Desolvation Penalty Research Articles

Free energy calculations of glycosaminoglycan–protein interactions

Glycosaminoglycans (GAGs) are complex highly charged linear polysaccharides that have a variety of roles in biological processes. We report the first use of molecular dynamics (MD) free energy calculations using the MM/PBSA method to investigate the binding of GAGs to protein molecules, namely the platelet endothelial cell adhesion molecule 1 (PECAM-1) and annexin A2. Calculations of the free energy of the binding of heparin fragments of different sizes reveal the existence of a region of low GAG-binding affinity in domains 5-6 of PECAM-1 and a region of high affinity in domains 2-3, consistent with experimental data and ligand-protein docking studies. A conformational hinge movement between domains 2 and 3 was observed, which allows the binding of heparin fragments of increasing size (pentasaccharides to octasaccharides) with an increasingly higher binding affinity. Similar simulations of the binding of a heparin fragment to annexin A2 reveal the optimization of electrostatic and hydrogen bonding interactions with the protein and protein-bound calcium ions. In general, these free energy calculations reveal that the binding of heparin to protein surfaces is dominated by strong electrostatic interactions for longer fragments, with equally important contributions from van der Waals interactions and vibrational entropy changes, against a large unfavorable desolvation penalty due to the high charge density of these molecules.

Physical Basis of Metal-Binding Specificity in Escherichia coli NikR

In Escherichia coli and other bacteria, nickel uptake is regulated by the transcription factor NikR. Nickel binding at high-affinity sites in E. coli NikR (EcNikR) facilitates EcNikR binding to the nik operon, where it then suppresses transcription of genes encoding the nickel uptake transporter, NikABCDE. A structure of the EcNikR-DNA complex suggests that a second metal-binding site is also present when NikR binds to the nik operon. Moreover, this co-crystal structure raises the question of what metal occupies the second site under physiological conditions: K(+), which is present in the crystal structure, or Ni(2+), which has been proposed to bind to low- as well as high-affinity sites on EcNikR. To determine which ion is preferred at the second metal-binding site and the physical basis for any preference of one ion over another in both the second metal-binding site and the high-affinity sites, we conducted a series of detailed molecular simulations on the EcNikR structure. Simulations that place Ni(2+) at high-affinity sites lead to stable trajectories with realistic ion-ligand distances and geometries, while simulations that place K(+) at these sites lead to conformational changes in the protein that are likely unfavorable for ion binding. By contrast, simulations on the second metal site in the EcNikR-DNA complex lead to stable trajectories with realistic geometries regardless of whether K(+) or Ni(2+) occupies this site. Electrostatic binding free energy calculations, however, suggest that EcNikR binding to DNA is more favorable when the second metal-binding site contains K(+). An analysis of the energetic contributions to the electrostatic binding free energy suggests that, while the interaction between EcNikR and DNA is more favorable when the second site contains Ni(2+), the large desolvation penalty associated with moving Ni(2+) from solution to the relatively buried second site offsets this favorable interaction term. Additional free energy simulations that account for both electrostatic and non-electrostatic effects argue that EcNikR binding to DNA is most favorable when the second site contains a monovalent ion the size of K(+). Taken together, these data suggest that the EcNikR structure is most stable when Ni(2+) occupies high-affinity sites and that EcNikR binding to DNA is more favorable when the second site contains K(+).

Experimentally based contact energies decode interactions responsible for protein–DNA affinity and the role of molecular waters at the binding interface

A major obstacle towards understanding the molecular basis of transcriptional regulation is the lack of a recognition code for protein–DNA interactions. Using high-quality crystal structures and binding data on the promiscuous family of C2H2 zinc fingers (ZF), we decode 10 fundamental specific interactions responsible for protein–DNA recognition. The interactions include five hydrogen bond types, three atomic desolvation penalties, a favorable non-polar energy, and a novel water accessibility factor. We apply this code to three large datasets containing a total of 89 C2H2 transcription factor (TF) mutants on the three ZFs of EGR. Guided by molecular dynamics simulations of individual ZFs, we map the interactions into homology models that embody all feasible intra- and intermolecular bonds, selecting for each sequence the structure with the lowest free energy. These interactions reproduce the change in affinity of 35 mutants of finger I (R2 = 0.998), 23 mutants of finger II (R2 = 0.96) and 31 finger III human domains (R2 = 0.94). Our findings reveal recognition rules that depend on DNA sequence/structure, molecular water at the interface and induced fit of the C2H2 TFs. Collectively, our method provides the first robust framework to decode the molecular basis of TFs binding to DNA.

A theoretical study on the metal cation-π complexes of Zn2+ and Cd2+ with benzene and cyclohexene

Transition metal cation-π complexes of Zn2+ and Cd2+ with benzene and cyclohexene have been studied with density functional B3PW91 method. Natural bond orbital (NBO) analyses on the direct cation-π complexes (with direct π-ion contact) indicate that the interaction between the π orbital of the π systems and the empty valance s orbital of the cation (πPI → s M2+ interactions) dominates the cation-π interactions. Energetical results indicate that formation of metal cation-π complexes in the gas phase is always energetically favourable and these complexes can thus be prepared and observed in experiments in the gas phase. However, in aqueous solution the desolvation penalty of Zn2+ and Cd2+ is so high that it is energetically unfavourable to form metal cation-π complexes from hydrated Zn2+ and Cd2+ at both 0 K and room temperature. It would thus be predicted that metal cation-π complexes of Zn2+ and Cd2+ are not likely to be observed in aqueous solution.

Study of MDM2 Binding to p53-Analogues: Affinity, Helicity, and Applicability to Drug Design

MDM2 is a key regulator of the p53 tumor-suppressor protein. Here we study the effect of modifications of a p53 N-terminal fragment on its binding to MDM2, using implicit-solvent MD and MM-GB/SA calculations. We provide interpretation of existing experimental data and predict the effect of mutations on binding. Notably 1) We analyze the effect of regulatory phosphorylations at Ser/Thr residues and suggest that a balance between favorable electrostatics and desolvation penalties determines the effect of phosphorylation; 2) We compare the helical stability in solution of p53 alanine mutants and propose a helix stabilizing role for several residues involved in hydrogen bonding and hydrophobic packing; 3) We obtain good correlations between calculated and experimental affinities for a set of peptidomimetic inhibitors, both alone and in combination with p53 analogues, demonstrating potential applicability to drug design. From the technical aspect, protocol optimization and selection of simulation tools are addressed in detail. To the best of our knowledge this is the first published example of MM-GB/SA calculations utilizing a conformational ensemble generated with implicit solvent MD. Our results suggest that this highly efficient variant of classical explicit-solvent MM-GB/SA may be used for studying protein-protein interactions and for the design of peptidomimetic drugs.

Assessment of QM/MM Scoring Functions for Molecular Docking to HIV-1 Protease

We explore the ability of four quantum mechanical (QM)/molecular mechanical (MM) models to accurately identify the native pose of six HIV-1 protease inhibitors and compare them with the AMBER force field and ChemScore and GoldScore scoring functions. Three QM/MM scoring functions treated the ligand at the HF/6-31G*, AM1d, and PM3 levels; the fourth QM/MM function modeled the ligand and active site at the PM3-D level. For the discrimination of native from non-native poses, solvent-corrected HF/6-31G*:AMBER and AMBER functions exhibited the best overall performance. While the electrostatic component of the MM and QM/MM functions appears important for discriminating the native pose of the ligand, the polarization contribution in the QM/MM functions was relatively insensitive to a ligand's binding mode and, for one ligand, actually hindered discrimination. The inclusion of a desolvation penalty, here using a generalized Born solvent model, improved discrimination for the MM and QM/MM methods. There appeared to be no advantage to binding mode prediction by incorporating active site polarization at the PM3-D level. Finally, we found that choice of the protonation state of the aspartyl dyad in the HIV-1 protease active site influenced the ability of scoring methods to determine the native binding pose.

Metal Preference At The Second Metal Binding Site Of E. coli NikR

Escherichia coli NikR (EcNikR) regulates cellular nickel uptake by binding free nickel ions at high affinity metal binding sites in the protein, which induces EcNikR binding to the nik operon - a process that leads to suppression of the gene encoding the nickel uptake transporter, NikABCDE. A structure of the EcNikR-DNA complex suggests that a second metal binding site is present in addition to the high affinity sites, and raises the question of which metal occupies this second site under physiologic conditions: K+, which is present in the crystal structure, or Ni+2. To determine which ion is preferred at the second metal binding site, and the reason for any preference of one ion over another, we calculated the electrostatic free energy of EcNikR binding to DNA with either K+ or Ni+2 in the second site. While the interaction between EcNikR and DNA is more favorable when the second site contains Ni+2, the large desolvation penalty associated with moving Ni+2 from solution to the relatively buried second site offsets this favorable interaction. Consequently, our data suggest that EcNikR binding to DNA is more favorable when the second site contains a K+ ion. Moreover, additional calculations indicate that the second site is best suited for an ion having the size of K+ and not Ni+2, suggesting that the second site is optimized for K+. Taken together, our data suggest that the second metal binding site contains K+ and explain why K+ is preferred over Ni+2 at this site.

Cluster Hydration Model for Binding Energy Calculations of Protein−Ligand Complexes

We present a new model for predicting the binding affinity in protein-ligand complexes based on an explicit solvent treatment. The model is referred to as the cluster hydration model. Test calculations were performed on complexes of FK506-binding protein (FKBP) and its six ligands. The calculated binding energies had a good correlation with experimental binding affinities; the correlation coefficient r(2) was 0.93. Moreover, we examined the competition between the residue-ligand interactions and the desolvation energies of residues. The results suggested that, for the stabilization of the complexes in solution, the contributions of nonpolar residues at the binding site are larger than those of charged and polar ones because the electrostatic interaction energies between the residues and the ligand were mostly canceled by the desolvation penalties.

Prediction and analysis of binding affinities for chemically diverse HIV‐1 PR inhibitors by the modified SAFE_p approach

One of the biggest challenges in the "in silico" screening of enzyme ligands is to have a protocol that could predict the ligand binding free energies. In our group we have developed a very simple screening function (referred to as solvent accessibility free energy of binding predictor, SAFE_p) which we have applied previously to the study of peptidic HIV-1 protease (HIV-1 PR) inhibitors and later to cyclic urea type HIV-1 PR inhibitors. In this work, we have extended the SAFE_p protocol to a chemically diverse set of HIV-1 PR inhibitors with binding constants that differ by several orders of magnitude. The resulting function is able to reproduce the ranking and in many cases the value of the inhibitor binding affinities for the HIV-1 PR, with accuracy comparable with that of costlier protocols. We also demonstrate that the binding pocket SAFE_p analysis can contribute to the understanding of the physical forces that participate in ligand binding. The analysis tools afforded by our protocol have allowed us to identify an induced fit phenomena mediated by the inhibitor and have demonstrated that larger fragments do not necessarily contribute the most to the binding free energy, an outcome partially brought about by the substantial role the desolvation penalty plays in the energetics of binding. Finally, we have revisited the effect of the Asp dyad protonation state on the predicted binding affinities.

Salt Bridges in the Hyperthermophilic Protein Ssh10b Are Resilient to Temperature Increases

A double mutant cycle (DMC) approach was employed to estimate the effect of temperature on the contribution of two highly conserved salt bridges to protein stability in the hyperthermophilic protein Ssh10b. The coupling free energy were 2.4 +/- 0.4 kJ/mol at 298 K and 2.2 +/- 0.4 kJ/mol at 353 K for Glu-54/Arg-57, and 6.0 +/- 0.2 kJ/mol at 298 K and 5.9 +/- 0.6 kJ/mol at 353 K for Glu-36/Lys-68. The stability free energy of Ssh10b decrease greatly with increasing temperature, while the direct contribution of these two salt bridges to protein stability remain almost constant, providing evidence supporting the theoretical prediction that salt bridges are extremely resilient to temperature increases and thus are specially suited to improving protein stability at high temperatures. The reason for the difference in coupling free energy between salt bridges Glu-54/Arg-57 and Glu-36/Lys-68 is discussed. Comparing our results with published DMC data for the contribution of salt bridges to stability in other proteins, we found that the energy contribution of a salt bridge formed by two charged residues far apart in the primary sequence is higher than that of those formed between two very close ones. Implications of this finding are useful for engineering proteins with enhanced thermostability.

The origins of femtomolar protein-ligand binding: hydrogen-bond cooperativity and desolvation energetics in the biotin-(strept)avidin binding site.

The unusually strong reversible binding of biotin by avidin and streptavidin has been investigated by density functional and MP2 ab initio quantum mechanical methods. The solvation of biotin by water has also been studied through QM/MM/MC calculations. The ureido moiety of biotin in the bound state hydrogen bonds to five residues, three to the carbonyl oxygen and one for each--NH group. These five hydrogen bonds act cooperatively, leading to stabilization that is larger than the sum of individual hydrogen-bonding energies. The charged aspartate is the key residue that provides the driving force for cooperativity in the hydrogen-bonding network for both avidin and streptavidin by greatly polarizing the urea of biotin. If the residue is removed, the network is disrupted, and the attenuation of the energetic contributions from the neighboring residues results in significant reduction of cooperative interactions. Aspartate is directly hydrogen-bonded with biotin in streptavidin and is one residue removed in avidin. The hydrogen-bonding groups in streptavidin are computed to give larger cooperative hydrogen-bonding effects than avidin. However, the net gain in electrostatic binding energy is predicted to favor the avidin-bicyclic urea complex due to the relatively large penalty for desolvation of the streptavidin binding site (specifically expulsion of bound water molecules). QM/MM/MC calculations involving biotin and the ureido moiety in aqueous solution, featuring PDDG/PM3, show that water interactions with the bicyclic urea are much weaker than (strept)avidin interactions due to relatively low polarization of the urea group in water.

Interaction of heteroboranes with biomolecules : Part 2. The effect of various metal vertices and exo-substitutions

Icosahedral heteroboranes and especially metallacarboranes, which have recently been shown to act as potent HIV-1 protease inhibitors, are a unique class of chemical compounds with unusual properties, one of which is the formation of dihydrogen bonds with biomolecules. In this study, we investigate the effect of various metal vertices and exo-substitutions on several series of heteroboranes, including 11-vertex carborane cages [nido-7,8-C2B9Hn]n-13(n= 11,12,13), closo-1-SB11H11, closo-1-NB11H12, metal bis(dicarbollides)[3,3'-M (1,2-C2B9H11)2]n(M/n=Fe/2-, Co/1-, Ni/0) and fluoro (F), amino (NH2) and hydroxo (OH) derivatives of the metal bis(dicarbollides). Besides the properties of isolated systems (geometries, electronic properties and hydration), we study their interactions with a tetrapeptide, which models their biomolecular partner. Calculations have confirmed that the extra hydrogen in [nido-7,8-C2B9H12]- forms a bridge, which fluctuates between two stationary states. Using RESP-derived charges, it was ascertained that the negative charge of heteroboranes is located mainly on boron-bound hydrogens. An increase of the negative total charge (from 0 to -1 or -2) of heteroboranes yields an increase in the stabilisation energies of heteroborane[dot dot dot]peptide complexes and also a substantial increase in the hydration free energies of heteroboranes. Compared to the substitutions of metal vertices, the exo-substitutions of metallacarboranes cause a larger increase in stabilisation energies and a smaller increase in desolvation penalties. These two terms, stabilisation energies and desolvation penalties, contribute in opposite directions to the total heteroborane-biomolecule binding energy and must both be taken into account when designing new HIV-1 protease inhibitors.

The role of charged surface residues in the binding ability of small hubs in protein-protein interaction networks.

Hubs are highly connected proteins in a protein-protein interaction network. Previous work has implicated disordered domains and high surface charge as the properties significant in the ability of hubs to bind multiple proteins. While conformational flexibility of disordered domains plays an important role in the binding ability of large hubs, high surface charge is the dominant property in small hubs. In this study, we further investigate the role of the high surface charge in the binding ability of small hubs in the absence of disordered domains. Using multipole expansion, we find that the charges are highly distributed over the hub surfaces. Residue enrichment studies show that the charged residues in hubs are more prevalent on the exposed surface, with the exception of Arg, which is predominantly found at the interface, as compared to non-hubs. This suggests that the charged residues act primarily from the exposed surface rather than the interface to affect the binding ability of small hubs. They do this through (i) enhanced intra-molecular electrostatic interactions to lower the desolvation penalty, (ii) indirect long – range intermolecular interactions with charged residues on the partner proteins for better complementarity and electrostatic steering, and (iii) increased solubility for enhanced diffusion-controlled rate of binding. Along with Arg, we also find a high prevalence of polar residues Tyr, Gln and His and the hydrophobic residue Met at the interfaces of hubs, all of which have the ability to form multiple types of interactions, indicating that the interfaces of hubs are optimized to participate in multiple interactions.

Kinetics and Mechanism of Proton Transport across Membrane Nanopores

We use computer simulations to study the kinetics and mechanism of proton passage through a narrow-pore carbon-nanotube membrane separating reservoirs of liquid water. Free energy and rate constant calculations show that protons move across the membrane diffusively along single-file chains of hydrogen-bonded water molecules. Proton passage through the membrane is opposed by a high barrier in the effective potential, reflecting the large electrostatic penalty for desolvation and reminiscent of charge exclusion in biological water channels. At neutral pH, we estimate a translocation rate of about 1 proton per hour and tube.

Dynamics of Asp23−Lys28 Salt-Bridge Formation in Aβ10-35Monomers

In the amyloid fibrils formed from long fragments of the amyloid beta-protein (Abeta-protein), the monomers are arranged in parallel and lie perpendicular to the fibril axis. The structure of the monomers satisfies the amyloid self-organization principle; namely, the low free energy state of the monomer maximizes the number of intra- and interpeptide contacts and salt bridges. The formation of the intramolecular salt bridge between Asp(D)23 and Lys(K)28 ensures that unpaired charges are not buried in the low-dielectric interior. We have investigated, using all-atom molecular dynamics simulations in explicit water, whether the D23-K28 interaction forms spontaneously in the isolated Abeta10-35 monomer. To validate the simulation protocol, we show, using five independent trajectories spanning a total of 100 ns, that the pKa values of the titratable groups are in good agreement with experimental measurements. The computed free energy disconnectvity graph shows that broadly the ensemble of compact random coil conformations can be clustered into four basins that are separated by free energy barriers ranging from 0.3 to 2.7 kcal/mol. There is significant residual structure in the conformation of the peptide in each of the basins. Due to the desolvation penalty, the structural motif with a stable turn involving the residues VGSN and a preformed D23-K28 contact is a minor component of the simulated structures. The extent of solvation of the peptides in the four basins varies greatly, which underscores the dynamical fluctuations in the monomer. Our results suggest that the early event in the oligomerization process must be the expulsion of discrete water molecules that facilitates the formation of interpeptide-interaction-driven stable structures with an intramolecular D23-K28 salt bridge and an intact VGSN turn.

Concepts in Receptor Optimization: Targeting the RGD Peptide

Synthetic receptors have a wide range of potential applications, but it has been difficult to design low molecular weight receptors that bind ligands with high, "proteinlike" affinities. This study uses novel computational methods to understand why it is hard to design a high-affinity receptor and to explore the limits of affinity, with the bioactive peptide RGD as a model ligand. The M2 modeling method is found to yield excellent agreement with experiment for a known RGD receptor and then is used to analyze a series of receptors generated in silico with a de novo design algorithm. Forces driving binding are found to be systematically opposed by proportionate repulsions due to desolvation and entropy. In particular, strong correlations are found between Coulombic attractions and the electrostatic desolvation penalty and between the mean energy change on binding and the cost in configurational entropy. These correlations help explain why it is hard to achieve high affinity. The change in surface area upon binding is found to correlate poorly with affinity within this series. Measures of receptor efficiency are formulated that summarize how effectively a receptor uses surface area, total energy, and Coulombic energy to achieve affinity. Analysis of the computed efficiencies suggests that a low molecular weight receptor can achieve proteinlike affinity. It is also found that macrocyclization of a receptor can, unexpectedly, increase the entropy cost of binding because the macrocyclic structure further restricts ligand motion.

Optimization of Small Molecule Drugs Binding to Highly Polar Target Sites: Lessons from the Discovery and Development of Neuraminidase Inhibitors

Binding affinity optimization of small molecules interacting with polar binding sites on target proteins is a formidable, but not uncommon challenge in drug discovery. The challenge relates to the difficulty of integrating favourable and unfavourable polar, non-polar and conformation contributions into overall favourable binding energies. This review describes the surprising breakthrough findings leading to the development of Tamiflu, a clinically efficacious orally bioavailable drug targeting the active site of influenza neuraminidase (NA). The NA active site is highly polar and formed mostly by arginine, aspartate and glutamate residues. This active site structure evolved for efficient interaction with charged sialic acid moieties on glycoproteins and stabilization of an oxocarbonium ion in the transition state of the neuraminidase reaction. The initial strategy of optimizing polar interactions in transition state analogs led to NA inhibitors (NAIs) with sub-nanomolar binding affinities, but such compounds were highly polar and lacked oral bioavailability. The realization of the possibility to achieve high affinity binding in a highly polar active site through optimization of non-polar and van-der-Waals interactions initially appeared counterintuitive and required a few serendipitous findings, but was key to reduce the polarity of drug candidates, avoid large desolvation penalties and achieve oral bioavailability.

Probing Molecular Docking in a Charged Model Binding Site

A model binding site was used to investigate charge-charge interactions in molecular docking. This simple site, a small (180A(3)) engineered cavity in cyctochrome c peroxidase (CCP), is negatively charged and completely buried from solvent, allowing us to explore the balance between electrostatic energy and ligand desolvation energy in a system where many of the common approximations in docking do not apply. A database with about 5300 molecules was docked into this cavity. Retrospective testing with known ligands and decoys showed that overall the balance between electrostatic interaction and desolvation energy was captured. More interesting were prospective docking scre"ens that looked for novel ligands, especially those that might reveal problems with the docking and energy methods. Based on screens of the 5300 compound database, both high-scoring and low-scoring molecules were acquired and tested for binding. Out of 16 new, high-scoring compounds tested, 15 were observed to bind. All of these were small heterocyclic cations. Binding constants were measured for a few of these, they ranged between 20microM and 60microM. Crystal structures were determined for ten of these ligands in complex with the protein. The observed ligand geometry corresponded closely to that predicted by docking. Several low-scoring alkyl amino cations were also tested and found to bind. The low docking score of these molecules owed to the relatively high charge density of the charged amino group and the corresponding high desolvation penalty. When the complex structures of those ligands were determined, a bound water molecule was observed interacting with the amino group and a backbone carbonyl group of the cavity. This water molecule mitigates the desolvation penalty and improves the interaction energy relative to that of the "naked" site used in the docking screen. Finally, six low-scoring neutral molecules were also tested, with a view to looking for false negative predictions. Whereas most of these did not bind, two did (phenol and 3-fluorocatechol). Crystal structures for these two ligands in complex with the cavity site suggest reasons for their binding. That these neutral molecules do, in fact bind, contradicts previous results in this site and, along with the alkyl amines, provides instructive false negatives that help identify weaknesses in our scoring functions. Several improvements of these are considered.

Investigation of Salt Bridge Stability in a Generalized Born Solvent Model.

Potentials of mean force (PMFs) of salt bridge formation between oppositely charged amino acid side chains were calculated both in explicit solvent and in a Generalized Born (GB) continuum solvent model to quantify the potential overstabilization of side chain ion pairs in GB relative to explicit solvation. These show that salt bridges are too stable by as much as 3-4 kcal/mol in the GB solvent models that we tested, consistent with previously reported observations of significantly different structural ensembles in GB models and explicit solvent for proteins containing ionizable groups. We thus investigated a simple empirical correction, wherein the intrinsic GB radii of hydrogen atoms bound to charged nitrogen atoms are reduced, effectively increasing the desolvation penalty of the positively charged groups. The thermodynamics of salt bridge formation were considerably improved, as exemplified by the close match of the corrected GB PMF to the reference explicit solvent PMF, and more significantly by our ability to closely reproduce the experimental temperature melting profile of the TC5b Trp-cage miniprotein, which is otherwise highly distorted by prevalent non-native salt bridges when using standard GB parameters.

Relative Strengths of NH··O and CH··O Hydrogen Bonds between Polypeptide Chain Segments

Correlated ab initio calculations are used to compare the energetics when the CH and NH groups of the model dipeptide CHONHCH2CONH2 are each allowed to form a H-bond with the proton acceptor O of a peptide group. When the dipeptide is in its C7 conformation, the NH..O H-bond energy is found to be 7.4 kcal/mol, as compared to only 2.8 kcal/mol for the CH..O interaction. On the other hand, the situation reverses, and the CH..O H-bond becomes stronger than NH..O, when the dipeptide adopts a C5 structure. This reversal is important as C5 is nearly equal in stability to C7 for the dipeptide, and is representative of the commonly observed beta-sheet structure in a protein. Immersing the dipeptide-peptide pair in a model solvent weakens both sorts of H-bonds, and in a fairly uniform manner. Consequently, the trends observed in the in vacuo situation retain their validity in either aqueous solution or the protein interior. Likewise, the desolvation penalty, suffered by removing a H-bonded complex from water and placing it in the less polar interior of a protein, is quite similar for the NH..O and CH..O bonds.