Distribution of amino acids in functional sites of proteins with high melting temperature

The stability of proteins in its native state has an important implication on its function and evolution. The functional site analysis may lead to better understanding of how these amino acid distributions influence the melting temperature of proteins. It has been reported that increasing the fraction of hydrophobic contacts in a protein tends to raise melting temperature; increasing the fraction of repulsive charge contacts decrease the melting temperature and consistent with a destabilizing effect. The role of amino acid distribution as hydrophobic, charged and polar residues in proteins and mainly in its functional sites has been studied. Due to limited data availability, redundancy check and controlled environment parameters, the study was carried out with ten single chain-wild proteins having melting temperature above 80°C at pH 7. The analysis depicts that, the entire protein, hydrophobic residues are more frequent in single chain proteins and charged residues are more frequent in multi-chains proteins. In functional sites of these proteins, hydrophobic and charged residues are equally frequent in single chain proteins and charged residues are very high in multi-chains proteins. But, the polar residue distribution remains same for both single chain and multi-chain proteins and its functional sites.     

Side chain dynamics and alternative hydrogen bonding in the mechanism of protein thermostabilization

To elucidate the mechanism of protein thermostabilization, the thermodynamic properties of small monomeric proteins from mesophilic and thermophilic organisms have been analyzed. Molecular dynamics simulations were employed in the study of dynamic features of charged and polar side chains of amino acid residues. The basic conclusion has been made: surface charged and polar side chains with high conformational mobility can form alternative hydrogen bonded (H-bonded) donor-acceptor pairs. The correlation between the quantitative content of alternative H-bonds per residue and the temperature of maximal thermostability of proteins has been found. The proposed mechanism of protein thermostabilization suggests continuous disruption of the primary H-bonds and formation of alternative ones, which maintain constant the enthalpy value in the native state and prevent a rapid increase of the conformational entropy with the rising temperature. The analysis of the results show that the more residues located in the N- and C-terminal regions and in the extended loops that are capable of forming alternative longer-range H-bonded pairs, the higher the protein thermostability.     

A hydrophilic microenvironment in the substrate-translocating groove of the YidC membrane insertase is essential for enzyme function

The YidC family of proteins are membrane insertases that catalyze the translocation of the periplasmic domain of membrane proteins via a hydrophilic groove located within the inner leaflet of the membrane. All homologs have a strictly conserved, positively charged residue in the center of this groove. In Bacillus subtilis, the positively charged residue has been proposed to be essential for interacting with negatively charged residues of the substrate, supporting a hypothesis that YidC catalyzes insertion via an early-step electrostatic attraction mechanism. Here, we provide data suggesting that the positively charged residue is important not for its charge but for increasing the hydrophilicity of the groove. We found that the positively charged residue is dispensable for Escherichia coli YidC function when an adjacent residue at position 517 was hydrophilic or aromatic, but was essential when the adjacent residue was apolar. Additionally, solvent accessibility studies support the idea that the conserved positively charged residue functions to keep the top and middle of the groove sufficiently hydrated. Moreover, we demonstrate that both the E. coli and Streptococcus mutans YidC homologs are functional when the strictly conserved arginine is replaced with a negatively charged residue, provided proper stabilization from neighboring residues. These combined results show that the positively charged residue functions to maintain a hydrophilic microenvironment in the groove necessary for the insertase activity, rather than to form electrostatic interactions with the substrates.     

Charge Composition Features of Model Single-span Membrane Proteins That Determine Selection of YidC and SecYEG Translocase Pathways in Escherichia coli

We have investigated the features of single-span model membrane proteins based upon leader peptidase that determines whether the proteins insert by a YidC/Sec-independent, YidC-only, or YidC/Sec mechanism. We find that a protein with a highly hydrophobic transmembrane segment that inserts into the membrane by a YidC/Sec-independent mechanism becomes YidC-dependent if negatively charged residues are inserted into the translocated periplasmic domain or if the hydrophobicity of the transmembrane segment is reduced by substituting polar residues for nonpolar ones. This suggests that charged residues in the translocated domain and the hydrophobicity within the transmembrane segment are important determinants of the insertion pathway. Strikingly, the addition of a positively charged residue to either the translocated region or the transmembrane region can switch the insertion requirements such that insertion requires both YidC and SecYEG. To test conclusions from the model protein studies, we confirmed that a positively charged residue is a SecYEG determinant for the endogenous proteins ATP synthase subunits a and b and the TatC subunit of the Tat translocase. These findings provide deeper insights into how pathways are selected for the insertion of proteins into the Escherichia coli inner membrane.     

Side Chain Dynamics and Alternative Hydrogen Bonding in the Mechanism of Protein Thermostabilization

Abstract

To elucidate the mechanism of protein thermostabilization, the thermodynamic properties of small monomeric proteins from mesophilic and thermophilic organisms have been analyzed. Molecular dynamics simulations were employed in the study of dynamic features of charged and polar side chains of amino acid residues. The basic conclusion has been made: surface charged and polar side chains with high conformational mobility can form alternative hydrogen bonded (H-bonded) donor-acceptor pairs. The correlation between the quantitative content of alternative H-bonds per residue and the temperature of maximal thermostability of proteins has been found. The proposed mechanism of protein thermostabilization suggests continuous disruption of the primary H- bonds and formation of alternative ones, which maintain constant the enthalpy value in the native state and prevent a rapid increase of the conformational entropy with the rising temperature. The analysis of the results show that the more residues located in the N- and C-terminal regions and in the extended loops that are capable of forming alternative longer-range H-bonded pairs, the higher the protein thermostability.     

Structural and genomic correlates of hyperthermostability

While most organisms grow at temperatures ranging between 20 and 50 degrees C, many archaea and a few bacteria have been found capable of withstanding temperatures close to 100 degrees C, or beyond, such as Pyrococcus or Aquifex. Here we report the results of two independent large scale unbiased approaches to identify global protein properties correlating with an extreme thermophile lifestyle. First, we performed a comparative proteome analyses using 30 complete genome sequences from the three kingdoms. A large difference between the proportions of charged versus polar (noncharged) amino acids was found to be a signature of all hyperthermophilic organisms. Second, we analyzed the water accessible surfaces of 189 protein structures belonging to mesophiles or hyperthermophiles. We found that the surfaces of hyperthermophilic proteins exhibited the shift already observed at the genomic level, i.e. a proportion of solvent accessible charged residues strongly increased at the expense of polar residues. The biophysical requirements for the presence of charged residues at the protein surface, allowing protein stabilization through ion bonds, is therefore clearly imprinted and detectable in all genome sequences available to date.     

Use of a potential of mean force to analyze free energy contributions in protein folding.

A method for calculation of the free energy of residues as a function of residue burial is proposed. The method is based on the potential of mean force, with a reaction coordinate expressed by residue burial. Residue burials are calculated from high-resolution protein structures. The largest individual contributions to the free energy of a residue are found to be due to the hydrophobic interactions of the nonpolar atoms, interactions of the main chain polar atoms, and interactions of the charged groups of residues Arg and Lys. The contribution to the free energy of folding due to the uncharged side chain polar atoms is small. The contribution to the free energy of folding due to the main chain polar atoms is favorable for partially buried residues and less favorable or unfavorable for fully buried residues. Comparison of the accessible surface areas of proteins and model spheres shows that proteins deviate considerably from a spherical shape and that the deviations increase with the size of a protein. The implications of these results for protein folding are also discussed.     

Protein-protein interactions; coupling of structurally conserved residues and of hot spots across interfaces. Implications for docking

Hot spot residues contribute dominantly to protein-protein interactions. Statistically, conserved residues correlate with hot spots, and their occurrence can distinguish between binding sites and the remainder of the protein surface. The hot spot and conservation analyses have been carried out on one side of the interface. Here, we show that both experimental hot spots and conserved residues tend to couple across two-chain interfaces. Intriguingly, the local packing density around both hot spots and conserved residues is higher than expected. We further observe a correlation between local packing density and experimental deltadeltaG. Favorable conserved pairs include Gly coupled with aromatics, charged and polar residues, as well as aromatic residue coupling. Remarkably, charged residue couples are underrepresented. Overall, protein-protein interactions appear to consist of regions of high and low packing density, with the hot spots organized in the former. The high local packing density in binding interfaces is reminiscent of protein cores.     

Electrostatic Environment of Proteorhodopsin Affects the pKa of Its Buried Primary Proton Acceptor

The protonation state of embedded charged residues in transmembrane proteins (TMPs) can control the onset of protein function. It is understood that interactions between an embedded charged residue and other charged or polar residues in the moiety would influence its pKa, but how the surrounding environment in which the TMP resides affects the pKa of these residues is unclear. Proteorhodopsin (PR), a light-responsive proton pump from marine bacteria, was used as a model to examine externally accessible factors that tune the pKa of its embedded charged residue, specifically its primary proton acceptor D97. The pKa of D97 was compared between PR reconstituted in liposomes with different net headgroup charges and equilibrated in buffer with different ion concentrations. For PR reconstituted in net positively charged compared to net negatively charged liposomes in low-salt buffer solutions, a drop of the apparent pKa from 7.6 to 5.6 was observed, whereas intrinsic pKa modeled with surface pH calculated from Gouy-Chapman predictions found an opposite trend for the pKa change, suggesting that surface pH does not account for the main changes observed in the apparent pKa. This difference in the pKa of D97 observed from PR reconstituted in oppositely charged liposome environments disappeared when the NaCl concentration was increased to 150 mM. We suggest that protein-intrinsic structural properties must play a role in adjusting the local microenvironment around D97 to affect its pKa, as corroborated with observations of changes in protein side-chain and hydration dynamics around the E-F loop of PR. Understanding the effect of externally controllable factors in tuning the pKa of TMP-embedded charged residues is important for bioengineering and biomedical applications relying on TMP systems, in which the onset of functions can be controlled by the protonation state of embedded residues.     

Effect of variations in the structure of a polyleucine-based alpha-helical transmembrane peptide on its interaction with phosphatidylglycerol bilayers

High-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy were used to study the interaction of a cationic alpha-helical transmembrane peptide, acetyl-Lys(2)-Leu(24)-Lys(2)-amide (L(24)), and members of the homologous series of anionic n-saturated diacyl phosphatidylglycerols (PGs). Analogues of L(24), in which the lysine residues were replaced by 2,3-diaminopropionic acid (L(24)DAP), or in which a leucine residue at each end of the polyleucine sequence was replaced by a tryptophan (WL(22)W), were also studied to investigate the roles of lysine side-chain snorkeling and aromatic side-chain interactions with the interfacial region of phospholipid bilayers. The gel/liquid-crystalline phase transition temperature of the host PG bilayers is altered by these peptides in a hydrophobic mismatch-dependent manner, as previously found with zwitterionic phosphatidylcholine (PC) bilayers. However, all three peptides reduce the phase transition temperature and enthalpy to a greater extent in anionic PG bilayers than in zwitterionic PC bilayers, with WL(22)W having the largest effect. All three peptides form very stable alpha-helices in PG bilayers, but small conformational changes are induced in response to a mismatch between peptide hydrophobic length and gel-state lipid bilayer hydrophobic thickness. Moreover, electrostatic and hydrogen-bonding interactions occur between the terminal lysine residues of L(24) and L(24)DAP and the polar headgroups of PG bilayers. However, such interactions were not observed in PG/WL(22)W bilayers, suggesting that the cation-pi interactions between the tryptophan and lysine residues predominate. These results indicate that the lipid-peptide interactions are affected not only by the hydrophobic mismatch between these peptides and the host lipid bilayer, but also by the tryptophan-modulated electrostatic and hydrogen-bonding interactions between the positively charged lysine residues at the termini of these peptides and the negatively charged polar headgroups of the PG bilayers.     

37 A comparative study of ribosomal proteins: linkage between amino acid distribution and ribosomal assembly

Assembly of the ribosome from its protein and RNA constituents has been studied extensively over the past 50?years, and here we utilize a comparative analysis approach to relate the composition of ribosomal proteins (r-proteins) to their role in the assembly process. We computed the amino acid distributions for the 30S subunit r-protein sequences from 560 bacterial species and compared this composition to those of other house-keeping proteins from the same species. We found that r-proteins have a significantly higher content of positively charged residues (Lysine, K, and Arginine, R) than do nonribosomal proteins (10% for R and 11% for K in r-proteins, vs. 4.7% R and 5.9% K in non-ribosomal proteins), which is consistent with prior knowledge of net positive charges carried by r-proteins (Baker et al., 2001; Klein et al., 2004; Burton et al., 2012). Furthermore, these two residues are also highly represented at contact sites along the protein/RNA interface (contact enrichment factor (CEF)?>?1). These results provide further evidence of the importance of electrostatic interactions between the positively charged proteins and negatively charged ribosomal RNA (rRNA) during ribosome assembly. Other highly represented contact residues include polar and aromatic residues, which are likely to interact with rRNA via hydrogen bonds and base stacking interactions, respectively. Interestingly, the proportion of K residues generally decreases with r-protein size, reflecting a negative correlation between protein lengths and the proportion of K (Spearman’s rank correlation, ρ?=??0.802, p?=?2.60e???5). We suggest that this trend helps the smaller r-proteins, which experience higher translational entropy than large proteins, overcome the increased free energy barrier during assembly. When the r-protein sequences were categorized according to the species’ optimal growth temperature, we found that thermophiles show increased R, Isoleucine (I), and Tyrosine (Y) content, whereas mesophiles have increased proportions of Serine (S) and Threonine (T). These results reflect one typical distinction between thermophiles and mesophiles (Kumar and Nussinov 2001), yet these differences in amino acid distributions do not extend to their respective contact sites. That is, the makeup of thermophilic and mesophilic r-protein contact residues are not significantly different (p?>?0.01). This indicates that, while the percent compositions of amino acids relating to qualities such as thermostability and protein folding are expected to vary with environmental temperature, the distributions of residues in contact with rRNA are comparable for all bacterial species. From this, we conclude that the electrostatic interactions that guide ribosome assembly are independent of temperature.     

Control of a tyrosyl radical mediated protein cross-linking reaction by electrostatic interaction

Herein, we demonstrate the control of protein heteroconjugation via a tyrosyl coupling reaction by using electrostatic interaction. Aspartic acid and arginine were introduced to a tyrosine containing peptide tag (Y-tag) to provide electrostatic charge. Designed negatively or positively charged Y-tags were tethered to the C-terminus of Escherichia coli alkaline phosphatase (BAP) and streptavidin (SA), and these model proteins were subjected to horseradish peroxidase (HRP) treatment. The negatively charged Y-tags showed low reactivity due to repulsive interactions between the Y-tags with the negatively charged BAP and SA. In contrast, the positively charged Y-tags showed high reactivity, indicating that the electrostatic interaction between Y-tags and proteins significantly affects the tyrosyl radical mediated protein cross-linking. From the heteroconjugation reaction of BAP and SA, the SA with the positively charged Y-tags exhibited favorable cross-linking toward negatively charged BAP, and the BAP-SA conjugates prepared from BAP with GY-tag (GGGGY) and SA with RYR-tag (RRYRR) had the best performance on a biotin-coated microplate. Encompassing the reactive tyrosine residue with arginine residues reduced the reactivity against HRP, enabling the modulation of cross-linking reaction rates with BAP-GY. Thus, by introducing a proper electrostatic interaction to Y-tags, it is possible to kinetically control the heteroconjugation behavior of proteins, thereby maximizing the functions of protein heteroconjugates.     

Membrane location of apocytochrome c and cytochrome c determined from lipid-protein spin exchange interactions by continuous wave saturation electron spin resonance.

Apocytochrome c derived from horse heart cytochrome c was spin-labeled on the cysteine residue at position 14 or 17 in the N-terminal region of the primary sequence, and cytochrome c from yeast was spin-labeled on the single cysteine residue at sequence position 102 in the C-terminal region. The spin-labeled apocytochrome c and cytochrome c were bound to fluid bilayers composed of different negatively charged phospholipids that also contained phospholipid probes that were spin-labeled either in the headgroup or at different positions in the sn-2 acyl chain. The location of the spin-labeled cysteine residues on the lipid-bound proteins was determined relative to the spin-label positions in the different spin-labeled phospholipids by the influence of spin-spin interactions on the microwave saturation properties of the spin-label electron spin resonance spectra. The enhanced spin relaxation observed in the doubly labeled systems arises from Heisenberg spin exchange, which is determined by the accessibility of the spin-label group on the protein to that on the lipid. It is found that the labeled cysteine groups in horse heart apocytochrome c are located closest to the 14-C atom of the lipid acyl chain when the protein is bound to dimyristoyl- or dioleoyl-phosphatidylglycerol, and to that of the 5-C atom when the protein is bound to a dimyristoylphosphatidylglycerol/dimyristoylphosphatidylcholine (15:85 mol/mol mixture. On binding to dioleoylphosphatidylglycerol, the labeled cysteine residue in yeast cytochrome c is located closest to the phospholipid headgroups but possibly between the polar group region and the 5-C atom of the acyl chains. These data determine the extent to which the different regions of the proteins are able to penetrate negatively charged phospholipid bilayers.     

Amino Acid Substitutions in Cold-Adapted Proteins from Halorubrum lacusprofundi,an Extremely Halophilic Microbe from Antarctica

The halophilic Archaeon Halorubrum lacusprofundi, isolated from the perennially cold and hypersaline Deep Lake in Antarctica, was recently sequenced and compared to 12 Haloarchaea from temperate climates by comparative genomics. Amino acid substitutions for 604 H. lacusprofundi proteins belonging to conserved haloarchaeal orthologous groups (cHOGs) were determined and found to occur at 7.85% of positions invariant in proteins from mesophilic Haloarchaea. The following substitutions were observed most frequently: (a) glutamic acid with aspartic acid or alanine; (b) small polar residues with other small polar or non-polar amino acids; (c) small non-polar residues with other small non-polar residues; (d) aromatic residues, especially tryptophan, with other aromatic residues; and (e) some larger polar residues with other similar residues. Amino acid substitutions for a cold-active H. lacusprofundi β-galactosidase were then examined in the context of a homology modeled structure at residues invariant in homologous enzymes from mesophilic Haloarchaea. Similar substitutions were observed as in the genome-wide approach, with the surface accessible regions of β-galactosidase displaying reduced acidity and increased hydrophobicity, and internal regions displaying mainly subtle changes among smaller non-polar and polar residues. These findings are consistent with H. lacusprofundi proteins displaying amino acid substitutions that increase structural flexibility and protein function at low temperature. We discuss the likely mechanisms of protein adaptation to a cold, hypersaline environment on Earth, with possible relevance to life elsewhere.     

Characterization of the interaction of GABARAPL-1 with the LIR motif of NBR1

Selective autophagy requires the specific segregation of targeted proteins into autophagosomes. The selectivity is mediated by autophagy receptors, such as p62 and NBR1, which can bind to autophagic effector proteins (Atg8 in yeast, MAP1LC3 protein family in mammals) anchored in the membrane of autophagosomes. Recognition of autophagy receptors by autophagy effectors takes place through an LC3 interaction region (LIR). The canonical LIR motif consists of a WXXL sequence, N-terminally preceded by negatively charged residues. The LIR motif of NBR1 presents differences to this classical LIR motif with a tyrosine residue and an isoleucine residue substituting the tryptophan residue and the leucine residue, respectively. We have determined the structure of the GABARAPL-1/NBR1-LIR complex and studied the influence of the different residues belonging to the LIR motif for the interaction with several mammalian autophagy modifiers (LC3B and GABARAPL-1). Our results indicate that the presence of a tryptophan residue in the LIR motif increases the binding affinity. Substitution by other aromatic amino acids or increasing the number of negatively charged residues at the N-terminus of the LIR motif, however, has little effect on the binding affinity due to enthalpy-entropy compensation. This indicates that different LIRs can interact with autophagy modifiers with unique binding properties.     

The crystal structure of the substrate-binding protein OpuBC from Bacillus subtilis in complex with choline

Bacillus subtilis can synthesize the compatible solute glycine betaine as an osmoprotectant from an exogenous supply of the precursor choline. Import of choline is mediated by two osmotically inducible ABC transport systems: OpuB and OpuC. OpuC catalyzes the import of various osmoprotectants, whereas OpuB is highly specific for choline. OpuBC is the substrate-binding protein of the OpuB transporter, and we have analyzed the affinity of the OpuBC/choline complex by intrinsic tryptophan fluorescence and determined a K_d value of about 30 μM. The X-ray crystal structure of the OpuBC/choline complex was solved at a resolution of 1.6 Å and revealed a fold typical of class II substrate-binding proteins. The positively charged trimethylammonium head group of choline is wedged into an aromatic cage formed by four tyrosine residues and is bound via cation-pi interactions. The hydroxyl group of choline protrudes out of this aromatic cage and makes a single interaction with residue Gln19. The substitution of this residue by Ala decreases choline binding affinity by approximately 15-fold. A water network stabilizes choline within its substrate-binding site and promotes indirect interactions between the two lobes of the OpuBC protein. Disruption of this intricate water network by site-directed mutagenesis of selected residues in OpuBC either strongly reduces choline binding affinity (between 18-fold and 25-fold) or abrogates ligand binding. The crystal structure of the OpuBC/choline complex provides a rational for the observed choline specificity of the OpuB ABC importer in vivo and explains its inability to catalyze the import of glycine betaine into osmotically stressed B. subtilis cells.     

Rearrangement of charge-charge interactions in variant ubiquitins as detected by double-mutant cycles and NMR

Previous studies of ubiquitin disclosed numerous charge-charge interactions on the protein's surface. To investigate how neighboring residues influence the strength of these interactions, double-mutant cycles are combined with pK(a) determinations by 2D NMR. More specifically, the environment around the Asp21-Lys29 ion pair has been altered through mutations at position 25, which is an asparagine in mammalian ubiquitin and a positively-charged residue in many other ubiquitin-like proteins. The pK(a) value of Asp21 decreases by 0.4 to 0.7 pH unit when Asn25 is substituted with a positively charged residue, suggesting a new and favorable ion pair interaction between positions 21 and 25. However, analysis of double mutants reveals that the favorable interaction between Asp21 and Lys29 is weakened when position 25 is a positively charged residue. Interestingly, while the pK(a) value of His25 in the N25H variant agrees with model compound values, additional mutants reveal that this agreement is fortuitous, resulting from a balance of favorable and unfavorable interactions; similar results were observed previously for Glu34 in ubiquitin and His8 in staphylococcal nuclease. Ionizable groups may thus have pK(a) values similar to model compound values and yet still be involved in significant interactions with other protein groups. One surprising result of introducing positively charged residues at position 25 is a new interaction between Lys29 and Glu18, an interaction not present in wild-type ubiquitin. This unanticipated result illustrates a key advantage of using NMR to determine pK(a) values for many residues simultaneously in the variant proteins. Overall, the strength of an interaction between two residues at the surface of ubiquitin is sensitive to the identity of neighboring residues. The results also demonstrate that relatively conservative and common point mutations such as substitutions of polar with charged residues and vice versa can have effects on interactions beyond the site of mutation per se.     

Differences in amino acids composition and coupling patterns between mesophilic and thermophilic proteins

Summary. Thermophilic proteins show substantially higher intrinsic thermal stability than their mesophilic counterparts. Amino acid composition is believed to alter the intrinsic stability of proteins. Several investigations and mutagenesis experiment have been carried out to understand the amino acid composition for the thermostability of proteins. This review presents some generalized features of amino acid composition found in thermophilic proteins, including an increase in residue hydrophobicity, a decrease in uncharged polar residues, an increase in charged residues, an increase in aromatic residues, certain amino acid coupling patterns and amino acid preferences for thermophilic proteins. The differences of amino acids composition between thermophilic and mesophilic proteins are related to some properties of amino acids. These features provide guidelines for engineering mesophilic protein to thermophilic protein. Authors’ addresses: Yuan-Jiang Pan, Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Zhejiang University Road 38, Hangzhou 310027, China; Wei-Fen Li, Microbiology Division, College of Animal Science, Zhejiang University, Hangzhou 310029, China     

Heat capacity of hydrogen-bonded networks: an alternative view of protein folding thermodynamics

Large changes in heat capacity (deltaCp) have long been regarded as the characteristic thermodynamic signature of hydrophobic interactions. However, similar effects arise quite generally in order-disorder transitions in homogeneous systems, particularly those comprising hydrogen-bonded networks, and this may have significance for our understanding of protein folding and other biomolecular processes. The positive deltaCp associated with unfolding of globular proteins in water, thought to be due to hydrophobic interactions, is also typical of the values found for the melting of crystalline solids, where the effect is greatest for the melting of polar compounds, including pure water. This suggests an alternative model of protein folding based on the thermodynamics of phase transitions in hydrogen-bonded networks. Folded proteins may be viewed as islands of cooperatively-ordered hydrogen-bonded structure, floating in an aqueous network of less-well-ordered H-bonds in which the degree of hydrogen bonding decreases with increasing temperature. The enthalpy of melting of the protein consequently increases with temperature. A simple algebraic model, based on the overall number of protein and solvent hydrogen bonds in folded and unfolded states, shows how deltaCp from this source could match the hydrophobic contribution. This confirms the growing view that the thermodynamics of protein folding, and other interactions in aqueous systems, are best described in terms of a mixture of polar and non-polar effects in which no one contribution is necessarily dominant.