arg 8 -Gly 9

of compound I.

The temperature coefficients of the amide protons (Fig. 2) of L-Igl 2 , Arg 8 and Gly 9 of [L- intramolecular and/or solute-solvent hydrogen bonds. However, the shielding of the amide protons from solvent exchange might be dominated by interaction between the peptide and micelle, rather than by intramolecular interactions [START_REF] De Luca | A cyclic CCK8 analogue selective for the cholecystokin type A receptor: design, synthesis, NMR structure and binding measurements[END_REF]. In the case of Gln 4 of compound III, the amide proton HN of Gln 4 is probably hydrogen bonding up to 30°C, but it becomes broken at higher temperatures, which is manifested by higher tilt of temperature gradient in the range of 30-50°C. Similar effect was observed for mesotocin in SDS/water solution [START_REF] Rodziewicz-Motowidło | Conformational studies of vasopressin and mesotocin using NMR spectroscopy and molecular modeling methods. Part II: Studies in the SDS micelle[END_REF].

The temperature coefficients of the remaining amide protons were more negative than -5 ppb/K, which rather excludes stable hydrogen bonds.

Conformational analysis of the calculated structures. The structures of the peptides are shown in The common conformational feature (Tab. 1) of all the analogues is a β-turn at position 2,3.

In the analogues I and III, this β-turn is stabilized by the HN 4 -CO 1 hydrogen bond. In turn, in the case of analogues II and IV, the β-turn at position 2,3 is not tight enough and the Gln 4 HN amide proton is not hydrogen bonding. Three of the analogues, namely II, III and IV create additional β-

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ACCEPTED MANUSCRIPT turn at position 3,4. Nevertheless, this β-turn is distorted. Therefore, their existence may not result directly from NMR data.

The analogue III, as the only one, adopts a β-turn at position 4,5. This β-turn is not closed by hydrogen bond, which is a consequence of distortion of either the ψ 4 or φ 5 dihedral angles. In addition, an inverse γ-turn over the Pro 7 was found for compounds III. In turn, the C-terminus of the peptide I is involved in a β-turn in the Cys 6 -Gly 9 fragment. While simultaneously, it assumes a hydrogen-bonded βIor βIII-turn at positions 6,7.

The averaged radii of gyration (Rg) (Tab.

Radial distribution functions and hydration numbers for the AVP analogues in SDS micelle.

During molecular dynamics simulations, the peptides diffusing from the hydrophobic core of the SDS micelle to the interface to adopt energetically more favorable conformation. The position of the peptides on the micellar surface becomes fixed after ca. 2 ns of MD. Thereafter, the peptides remain bound to the interface for a further 4 ns of LES simulations. Figure 5 presents the structures of the peptides with the highest statistical weight binding with the SDS micelle. The peptide-micelle interactions were analyzed by the radial distribution function (RDF) values for all the amino acids in each peptide (Figs. 6 and7). In addition, the hydration numbers were calculated to investigate interaction with water molecules (Tab. 2).

In each peptide the aromatic residues, Igl/D-Igl 2 and Phe 3 are deeply immersed into the hydrophobic micelle core as additionally confirmed by low hydration numbers calculated for their side chains. Nevertheless, the backbone oxygen atoms of both D-Igl 2 and Phe 3 in compound IV are strongly hydrated in contrast to the remaining peptides.

The positively charged centre on the Arg side chain shows the tendency to be associated with sulfate ions of SDS and at the same time, it is located near the hydrophobic part of the micelle.

In turn, the oxygen atom of Arg 8 is exposed to the aqueous environment in each case. The analogues I and II possess also the positively charged N-terminus, and consequently the stronger interactions between the N-terminus and negatively charged sulfate head groups of the SDS micelle is noticed than for the deamino analogues (Fig. 6). In turn, in the deamino analogues, III and IV, the Pro 7 side chain clearly penetrates interior of the SDS micelle (Fig. 7). Moreover, the hydration numbers (Tab.

2) indicate that also the oxygen atom of Pro is embedded into the hydrophobic micelle core. The common feature of the deamino analogues are also high hydration number calculated for a backbone oxygen atom of Gln 4 , which indicates that it is exposed to the aqueous phase. In the case

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of peptide III, the Gln 4 side chain is also characterized by a high hydration number.

The side chains of residues 1 and 6 of the D-Igl-modified analogues, II and IV, are evidently more deeply embedded into the micelle core than in the remaining ones and consequently disulfide bridges of both peptides are hidden from the aqueous phase.

Biological relevance of the modifications. It becomes clear that all the analogues exhibit the tendency to adopt β-turns at positions 2,3 and/or 3,4, which is characteristic of oxytocin-like peptides [START_REF] Liwo | Exploration of the conformational space of oxytocin and arginine-vasopressin using the electrostatically driven Monte Carlo and molecular dynamics methods[END_REF]. Thus, the high affinity of the analogues to the OT receptors might be due to these reverse structures. Moreover, it seems likely that the strong π-π interactions between aromatic side chains of the vasopressin-like peptides and the aromatic residue cluster, located in TM6 OT and V 1a receptor, might be responsible for antagonistic properties [START_REF] Ślusarz | Molecular docking-based study of vasopressin analogues modified at positions 2 and 3 with N-methylphenylalanine: influence on receptor-bound conformations and interactions with vasopressin and oxytocin receptors[END_REF]. It is also known that the amino acid at position 2 of a ligand plays a key role in the antagonistic effect. Insertion of a bulky lipophilic Damino acid at this position enhances often antioxytocic properties [START_REF] Jójárt | Comparative study of eight oxytocin antagonists by simulated annealing[END_REF], and at the same time alters the side chain orientation. Consequently, mutual arrangement of the side chains of neighbouring residues at positions 2 and 3 undergoes changes. A closer examination of the indanylglycine χ 1 space angle suggests that the common feature of analogues I and III is the gauche(-) conformation of the Igl side chain, which may be crucial for selectivity in this series of analogues, given that both analogues exhibit only antiuterotonic activity. On the other hand, a comparison of the flat F Igl-Phe and dihedral D Igl-Phe angles (see Conformation of the aromatic side chains section) of the analogues has shown that the latter adopts greater values for analogues II and IV, which are blockers of both the OT and V 1a receptors. A similar tendency was reported for AVP analogues modified with Nmethylpenylalanine enantiomers at positions 2 and 3 [START_REF] Sikorska | Conformational studies of vasopressin analogues modified with N-methylphenylalanine enantiomers in dimethyl sulfoxide solution[END_REF].

Earlier investigations have shown that replacement of Gln 4 in AVP reduced significantly the affinity toward V 1a receptors [START_REF] Sawyer | Structural changes in the arginine vasopressin molecule that enhance antidiuretic activity and specificity[END_REF][START_REF] Gillessen | The synthesis and pharmacological properties of 4-decarboxamide-8-1ysinevasopressin, 5-decarboxamide-8-1ysinevasopressin, and their l-deamino analogs[END_REF]. Moreover, the hydrogen bond between the Thr 7.38 333 hydroxyl of V 1a receptor and the carboxamide of the AVP Gln 4 was found to be important for AVP binding [START_REF] Ślusarz | Analysis of interactions responsible for vasopressin binding to human neurohyphophyseal hormone receptors -molecular dynamics study of the activated receptor-vasopressin-Gα systems[END_REF]. In addition, Thr 7.38 333 was proposed as the residue controlling the V 1a /V 2 binding selectivity

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for vasopressin antagonists [START_REF] Cotte | Conserved aromatic residues in the transmembrane region VI of the V1a vasopressin receptor differentiate agonist vs. antagonist ligand binding[END_REF]. Hence, the orientation of Gln 4 side chain of a ligand seems to be important for V 1a receptor affinity. In each of the peptides studied, the Gln 4 side chain is folded back over the ring moiety. However, it lies on the opposite face of the tocin moiety in analogues with L and D enantiomers of Igl, which can additionally explain their different activities towards the V 1a receptor.

CONCLUSIONS

The affinity of the vasopressin analogues to respective receptors is a consequence of their structure and conformation. For this reason, a correlation between the structure and conformation can provide valuable data to design new analogues with appropriate biological activity. However, short peptides, such as vasopressin, usually adopt unstable conformation and exist in many conformations at equilibrium. Multiple conformations occurring in a dynamic exchange produce averaged NMR parameters, which hamper unequivocal conformational analysis. Hence, all the available NMR data should be examined with caution.

The primary purpose of substitution of indanylglycine (Igl) at position 2 of the AVP analogues was to reduce their flexibility and to change pharmacological profile of the peptides.

Moreover, using the SDS micelle in the NMR studies may additionally restrict conformational freedom of the peptides and probably induces a conformation, which is supposed to be bound to the receptor.

In summary, the new AVP analogues should be designed in such a way, as to preserve specific conformation of a peptide and the proper mutual arrangement of the side chains. Moreover, it should be remembered that the structurally restricted modifications reduce the flexibility of a peptide thus enabling a more precise conformational analysis to be made. Introduction of a D-amino acid in the second position of the AVP analogues changes the side chain orientation and enforces the possibility of β-turn formation in a 1-4 fragment, which is characteristic of oxytocin-like peptides.

Furthermore, the specific orientation of the side chains at positions 2 and 3 is important for

  ) at position 2, [L-Igl 2 ]AVP (I), [D-Igl 2 ]AVP (II), [Mpa 1 ,L-Igl 2 ]AVP (III) and [Mpa 1 ,D-Igl 2 ]AVP (IV) embedded in a sodium A C C E P T E D M A N U S C R I P T

  connectivities in the NOESY spectra of analogues II and IV implies a β-turn at position 2,3. In the case of compound IV, the d αN (1,4) NOE cross-peak found may also indicate β-turn at this position. The 3 J HNHα coupling constants (Fig.2) measured for the peptides are characteristic of either extended or unfolded states. Nevertheless, an averaging over at least two different conformations cannot be excluded. With analogues I and III, the 3 J HNHα coupling constants recorded for the L-Igl 2 and Phe 3 neighbouring residues, (6.4 and 9.7; 7.4 and 9.7 for peptides I and III, respectively) suggest β-turn at position 2,3 (numbers correspond to the two central residues of the β-turn). This is the consequence of the steric hindrance imposed by the β-turn, which causes that the corner residues at positions i+1 and i+2 of the β-turn are characterized by low and high the 3 J HNHα coupling constant values, respectively.

Igl 2 ]

 2 AVP (I), D-Igl 2 , Phe 3 and Gly 9 of [D-Igl 2 ]AVP (II), Arg 8 and Gly 9 of [Mpa 1 ,L-Igl 2 ]AVP (III) and Gln 4 of [Mpa 1 ,D-Igl 2 ]AVP (IV) were in the range of -5 < Δδ/ΔT < -3 ppb/K, indicating weak

Figure 3

 3 Figure 3 and are aligned to their mostly populated conformations using backbone atoms of the cyclic part of the molecules. The RMSD values for the ensemble of the structures are 0.288, 0.276, 0.226 and 0.195 Å for backbone atoms of cyclic part of analogues I, II, III and IV, respectively.

1 )

 1 calculated with all of the heavy atoms for each analogue indicate that the conformation of the analogue II is noticeably more extended than the remaining analogues, this being due to a more outstretched structure of the acyclic part of the molecule. The differences in the Rg values obtained for the cyclic part of each analogue are comparable, indicating similar sizes of the tocin rings. Conformation of the aromatic side chains. All the structures were submitted to statistical study of relative orientation of the Igl/D-Igl 2 and Phe 3 aromatic side chains. It is clearly seen (Fig. 4a) that the preferred rotamer about the χ 1 torsion angle of Igl is gauche(-) for both analogues with L-Igl, I and III, and either trans or gauche(+) for II and IV, respectively. A relationship has been established between the flat angle between the planes of aromatic rings (F Igl-Phe ) and a dihedral angle between two planes, where the former is determined by the mass centre of aromatic part of Igl/D-Igl 2 , Cα of Igl/D-Igl 2 and Cα of Phe 3 , whereas the latter by Cα of Igl/D-Igl 2 , Cα of Phe 3 and the centre of mass of aromatic ring of Phe 3 (D Igl-Phe ) (Fig. 4b). Taking into account the flat angle, F Igl-Phe , the structure of all the peptides, with the exception of analogue IV, can be divided into two groups based on different orientation of the Phe 3 side chain caused by changes of its χ 2 dihedral angle (Fig. 4a). In turn, the dihedral angle values, D Igl-Phe , are clustered about horizontal lines, one for each peptide, D Igl-Phe = 15°, 85°, 13° and 65° for analogues I, II, III and IV, respectively, where the angles correspond to average values of D Igl-Phe . As seen, the D Igl-Phe values are noticeably greater for analogues modified A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT with the D enantiomer of Igl. The differences in the D Igl-Phe values are most of all the consequence of the change of Igl configuration from L to D, which evidently alters the side chain orientation.

Figure 1 .

 1 Figure captions

Figure 2 .

 2 Figure 2. The NOE effects corresponding to the interproton distances, 3 J HNHα coupling constants and the temperature coefficients of the backbone amide atoms of (A) [L-Igl 2 ]AVP (I), (B) [D-Igl 2 ]AVP (II), (C) [Mpa 1 ,L-Igl 2 ]AVP (III) and (D) [Mpa 1 ,D-Igl 2 ]AVP (IV).

Figure 3 .

 3 Figure 3. Stereoview showing the conformational ensembles of (A) [L-Igl 2 ]AVP (I), (B) [D-Igl 2 ]AVP (II), (C) [Mpa 1 ,L-Igl 2 ]AVP (III) and (D) [Mpa 1 ,D-Igl 2 ]AVP (IV) obtained by fitting the statistical weights of MD-generated conformations. RMSD= 0.288, 0.276, 0.226 and 0.195 for backbone atoms of cyclic part, respectively.

Figure 4 .

 4 Figure 4. (A) Scatter plots of the side chain dihedral angles (χ 1 :N-Cα-Cβ-Cγ1 of Igl/D-Igl 2 , χ 1 :N-Cα-Cβ-Cγ and χ 2 :Cα-Cβ-Cγ-Cδ1 of Phe 3 ) of aromatic residues of [L-Igl 2 ]AVP (I), [D-Igl 2 ]AVP (II), [Mpa 1 ,L-Igl 2 ]AVP (III) and [Mpa 1 ,D-Igl 2 ]AVP (IV) and (B) the relationship between the flat angle between the planes of aromatic rings (F Igl-Phe ) and dihedral angle between two planes, where

Figure 5 .

 5 Figure 5. The complexes of the structure with the highest statistical weight of (A) [L-Igl 2 ]AVP (I), (B) [D-Igl 2 ]AVP (II), (C) [Mpa 1 ,L-Igl 2 ]AVP (III) and (D) [Mpa 1 ,D-Igl 2 ]AVP (IV) with SDS micelles. The positively charged residues are displayed in cyan, the uncharged polar residue in blue, the hydrophobic residues in red. Additionally, the sulfur atoms are presented in yellow.

Figure 6 .

 6 Figure 6. Radial distribution functions g(r) between the sulfate head groups on SDS micelle and the side chain (heavy atoms) of each residue. The r parameter defines the distance in Å.

Figure 7 .

 7 Figure 7. Radial distribution functions g(r) between the micelle core and the side chain (heavy atoms) of each residue. The r parameter defines the distance in Å. Note different scale for residues 3 and 7.

  7 ml of a partially deuterated phosphate buffer (90% H 2 O and 10% D 2 O) of pH=7.4 containing about 35 mg of SDSd 25 . The SDS-d 25 :peptide ratio was adjusted to approximately 1:40. The SDS-d 25 concentration exceeded considerably the critical micelle concentration of SDS (8.3 mM), to be sure that the peptides were indeed micelle-bound.

	NMR measurements. All the NMR experiments were recorded on a 500 MHz Varian spectrometer
	equipped with a Performa II gradient generator unit, WFG, Ultrashims, a high stability temperature
	unit and a 5 mm

1

H{

13

C/

15

N} PFG triple resonance inverse probe head (Intercollegiate NMR Laboratory, Faculty of Chemistry, Gdansk University of Technology, Poland). The 2D NMR spectra were taken at 30°C. The temperature coefficients of the amide proton chemical shifts were measured from 1D NMR spectra at 22, 25, 30, 35, 40, 45 and 50 o C. Proton

Table 1 .

 1 Structural statistics for the set of the conformations of [Igl 2 ]AVP (I), [D-Igl 2 ]AVP (II), [Mpa 1 ,Igl 2 ]AVP (III) and [Mpa 1 ,D-Igl 2 ]AVP (IV) constituting 60% of the ensemble.

	Peptide	I		II		III		IV
	Statistic
	NOE connectivities:
	Intraresidual	56		60		65		75
	Sequential	14		19		23		28
	Medium-range	5		4		7		12
	Long-range	0		0		1		0
	Sd in peak volume:	1.4		0.6		0.9		2.9
	3 JHNHα:	Jexp	Jcalc	Jexp	Jcalc	Jexp	Jcalc	Jexp	Jcalc
	Cys 1 /Mpa 1	n.o.	n.o.	n.o.	n.o.	-	-	-	-
	L-Igl 2 /D-Igl 2	6.4	6.6	n.o.	n.o.	7.4	5.9	6.5	5.0
	Phe 3	9.7	9.4	8.0	8.9	9.7	9.4	n.o.	n.o.
	Gln 4	6.9	7.7	7.7	8.4	n.o.	n.o.	8.4	7.9
	Asn 5	6.9	6.6	n.o.	n.o.	n.o.	n.o.	n.o.	n.o.
	Cys 6	n.o.	n.o.	9.6	9.0	n.o.	n.o.	6.5	7.8
	Pro 7	-	-	-	-	-	-	-	-
	Arg 8	10.2	10.3	8.2	7.9	7.7	8.2	8.5	8.8
	Gly 9	5.4	5.2	5.6	5.2	5.6	5.1	5.7	5.3
	Sd in coupling constant:	0.4		0.7		0.9		1.0
	The numbers of conformations	194		111		23		42
	constituting 60% of the ensemble
	Atomic r.m.s. differences (Å):
	Backbone atoms 1-6	0.288		0.276		0.226		0.195
	Heavy atoms 1-6	0.908		0.680		0.786		0.746
	Conformational properties:
	Reverse structures	2,3 βI 6,7 βI or III 7,8 βI or IV	2,3 βI or IV 3,4 βI or IV	2,3 βII 3,4 βIII' 7 γ* 4,5 β I' or II'	2,3 βII' 3,4 βI
	Hydrogen bonds	HN 4 -CO 1 HN 8 -CO 5	HN 2,3 -εCO 4	HN 5,6 -CO 2 HN 4 -CO 1	s.c. 8 -s.c. 5
		HN 9 -CO 5			HN 8 -CO 6
		s.c. 8 -s.c. 5
	Radius of gyration (Å):
	Heavy atoms	6.3		7.1		6.2		6.4
	Heavy atoms 1-6	5.3		5.3		4.9		5.0
	Sd -standard deviation
	n.o. -not observed
	s.c. -side chain

Table 2 .

 2 Hydration numbers for each residue of [Igl 2 ]AVP (I), [D-Igl 2 ]AVP (II), [Mpa 1 ,Igl 2 ]AVP (III) and [Mpa 1 ,D-Igl 2 ]AVP (IV) in SDS micelle/water system. The hydration numbers were averaged over the conformations with significant weights -comprising 60% of the ensemble for each analogue.

			Carbonyl oxygen			Side chains
	Residue	I	II	III	IV	I	II	III	IV
	Cys 1 /Mpa 1	0.21	0.15	0.55	0.15	0.34	0.21	0.30	0.16
	L-Igl 2 / D-Igl 2	0.09	0.00	0.02	0.40	0.05	0.01	0.01	0.05
	Phe 3	0.20	0.35	0.21	0.57	0.01	0.15	0.01	0.00
	Gln 4	0.36	0.19	0.56	0.64	0.27	0.25	0.48	0.17
	Asn 5	0.01	0.56	0.59	0.0	0.08	0.35	0.28	0.33
	Cys 6	0.55	0.32	0.33	0.21	0.33	0.20	0.27	0.02
	Pro 7	0.78	0.57	0.12	0.00	0.24	0.31	0.00	0.00
	Arg 8	0.55	0.77	0.47	0.47	0.28	0.20	0.20	0.28
	Gly 9	0.61	0.88	0.81	0.58	-	-	-	-

Acknowledgements. This work was supported by the Polish Scientific Research Committee Grant

No. N N204 181736 . The calculations were carried out in the Academic Computer Centre (TASK) in Gdańsk, Poland.

Appendix A. Supplementary materials. Supplementary data associated with this article can be found, in the online version.


WestChem Alan Cooper
email: alanc@chem.gla.ac.uk

Malcolm W Kennedy

Biofoams and natural protein surfactants

Keywords: Protein, Foam, Surfactant, Frog, Nest, Latherin, Ranaspumin, Ranasmurfin

Naturally occurring foam constituent and surfactant proteins with intriguing structures and functions are now being identified from a variety of biological sources. The ranaspumins from tropical frog foam nests comprise a range of proteins with a mixture of surfactant, carbohydrate binding and antimicrobial activities that together provide a stable, biocompatible, protective foam environment for developing eggs and embryos. Ranasmurfin, a blue protein from a different species of frog, displays a novel structure with a unique chromophoric crosslink. Latherin, primarily from horse sweat, but with similarities to salivary, oral and upper respiratory tract proteins, illustrates several potential roles for surfactant proteins in mammalian systems. These proteins, together with the previously discovered hydrophobins of fungi, throw new light on biomolecular processes at air-water and other interfaces. This review provides a perspective on these recent findings, focussing on structure and biophysical properties.

A C C E P T E D M A N U S C R I P T Introduction

Fluid-based froths and foams are inherently unstable entities and relatively rare in biology. Formation of bubbles in liquids, and particularly in water, requires considerable energy input to overcome high surface tension and increased surface energy at the exposed gas-liquid interface. Consequently, foams are energetically expensive to make and difficult to maintain, with a tendency to collapse over time unless stabilised mechanically or kinetically by additional processes. Stability at the molecular level is an additional major issue for foams made out of biological macromolecules, since the surface tension forces at the air-water interface are often sufficient to disrupt macromolecular conformations . Most proteins are potentially susceptible to surface effects and inadvertent foaming will often lead to denaturation.

As a result, denatured proteins often display surfactant properties, presumably due to aberrant exposure of hydrophobic groups. In the biological context, resistance to microbial degradation, predation, and other environmental challenges is a significant issue. Moreover, for biofoams that come into contact with delicate biological tissues, damage to cell membranes that might result from conventional surfactant activity must somehow be avoided or averted. It is perhaps therefore not surprising that biological foam and surfactant activity is relatively uncommon, except in special instances. The purpose of this review is to examine some such special instances, and to summarize recent developments in the study of the structure(s) and function(s) of a range of proteins with natural foam and surfactant activities in instructive biological contexts. Relevant earlier work on surfactant proteins and peptides has been reviewed by others elsewhere and will be only briefly summarized here, allowing us to focus on more recent studies, mainly from our own group together with the beginnings of similar investigations in other systems.

The general physics of foams and surfactants

Soap bubbles, foams and related surfactant activities are not only a source of childhood (and childish adult) fascination, but also a rich source of intriguing physics and physical chemistry . William Thomson (Lord Kelvin) speculated that the structure of the ether could be likened to that of a foam . Using geometric arguments, based partly on earlier empirical rules of Plateau, he showed that an ideal 3-dimensional foam would comprise body-centred cubic packing of 14-sided polyhedra

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(tetrakaidecahedron, or "Kelvin cell" lattices) which minimises the total surface area in a space packed with identical units. More recently, using computational methods, Weaire and Phelan showed that a more complicated polyhedral arrangement satisfied this criterion slightly better. But reality is a little more complicated, and Kelvin cells or Weaire-Phelan structures are rarely seen in practice. When first formed, "wet" foams are usually made up of spherical (air) bubbles separated by relatively thick films of liquid (water), giving the traditional "kugelschaum" (spherical bubble) structure. As the liquid drains (under gravity or by capillary action) the lamellae between bubbles get thinner and the "dry" foam takes on a more irregular polyhedral ("polyederschaum") structure. But such foams are physically unstable. The excess pressure inside a bubble is inversely proportional to its radius. For a spherical bubble the excess pressure (∆P) is given by: ∆P = 2γ/r , where γ is the surface tension of the liquid. Also, and not unrelated, because surface area-to-volume ratios are higher, the excess surface free energy of liquid (water) molecules exposed to the air interface is higher for small bubbles compared to large. Consequently, given the opportunity, bubbles will tend to burst (at the surface of the foam), and smaller bubbles (with higher excess pressure and surface energies) will tend to coalesce to form larger bubbles. Air may also diffuse across thin liquid films from smaller to larger bubbles, likewise leading to eventual collapse of the foam.

Foam stability therefore depends on numerous kinetic and non-equilibrium processes related to viscosity, surface tension, drainage, diffusion, capillarity, and so forth.

Initial formation of foams or bubbles is facilitated by reduction in surface tension, and this is the basis for the everyday experience with soaps and detergents. Water is acknowledged to be an unusual liquid in almost all respects, and has an unusually high surface tension (excess surface energy) compared to most other fluids, related to the characteristic tetrahedral hydrogen-bonded structure in the bulk liquid. Water molecules at the air-water interface must adopt a less satisfactory packing arrangement that, crudely speaking, leaves fewer intermolecular H-bonds intact.

Soaps, detergents, lipids and other amphiphilic molecules can reduce this effect by forming (mono)layers at the interface, exposing less polar functional groups to the air whilst presenting a more water-compatible surface to the bulk liquid. interface with depth resolution of order 1-3 Å along an axis normal to the plane of the interface.

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Slightly more molecular detail can be provided by surface spectroscopy techniques such as infrared reflection absorption spectroscopy (IRRAS) . In favourable circumstances this can give information about the structure and orientation of molecules in the surface layers, based on characteristic IR absorption bands. With peptides and proteins, for example, this can give estimates of secondary structure (helix, sheet) content and relative orientation at the air-water interface.

Foams and surfactants in biology

Probably the largest foam masses of natural biological origin are those seen on the seashore or in turbulent freshwater streams, usually resulting from the adventitious agitation of natural organic materials or detritus, but without any obvious function relevant to the organisms involved in producing the foam constituents . One exception is the recently described foam accumulations associated with the synchronous reproductive stages of a species of marine tunicate (sea squirt) that appears to enhance fertilization of eggs and assist in the settling and retention of the larvae during

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spawning . These foams are found on rocky beaches and tidal channels in inter-tidal regions in Chile, where tunicate eggs and larvae would otherwise be dispersed by wave activity. The foam appears to be formed by the action of turbulent aerated seawater on materials released in large quantities by the tunicate colonies during spawning, though the precise composition of this material has not yet been described.

Consequently, the energy invested by the tunicates is confined to the synthesis and release of the foam precursor materials, whilst the foaming stage itself relies on wave energy. This is in contrast to most other cases of biofoam production, where both stages of foam production require direct action by the animals concerned.

The largest foam masses created by land or semi-aquatic animals are the foam nests of various species of tropical and sub-tropical frog (see Figure 1 for example). These are remarkable biological materials that, depending on the species, are adapted to persist intact in underground burrows, floating on microbe-infested temporary pools, or higher up in vegetation overhanging water. These foams are stable, resilient to physical and biological environmental challenges yet must be compatible with the membranes of delicate reproductive stages. Because frogs are external fertilisers, this biocompatibility must apply to both naked eggs and spermatozoa, as well as to developing embryos. As we shall see, these frog nest foams are not based on conventional small molecule surfactants, but rather depend on specialised surfactant proteins in synergy with a range of other proteins that can act together to protect the foams against microbial and parasitic attack, as well as providing structural stability for the foam.

As with the frogs and tunicates, biofoams are commonly associated with animal reproduction, often in the protection of untended stages such as fertilised eggs or sensitive juvenile phases. Examples would include the several species of freshwater fish (armoured catfish, Japanese fighting fish) that produce floating layers of foam to protect their eggs, apparently using mucus from their gills or oral cavities . Amongst land animals, locusts and preying mantids lay their eggs in foams, but the most commonly seen foams are those enclosing the larvae of Hemipteran insects (leafhopper; froghopper; spittle bugs; "cuckoo spit"). Little is yet known of the composition and molecular structures of any of these foams, though the spittlebug froth is reported to be made up of a complex and poorly characterized mixture of

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glycoproteins and proteoglycans , and the mineral composition of the foam resembles that of the xylem sap upon which the insect feeds .

Surfactant (as opposed to foam) activity is seen in cases where wetting of non-polar surfaces is required, with protein-based systems including hydrophobins, latherin and lung surfactants to be described below. Other natural surfactants, mostly of plant or microbial origin, are predominantly lipid-based or conform to established concepts of low molecular weight detergents and have been extensively reviewed elsewhere .

Non-specific or adventitious foam or froth formation is often seen with mucins, for example in saliva, slimes and egg jellies , but here we will concentrate on more definitive protein surfactants produced by vertebrate animals.

Protein foams and surfactants

The relatively non-specific foaming of denatured proteins is commonplace and widely exploited in food technology and other processes . However, this usually requires much higher protein concentrations (typically > 10 mg ml -1 ) and much more vigorous physical treatment (whipping and sparging) than is the case with the specialised surfactant and related proteins to be described here. The process is generally acknowledged to be associated with the higher hydrophobicity and/or increased viscosity of denatured protein in which physical entrapment of air bubbles is facilitated in concentrated viscous mixtures . This is usually the dominant mechanism in common culinary processes such as the whipping of cream or preparation of meringue from egg white, for example. Other familiar examples include the use of protein products to stabilise "instant whips", beer foam, and other products.

Denatured, fluorinated whey and soy proteins are also used on a large scale in firefighting foams .

By contrast, natural surfactant activity and deliberate foaming appears relatively rarely in biology. But there are some notable exceptions, including the lung surfactants that allow alveolar expansion and also act as a defence against inhaled pathogens , surfactin and other microbial lipopeptide surfactants , milk caseins , and, most notably, the hydrophobins of filamentous fungi that reduce surface tension and facilitate mycelial growth in thin water layers and at air-water interfaces . Pulmonary surfactants are made up of a mixture of proteins (ca. 10%) and phospholipids (ca. Hydrophobins have been described as the most powerful surface-active proteins known and, like the proteins from frogs and horses that we discuss below, their activity is intrinsic to the proteins themselves, independent of any obligatory association with lipids or carbohydrates. They are small proteins (7-9kDa), unique to filamentous fungi where they are secreted during growth and spread of these fungi.

By lowering the water surface tension, the hydrophobins make it easier for the growing hyphae to penetrate through the air-water interface , subsequently forming a protective coating on the aerial structures and spores. Hydrophobins are also involved in attachment of fungi to surfaces such as plant leaves or insect cuticles . The proteins exhibit a characteristic four-disulphide bridge motif and a distinct amphipathic tertiary structure related to their self-assembly and surfactant properties . Each monomer has a discrete hydrophobic patch that is thought to be involved in interaction with an identical partner protein that obscures the hydrophobic region. This permits miscibility with the bulk water phase until reaching an air-water interface or other non-polar surface, where, they probably dissociate and re-orient with the hydrophobic surface exposed to the interface, in a process similar to the micellar rearrangement mode of action assumed for conventional small molecule amphiphilic detergents. This

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process would not necessarily require any significant conformational change in the protein, which in any case would likely be precluded by the stability imposed by intra-molecular disulphide bridging in these proteins .

Although not strictly a surfactant in terms of this current review, it is worth making reference to the flocculant activity of Moringa oleifera seed protein that is attracting interest as a means of water treatment in deprived areas . The seeds of this tropical tree have been used in traditional water cleaning processes in parts of Africa, and recent biophysical work has characterized one of the small (6.5-13 kDa) proteins present in the seed extracts that can adsorb to hydrophilic surfaces .

Foam nest proteins

As might be gathered from the brief summary so far, with the notable exception of the hydrophobins, there appear to be few examples of proteins evolved specifically to have functionally significant foam or surfactant properties. However, recent work has identified interesting and previously unexplored occurrences of protein surfactants in natural foams, particularly in the protein foams that occur most frequently as a means of protecting delicate organisms in potentially adverse conditions . An interesting outcome of this work has been the realisation that biofoams usually require not only surfactant proteins, but also a cocktail of other molecules that can act synergistically to give longer term physical and biochemical stability in the natural environment .

Ranaspumins -a multifunctional mixture of surfactant and protective proteins

The use of protein-based foams is most evident in tropical frogs, where the amphibian lifestyle usually requires a moist, bio-compatible, protective environment for the development of eggs and embryos. This can be difficult in tropical climates, and numerous strategies have evolved. Foam nesting is one such strategy developed as a way to protect eggs and tadpoles against environmental challenges. For example, Engystomops pustulosus (previously named Physalaemus pustulosus) -the common mud puddle "túngara" frog of Central/South America and parts of the Caribbeanproduces voluminous protein foam nests containing fertilized eggs. These foam nests are stable for several days under exposed tropical conditions, and protect the developing embryos and juveniles against dehydration, predation and microbial

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degradation. They also provide a more stable temperature environment and act as mini-incubators to facilitate rapid development of eggs and tadpoles . Measurements in the wild (see supplementary material to ) show that temperatures within the nest are usually slightly higher than the surroundings, most likely due to a local greenhouse effect as incident solar radiation is trapped within the insulating foam. The trapped air bubbles and restricted convection within the foam will reduce thermal losses and, as also suggested for the bubble nests of some fish , this insulation might serve to buffer the developing eggs and larvae against extremes of temperature fluctuation.

Foam nests of E. pustulosus are produced overnight at the edges of puddles, ditches or other temporary standing water after rainfall. During mating, the female produces clutches of eggs together with foam precursor fluid which the male, clinging to the back of the female and using his back legs in a rapid "egg-beater" motion, whips into a white foamy mass incorporating the fertilized eggs . These nests, often in larger communal masses, remain attached to adjacent soils and/or vegetation as water levels subside, and the parents take no further interest in subsequent development.

Embryogenesis takes place over the next 1-2 days, and tadpoles are ready to leave the nest at about 3 days under normal conditions -though they can remain longer if water is not available. In the absence of developing eggs or tadpoles, the foams remain stable and intact for at least 10 days in tropical conditions, with only marginal dehydration and no sign of bacterial or fungal degradation. This is surprising considering the microbial content of the waters in which these nests are produced.

Microbiological analysis (unpublished) shows that the foams produced in the wild are contaminated with a rich variety of organisms, growth of which seems to be inhibited in the foam. The compatibility of the foam with eggs and sperm suggests that this microbial resistance is not due to the membrane disruption that might be expected from simple detergent activity, but is a more complex property of the foam components. Why should the nest foam contain so many putative lectins? One role may be to assist in long-term foam stabilization by formation of a cross-linked carbohydrate network at the air-water interface. As described above, foams are inherently unstable entities, and reduction in surface tension alone is insufficient to preserve aqueous foam structures over long periods. Most liquid foams collapse within minutes or hours, not the many days required of frog foam nests, except when stabilized by crosslinking or high viscosity. None of the foam proteins investigated so far is glycosylated, nor are there any consensus N-glycosylation sites present in their amino acid sequences, yet the foam fluid contains separately a significant amount of complex carbohydrate.

Non-covalent binding of carbohydrate chains to lectins in the interface layer could provide a stabilizing matrix that would aid both foam stability and water retention, as pictured in Figure 2. Other macromolecules (in addition to the lectins) with which the carbohydrates may be associated have not yet been identified, but could be mucins, which would serve also to increase the viscosity of the matrix of foam nests, particularly in those of frogs that produce the more rigid aerial nests. Mucins are common to frog egg jellies, and may additionally act to restrain microbial colonisation.

Carbohydrate binding proteins may also form part of the antimicrobial defence system in the nest. Lectins are normally unable to kill bacteria in the absence of accessory proteins, but can agglutinate particles bearing their target sugars. Consequently, their role in frog nest foams may be to restrain microbial dissemination and colonisation of the foam and eggs and to inhibit microbial activity by blocking cell surface receptors.

Exactly this function is thought to apply to the fish fucolectins, which are present in large amounts in gills, eggs and blood . However, as a possible exception to this rule, killing of bacteria by specific lectins in the absence of accessory proteins has recently been demonstrated for E. coli expressing human blood group antigens . This raises the possibility that foam lectins may have a more direct antimicrobial role than has currently been observed. Furthermore, it is also worth noting that plant lectins, most

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notably in seeds and beans, can also act as anti-feedants and deter parasitism and predation by insects, birds and mammals through the disruptive effects that lectins can have on the epithelium of the gut. The role of surfactants as a defence against predators, recently demonstrated for insects , may also be relevant here.

The structure of one of the proteins in this mixture (RSN-2) has been examined in detail, both in solution by high-resolution NMR and (to much lower resolution) by neutron reflectivity and IRRAS at the air-water interface . As mentioned above, RSN-2 was predicted to have surfactant properties on the basis of its unusual amphiphilic amino acid sequence. This has turned out to be correct, but perhaps not for the reasons first imagined. The relatively non-polar N-terminal sequence (LILDGDLLK-) coupled with the remarkably polar C-terminus (-RKDDDDDDGY) is reminiscent of the polar head/non-polar tail motif of traditional small-molecule detergents, though on a somewhat larger scale. Recombinant RSN-2 reduces surface tension markedly at concentrations an order of magnitude lower than the natural foam mixture. Also, upon agitation, it produces a foam similar to that seen in the natural material, though this foam collapses quite quickly, supporting the notion that other components of the mix are required for longer-term stability. But, the amphiphilicty that might be expected from both primary structure and surfactant properties is not immediately apparent in the 3-dimensional structure of the molecule, as determined by high-resolution NMR in solution (Figure 3). More specifically, there are no significant discrete patches of polar or nonpolar amino acids on the protein's surface in bulk solution. Nor, unlike the hydrophobins does RSN-2 show any tendency to aggregate or self-assemble in solution. Furthermore, despite its significant surfactant More generally, the picture that is revealed here from the túngara frog foam is of a fascinating synergy involving a range of specialized proteins with a mix of useful properties that work together to meet the requirements of a biocompatible foam, sufficiently robust and biochemically stable to act as a temporary nest. As illustrated in Figure 2, the surfactant activity of ranaspumins is just one part of a possible mechanism in which initial foam formation is further stabilized by self-assembly of a protein-carbohydrate matrix at the interface, providing longer term physical stability

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and water retention. And several of these proteins appear to have dual/multiple functions including protease inhibition and other roles in inhibiting predation and microbial degradation.

Ranasmurfin -a blue protein with a new type of protein chromophore Foam nest components from other frog species have not yet been studied

systematically. However, one Asian species, Polypedates leucomystax (striped tree frog, or Java whipping frog), has been examined in some detail and shows significant differences when compared to the ranaspumins described above. P. leucomystax seem to rely more on viscosity than surfactant activity for initial stability of the nest foam.

These frogs, common and widespread in south and east Asia, produce a sticky, syrupy fluid (from the female, together with eggs) that is whipped up by the mating pair in much the same way as described above for E. pustulosus to form a protective environment for developing eggs and tadpoles. However, unlike the túngara frog, these nests are produced out of water, attached to overhanging vegetation or structures so that tadpoles may drop into underlying pools when ready. Thus, almost the entire burden of the nest (foam fluid plus eggs) is carried by the female, and this is possibly why the female is generally much larger than the male in this species. (Similar behaviour is seen in the African foam nesting tree frog, Chiromantis xerampelina , though the proteins of this species have yet to be examined.) The total protein concentration in P. leucomystax foam fluid is 2-4 mg ml -1 , depending on sample, with about 1-1.5 mg ml -1 carbohydrate (Rosalind Tan, Malcolm Kennedy & Alan Cooper, unpublished).

Although full analysis of the protein components of the P. leucomystax nest foam is not yet complete, one particularly intriguing protein has been examined in some detail at the molecular level . An unusual feature is that, although unpigmented or pale creamy pink/orange when first produced, some of these nests subsequently develop a streaky blue/green pigmentation that is more pronounced when nests are physically disrupted. The purpose (if any) of this pigmentation is not yet known, nor is it clear why not all nests undergo this colour change in the wild, but the colour is associated with a specific and quite unusual protein, designated ranasmurfin. This was first observed by SDS PAGE analysis of natural nest material, during which a brilliant turquoise blue band, corresponding to a protein of around 28 kDa, was observed

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migrating on the (unstained) electrophoresis gels. Subsequent purification and characterization of this protein from natural material has confirmed its uniqueness.

Although cDNA encoding this protein has not yet been isolated, a combination of fortuitous circumstances allowed high resolution structure determination. The purified natural protein crystallised readily as bright blue crystals that diffracted well; the protein was found to contain a heavy metal atom -identified as zinc by X-ray fluorescence and metal analysis -that facilitated phasing of the X-ray diffraction data and structure determination. The resulting electron density map was of sufficient quality (1.1 Å resolution) that, together with mass spectrometry of peptide fragments, the amino acid sequence could be determined directly.

The structure of ranasmurfin is shown in Figure 4, revealing an unusual dimeric structure with a novel fold . Also apparent in the structure are several posttranslational modifications, including an unusual extended chromophoric co-factor confirmed by chemical and spectroscopic evidence to be an N-linked indophenol-type moiety of a type not previously observed and comprising a Lys-Tyr-Tyr-Lys crosslink that unites the dimeric protein structure. This, together with two histidine sidechains (one from each monomer), coordinates the zinc (presumably Zn 2+ ) to form the blue chromophore.

By analogy with similar co-factors found in other systems, it is possible that ranasmurfin is involved in an extensive protein crosslinking function, particularly at its exposed surface, to promote long-term stabilization of the foam nest. Alternatively, or simultaneously, because of its unusual spectral properties, it may also be part of a sunscreen mechanism that protects the unpigmented eggs and embryos in nests exposed to tropical sunlight, or simply provides camouflage for the otherwise highly noticeable nests.

The other components of this foam have yet to be analysed but, interestingly, the natural, unfractionated nest material shows protease inhibition (cystatin-like) activity as potent as that found in foam nests of the túngara frog . This suggests that a similar cocktail of anti-microbial and anti-feedant components may also be present in the foam, providing short-to medium-term protection against biochemical degradation as well as mechanical stability.

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Latherin -a mammalian surfactant protein One of the first proteins shown to have strong surfactant properties in its native state is latherin, which is found in the sweat of horses, and recently shown also to occur in horse saliva . It is latherin's significant surfactant activity that gives rise to the familiar foam in the horse pelt, formed by friction during vigorous exercise . The protein is characterised by an unusually high leucine content (ca. 24% leucine, compared to an average of around 10% for most other proteins), which may directly relate to its surface properties. Bacterial recombinant latherin is also strongly surface active, so this surfactance is intrinsic to the protein and not dependent on association with lipids or carbohydrate. Foaming is probably merely a side effect of its surfactant properties, which more likely have evolved to enhance the wetting of the horsehair and facilitate the rapid translocation of sweat water from the skin to the oily surface of the pelt to improve evaporative cooling. The reason for the presence of the protein in saliva is not quite so obvious, though it is speculated that it could be involved in the prevention of adhesion of the surfaces of muco-cutaneous surfaces in the oral cavity, throat and associated structures, like the pulmonary surfactant proteins in the lower respiratory tract. It could also be that salivary latherin acts to facilitate wetting of the dry forage for which equines are specialised, allowing more efficient mastication and penetration by digestive enzymes. The amino acid sequence of latherin shows that it is related to the palate, lung, and nasal epithelium carcinoma associated proteins (PLUNC) proteins that are relatively abundant in the salivary glands and oral cavities of humans and mice . The biological function of the PLUNCs is not understood, but it has been noticed that some of them have leucine compositions very similar to that of latherin, such that they may also have surface active properties that may be relevant to their functions . Subsequently, one of the leucine-rich PLUNCs has indeed been shown indeed to be highly surface active, and this property may explain the ability of the protein to inhibit the growth of biofilms . It follows, therefore, that the surfactant activity of latherin may have a dual role in enhancing the speed of evaporative heat loss in an exercising horse and protection of its pelt from microbial growth.

Evolutionary aspects

There is as yet insufficient sequence information available to allow sensible conclusions regarding evolutionary relationships (if any) between the various foam

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and surfactant proteins examined so far. Latherins with only a few differences in amino acid sequence have been shown for all equids examined to date , and the similarities with salivary and PLUNC proteins clearly indicates a close evolutionary relationship. Other than in horses, there is no indications yet that latherins or PLUNCs are synthesised in the skin, so it therefore seems likely that equine sweat latherin is descended from a salivary protein.

The situation with frog foam proteins is even less clear, and the recently announced Xenopus tropicalis genome is not yet sufficiently annotated to be helpful here (and Xenopus are, in any case, entirely aquatic and not foam producing species). Foam nesting frogs are found in many tropical regions, both Old and New World, but we can only speculate as to what extent these biogeographically separated and distantly related species have evolved similar strategies, or utilize similar proteins. However, there is no sequence similarity between the E. pustulosus ranaspumins (Americas) and P. leucomystax ranasmurfin (Afro-Asia), nor with putative ranaspumins from other frog species , and preliminary evidence suggests that this lack of similarity persists, at least at the protein level . SDS-PAGE analysis of foam nest components from a number of species shows quite diverse protein signatures even with foam nesting species from the same locality (Caribbean), let alone different continents (S. America, Asia, Africa). This diversity also applies at the macroscopic level, with foam nests from different species having different morphological and rheological propertiessome lightweight and floating on water, others more sticky, viscous, etc.. It is also intriguing to note that the combination of lectin and cystatin activities found in at least one type of frog foam nest is similar to that comprising part of the anti-microbial and anti-insect protection system of plant seeds, albeit achieved with unrelated proteins, representing an intriguing form of convergent evolution. We anticipate that closer analysis of the Xenopus genome will throw more light on the evolutionary aspects here. But, we should also bear in mind that the apparent diversity in the protein composition of the foams of different species might hide similarities in the biochemical activities present, adjusted for the different physical and biological challenges presented by nesting in sites that are diverse in their exposure to infections, parasitism, and biophysical hazards and imperatives.

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During the final stages of compiling this review, an interesting prospect arose relating to the origin of ranasmurfin -the blue protein from foam nests of the Asian frog, P.

Implications and applications

There are already numerous applications of proteins as foams and surfactants in food processing and related activities , where the empirical science of soft matter has been exploited (mostly unawares) for centuries. The food science literature on this topic has been covered in numerous reviews and reports available elsewhere (e.g. ). Such applications are, however, based mainly on the adventitious properties arising from denaturation or other modifications of proteins that are not normally surfactants in the native state. For more natural, specific protein foams and surfactants, although it neither feasible nor desirable that natural sources be exploited for commercial or other bulk applications, the principles derived from the study of these molecules may well

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  coat the narrow airways in the lung. The surfactant activity arises mainly from the phospholipid component, with a range of surfactant and plasma proteins to facilitate spreading and other functions. The four proteins that predominate in mammalian pulmonary surfactant (SP-A, B, C, D) are a mixture of phospholipid and carbohydrate-associated peptides/proteins with monomer masses in the 4-45 kDa range, some of them membrane-associated, that self-assemble in various higher order structures. SP-A and -D are C-type lectins that are probably involved in innate immunity to infections, and the major burden of surfactant activity is attributed to SP-B and -C, which are hydrophobic membrane-associated proteins that enhance the rate at which the surfactant mixture spreads over the surface .Caseins are a heterogeneous class of surfactant/micelle-forming proteins that are now regarded as examples of the growing range of "natively unfolded" proteins with flexible open structures, and with conformational flexibility related to their surfactant and micelle-forming properties . Their surfactant/micelle behaviour remains somewhat controversial and is complicated by significant calcium-binding activity .

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  . pustulosus nest foams are mechanically very stable; they resist both mechanical compression and extension, and do not shear easily, yet are sufficiently elastic to conform to different shapes. When observed under low magnification, the material shows the classic wet-foam/dry-foam (kugelschaum/polyederschaum) structures characteristic of foams in general. Depending on age, extent of drainage, and locationA C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPTwithin the nest, the polyhedral cell structure generally predominates. The overall density of the foam is around 0.1 g cm-3 , so approximately 90% of the structure is air, with the fluid phase made up mainly of water and frog secretions.The foam liquid obtained from natural túngara frog nests by drainage, centrifugation, or sonication of isolated foam shows strong surfactant properties. Contact angle measurements using small droplets on a hydrophobic surface illustrate the excellent wetting and surface tension characteristics of this material, which presumably aids in the attachment of foam nests to the waxy surfaces of adjacent vegetation. Surface tension measurements with serial dilutions of foam fluids show a dramatic reduction in surface tension from the pure water value of around 74 mN m -1 to below 55 mN m -1 at total protein concentrations as low as 10 µg ml -1 . Given that the foam fluid is composed of a mixture of proteins (see below), not all of which necessarily have surfactant properties, this indicates that some of the foam components have much more specific surface tension reduction capability than normally observed even with denatured proteins. This reduction in surface tension is time-dependent, taking several minutes to develop on a freshly exposed surface (foam nesting itself usually takes 1-2 hours). Such kinetic effects are well known from studies of other detergent systems , where static surface tension effects can take some time to mature. With frog foam mixtures, this lag time probably reflects the kinetics of organization of macromolecular components in the air-water interface, or chemical or conformational changes taking place upon arrival at the interface.The foam fluid contains 1-2 mg ml -1 total protein and similar quantities of carbohydrate, predominantly complex crosslinked mixtures of O-and N-glycans (Simon Parry, Jaspinder Bhandal, Stuart Haslam and Anne Dell, Imperial College, personal communication, 2003). There is no detectable fat or lipid, suggesting an absence of conventional small molecule surfactant species. Electrophoresis (SDS-PAGE) analysis of the natural material after removal of eggs shows a number of proteins in the 10-40 kDa range, none of them glycosylated . Intrinsic fluorescence and circular dichroism (CD) spectra of the protein mix are typical of folded proteins, with CD indicating predominantly β-sheet secondary structures, and tests for amyloid have proved negative, ruling out any major involvement of amyloid-like aggregate A C C E P T E D M A N U S C R I P TACCEPTED MANUSCRIPTstructures in these proteins, at least in the bulk phase. The foam fluid also shows interesting carbohydrate binding (lectin) and protease inhibition (cystatin) properties.Aniline-naphthalene sulfonic acid (ANS) fluorescence is enhanced and blue-shifted when mixed with the foam fluid in solution, characteristic of binding of this dye to non-polar regions in proteins . This indicates that at least some of the proteins present in the mixture may contain accessible hydrophobic patches that may be associated with surfactant properties. This has been exploited in two-photon fluorescence excitation microscopy imaging of ANS-treated foam demonstrating the partitioning of hydrophobic or amphipathic components at the air-water interface of foam bubbles.This also allows examination of bubble structure and packing within the bulk of the foam matrix .More detailed analysis of túngara nest foam and sequence analysis of E. pustulosus oviduct mRNA has led to the discovery of six major proteins in the foam, designated ranaspumins (RSN-1 to RSN-6), all of them previously unidentified. Database comparison of these sequences highlights a number of interesting features relating to possible structure and function. RSN-1 shows some sequence similarity with the cystatin (cysteine proteinase inhibitor) family , suggesting a possible antimicrobial role for this protein (though this is yet to be proven). RSN-2 has no counterpart in current databases, but its relative abundance and marked amphiphilic amino acid sequence suggested that this might be one of the major surfactant components in the foam. RSN-3, 4, and 5 are similar to each other and show significant sequence similarities to fucose binding proteins from other aquatic vertebrates (eels and the toad Xenopus) . Sequence-based conformational modelling indicates probable structural similarity with the agglutinin fucolectin (AAA) from the European eel, Anguilla anguilla, whose crystal structure has been determined (PDB: 1K12) , and features of the AAA fucose binding site are preserved in these ranaspumins.Consistent with this prediction, recombinant RSN-4 exhibits lectin activity in agglutination assays using human erythrocytes. However, contrary to the anticipated fucose specificty, RSN-4 lectin activity is not inhibited by fucose, but rather by lactose and galactose, opening up the possibility of useful new specificities of these foam-derived lectins . The sequence of RSN-5 shows an unusual feature in which the hydrophobic N-terminal sequence, predicted to be the leader sequence normally removed during post-translational processing, is still present in intact foam-derived A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT protein. This suggests incorporation of oriented lectin-like activity by tethering of the macromolecule in the amphiphilic air-water interface. RSN-6 is different again, and shows sequence similarity with a class of galactose-binding proteins from aquatic organisms .

  activity, and unlike conventional detergents, trials with phospholipid vesicles (Steven Vance & Alan Cooper, unpublished) indicate that RSN-2 does not disrupt biological membranes, and foam fluid, of which RSN-2 is a major constituent, does not damage human blood erythrocytes (Rachel Fleming & Malcolm Kennedy, unpublished). However, the NMR structure of RSN-2 in solution does not necessarily reflect that at the air-water interface. Closer examination of the structure suggests a possible mechanism in which hinge-bending, clamshell opening of the compact globular structure at the interface would allow separation of the helix and sheet domains, exposing the hydrophobic interior of the protein to the air, whilst retaining more polar A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT surfaces to the water layer (Fig. 3). Experimental support for this model comes from neutron reflectivity and surface IR data . Neutron reflectivity profiles for dilute solutions of purified recombinant RSN-2 are consistent with formation of a relatively thin (8-10 Å) protein layer at the interface. Interestingly this is much thinner than the equivalent layer seen with the natural túngara foam mix , and is also thinner than would be anticipated from a monolayer formed from RSN-2 in its globular, bulk solution conformation. However, it is consistent with the smaller dimensions of the open clamshell model. Further support comes from polarised IRRAS measurements showing that the relative backbone orientations of α-helix and β-sheet components at the interface are consistent with the model. Interestingly, there seems to be no consensus emerging regarding the structural basis for specific protein surfactant behaviour. It would seem to be mandatory, on basic physical chemistry grounds, that surfactant proteins at air/water or other hydrophobic interfaces should present amphiphilic structures, with a polar surface exposed to water and a hydrophobic face to the non-polar phase. But this is generally incompatible with the solubility requirements of monomeric globular protein in aqueous solution. RSN-2 may solve this by being a relatively flexible monomer, burying the hydrophobic surface within the compact globular fold, only to be revealed when required at the interface by hinge-bending, clam-shell opening. The hydrophobins, on the other hand, have much more rigid compact structures, stabilized by conserved networks of intramolecular disulfide bonds with insufficient flexibility to bury the exposed hydrophobic patch of the monomer in solution. This leads to the formation of dimers and higher oligomers in solution that obscure the hydrophobic regions until required at the interface .

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  photosynthesis . The biocompatibility of surfactant RSN-2 made possible the combination of high concentrations of lipid vesicles and coupled enzymes of the photosynthetic pathway in a cell-free foam structure that yielded exceptional photochemical efficiency. We anticipate further such innovative applications as the remarkable properties of these natural materials become more widely appreciated.

Fig. 1 :Fig. 2 :

 12 Fig. 1: Foam nests of the African foam nesting tree frog, Chiromantis xerampelina.Usually found adjacent to water after heavy rain, in overhanging vegetation or, as here, on old tree stumps (MalaMala, Mpumalanga Province, South Africa, January 2010. Photo: AlanCooper)

Fig. 3 :

 3 Fig. 3: (a) NMR solution structure of ranaspumin-2 (RSN-2), the surfactant protein from foam nests of the túngara frog. (b) Hypothetical "open" conformation of RSN-2 that might be adopted at the air-water interface. (Adapted from ). The colour coding signifies chain progression from N-(blue) to C-terminal (red).

Fig. 4 :Fig. 5 :

 45 Fig. 4: (a) X-ray structure of the ranasmurfin dimer, isolated from foam nests of the Malaysian tree frog, Polypedates leucomystax. (b) Expanded view of the unusual Lys-Tyr-N-Tyr-Lys chromophore linking the two subunits. (Adapted from )

Figure 1 A

 1 Figure 1

  Probing this interfacial layer is experimentally challenging and current techniques cannot yet reach the level of atomic or molecular resolution available in other systems. Macroscopic properties of surface films can be measured using traditional surface tension and Langmuir trough techniques, sometimes coupled with optical methods such as Brewster angle microscopy, but these give little direct information about molecular structures and packing arrangements at the interface. Small angle scattering of neutrons or X-rays is potentially more informative, though technically difficult because of the nature of the samples. Small angle neutron scattering (SANS) in particular has been shown to be a powerful method for determining packing densities and layer thicknesses at air-water interfaces , but gives little information about actual molecular structures or lateral assemblies. The technique is based on

measurement of the specular reflection of thermal neutrons in a beam directed at grazing incidence to the liquid surface. Taking advantage of the different neutron scattering length densities of protons and deuterons, experimental neutron reflectivity profiles in different D 2 O/H 2 O mixtures can be fitted to theoretical models yielding estimates of the thickness and volume fractions of surfactant layers at the air-water

  leucomystax. At the time of deposition of the ranasmurfin sequence (April 2008, SwissProt accession code P85511, see ref. ) there were no comparable sequences in available databases. Recently however (March 2010), a short, 49 amino acid sequence encoded in the genome of Methanobrevibacter smithii has appeared in the databases as being sufficiently similar to part of the frog protein to be given the name ranasmurfin (accession code D2ZS31). See Fig. 5 for sequence comparison. M. smithii is an archaean whose genome has been sequenced as part of the Human Microbiome Project (http://nihroadmap.nih.gov/hmp/). This raises the intriguing possibility that the frog ranasmurfin originates from a non-frog gene. There are several interesting possibilities here. Microbial contamination/colonization of nests in the wild by archaeans seems unlikely;the large concentrations of ranasmurfin found in the nests are not accompanied by other proteins sufficiently abundant to indicate a

substantial microbial presence. Instead we may be observing an example of symbiosis between an archaean and a species of frog, or even horizontal gene transfer from the former to the latter. cDNA for ranasmurfin has not been isolated, so we cannot yet determine whether it is encoded within the P. leucomystax genome. Interestingly however, there is at present no indication of a similar gene in the one species of amphibian whose genome has been sequenced (Xenopus tropicalis; ref ). This intriguing phenomenon clearly must be pursued further, as more information on the M. smithii and amphibian genomes and proteomes becomes available .

Acknowledgements

Most of the work described here would not have been feasible without the expertise and enthusiasm of numerous colleagues at home and abroad -too many to list here, but their names may be found in the original publications, and they have our gratitude.

We also gratefully acknowledge funding from the Wellcome Trust, the UK Biotechnology and Biological Sciences Research Council, The Royal Society, and the Carnegie Trust for the Universities of Scotland.