Amino acid conformational preferences and solvation of polar backbone atoms in peptides and proteins

Amino acids in peptides and proteins display distinct preferences for alpha-helical, beta-strand, and other conformational states. Various physicochemical reasons for these preferences have been suggested: conformational entropy, steric factors, hydrophobic effect, and backbone electrostatics; however, the issue remains controversial. It has been proposed recently that the side-chain-dependent solvent screening of the local and non-local backbone electrostatic interactions primarily determines the preferences not only for the alpha-helical but also for all other main-chain conformational states. Side-chains modulate the electrostatic screening of backbone interactions by excluding the solvent from the vicinity of main-chain polar atoms. The deficiency of this electrostatic screening model of amino acid preferences is that the relationships between the main-chain electrostatics and the amino acid preferences have been demonstrated for a limited set of six non-polar amino acid types in proteins only. Here, these relationships are determined for all amino acid types in tripeptides, dekapeptides, and proteins. The solvation free energies of polar backbone atoms are approximated by the electrostatic contributions calculated by the finite difference Poisson-Boltzmann and the Langevin dipoles methods. The results show that the average solvation free energy of main-chain polar atoms depends strongly on backbone conformation, shape of side-chains, and exposure to solvent. The equilibrium between the low-energy beta-strand conformation of an amino acid (anti-parallel alignment of backbone dipole moments) and the high-energy alpha conformation (parallel alignment of backbone dipole moments) is strongly influenced by the solvation of backbone polar atoms. The free energy cost of reaching the alpha conformation is by approximately 1.5 kcal/mol smaller for residues with short side-chains than it is for the large beta-branched amino acid residues. This free energy difference is comparable to those obtained experimentally by mutation studies and is thus large enough to account for the distinct preferences of amino acid residues. The screening coefficients gamma(local)(r) and gamma(non-local)(r) correlate with the solvation effects for 19 amino acid types with the coefficients between 0.698 to 0.851, depending on the type of calculation and on the set of point atomic charges used. The screening coefficients gamma(local)(r) increase with the level of burial of amino acids in proteins, converging to 1.0 for the completely buried amino acid residues. The backbone solvation free energies of amino acid residues involved in strong hydrogen bonding (for example: in the middle of an alpha-helix) are small. The hydrogen bonded backbone is thus more hydrophobic than the peptide groups in random coil. The alpha-helix forming preference of alanine is attributed to the relatively small free energy cost of reaching the high-energy alpha-helix conformation. These results confirm that the side-chain-dependent solvent screening of the backbone electrostatic interactions is the dominant factor in determining amino acid conformational preferences.     

High polar content of long buried blocks of sequence in protein domains suggests selection against amyloidogenic non-polar sequences

Native protein structures achieve stability in part by burying hydrophobic side-chains. About 75% of all amino acid residues buried in protein interiors are non-polar. Buried residues are not uniformly distributed in protein sequences, but sometimes cluster as contiguous polypeptide stretches that run through the interior of protein domain structures. Such regions have an intrinsically high local sequence density of non-polar residues, creating a potential problem: local non-polar sequences also promote protein misfolding and aggregation into non-native structures such as the amyloid fibrils in Alzheimer's disease. Here we show that long buried blocks of sequence in protein domains of known structure have, on average, a lower content of non-polar amino acids (about 70%) than do isolated buried residues (about 80%). This trend is observed both in small and in large protein domains and is independent of secondary structure. Long, completely non-polar buried stretches containing many large side-chains are particularly avoided. Aspartate residues that are incorporated in long buried stretches were found to make fewer polar interactions than those in short stretches, hinting that they may be destabilizing to the native state. We suggest that evolutionary pressure is acting on non-native properties, causing buried polar residues to be placed at positions where they would break up aggregation-prone non-polar sequences, perhaps even at some cost to native state stability.     

Solution and ion-complexed conformations of beauvericin determined by proton magnetic resonance spectroscopy

The conformational properties of the cyclohexadepsipeptide antibiotic Beauvericin have been investigated by 1H-NMR spectroscopy in polar (C2H3O2H) and non-polar (CCl4, C62H6, C2HCl3) solvents and in two solvent mixtures; one a mixture of a polar and non-polar solvent (C2H3O2H/CCl4) and the other an aromatic solvent in a non-polar environment (C62H6/CCl4). The ion-complexation properties of Beauvericin with alkali metal halides (Li+, Na+, K+, Cs+) have also been studied. It is demonstrated that changes in chemical shifts of Beauvericin with concentration, with polarity of solvent or with added alkali metal ion reflect changes not only in the solvent properties but also changes in backbone conformation and changes due to ion-complexation, where appropriate, and therefore cannot be used, by themselves, to determine the conformation of the molecule, its self-aggregation properties, or the stoichiometry of the metal ion-complex. The backbone conformations of Beauvericin in different environments are determined by methods that are independent of chemical shift analysis; i.e., by measurements of 5J(HH) magnitudes observed between the alpha-CH protons of the L-phenylalanine and D-hydroxyisovaleric acid (DHyIv) residues and by nuclear Overhauser effect measurements observed between alpha-CH(HyIv) and (N)-CH3(Phe) proton signals. In the knowledge of these results the chemical shifts of Beauvericin in different environments can then be rationalised. It is found that the conformation of Beauvericin in a polar solvent is different from that found in a non-polar solvent and from that found for the in the ion-complexed form is similar to that found in non-polar solvents. By taking into account the conformational properties of the L-phenylalanine and DHyIv side-chains, it is possible to assign unambiguously the magnetically non-equivalent beta-CH2(Phe) and gamma Me(HyIv) proton signals and so elucidate the complete conformational behaviour of the uncomplexed forms of Beauvericin in a polar and a non-polar environment, and of the ion-complexed form of Beauvericin in a polar solvent.     

Folding of polypeptide chains induced by the amino acid side-chains

Conformational calculations with the use of semi-empirical potential functions have been applied to the analysis of the folding of peptide chains. In particular, the part played by the amino acid side-chains in the adoption of folded conformations has been investigated.The results show that the preferred conformations of short peptides are mostly extended ones. However, from a given peptide chain-length, the side-chain to backbone and side-chain to side-chain interactions become strong enough so that definite sequences of amino acids can induce a transition from extended to folded conformations. We propose to call these folded structures “conformational nuclei”. The type of “nucleus” formed is dependent on both the amino acid composition and the sequence.Our results strongly support the hypothesis that folding of polypeptide chains can occur through a nucleation process that could be induced by the side-chains.     

An analysis of incorrectly folded protein models. Implications for structure predictions

Proteins with homologous amino acid sequences have similar folds and it has been assumed that an unknown three-dimensional structure can be obtained from a known homologous structure by substituting new side-chains into the polypeptide chain backbone, followed by relatively small adjustment of the model. To examine this approach of structure prediction and, more generally, to isolate the characteristics of native proteins, we constructed two incorrectly folded protein models. Sea-worm hemerythrin and the variable domain of mouse immunoglobulin K-chain, two proteins with no sequence homology, were chosen for study; the former is composed of a bundle of four alpha-helices and the latter consists of two 4-stranded beta-sheets. Using an automatic computer procedure, hemerythrin side-chains were substituted into the immunoglobulin domain and vice versa. The structures were energy-minimized with the program CHARMM and the resulting structures compared with the correctly folded forms. It was found that the incorrect side-chains can be incorporated readily into both types of structures (alpha-helices, beta-sheets) with only small structural adjustments. After constrained energy-minimization, which led to an average atomic co-ordinate shift of no more than 0.7 to 0.9 A, the incorrectly folded models arrived at potential energy values comparable to those of the correct structures. Detailed analysis of the energy results shows that the incorrect structures have less stabilizing electrostatic, van der Waals' and hydrogen-bonding interactions. The difference is particularly pronounced when the electrostatic and van der Waals' energy terms are calculated by modified equations that include an approximate representation of solvent effects. The incorrectly folded structures also have a significantly larger solvent-accessible surface and a greater fraction of non-polar side-chain atoms exposed to solvent. Examination of their interior shows that the packing of side-chains at the secondary structure interfaces, although corresponding to sterically allowed conformations, deviates from the characteristics found in normal proteins. The analysis of incorrectly folded structures has made it clear that the absence of bad non-bonded contacts, though necessary, is not sufficient to demonstrate the validity of model-built structures and that modeling of homologous structures has to be accompanied by a thorough quantitative evaluation of the results. Further, certain features that characterize native proteins are made evident by their absence in misfolded models.     

Straight-chain non-polar amino acids are good helix-formers in water

For comparison with earlier data on naturally occurring non-polar amino acids (Ala, Leu, Phe, Val, Ile), the comparative helix-forming tendencies have been measured for non-polar amino acid residues that have unbranched side-chains, with an ethyl, propyl or butyl group, and also for methionine. The substitutions are made in a 17-residue alanine-based peptide. The results show that straight-chain non-polar amino acids have high helix-forming tendencies compared to beta-branched non-polar amino acids. Restriction of side-chain conformations in the helix, with a corresponding reduction in conformational entropy, is the likely explanation. There is a small increase in helix-forming tendency as the side-chain increases in length from ethyl to butyl, which suggests that a helix-stabilizing hydrophobic interaction is being detected.     

A consistent set of statistical potentials for quantifying local side-chain and backbone interactions

The frequencies of occurrence of atom arrangements in high-resolution protein structures provide some of the most accurate quantitative measures of interaction energies in proteins. In this report we extend our development of a consistent set of statistical potentials for quantifying local interactions between side-chains and the polypeptide backbone, as well as nearby side-chains. Starting with phi/psi/chi1 propensities that select for optimal interactions of the 20 amino acid side-chains with the 2 flanking peptide bonds, the following 3 new terms are added: (1) a distance-dependent interaction between the side-chain at i and the carbonyl oxygens and amide protons of the peptide units at i +/- 2, i +/- 3, and i +/- 4; (2) a distance-dependent interaction between the side-chain at position i and side-chains at positions i + 1 through i + 4; and (3) an orientation-dependent interaction between the side-chain at position i and side-chains at i + 1 through i + 4. The relative strengths of these 4 pseudo free energy terms are estimated by the average information content of each scoring matrix and by assessing their performance in a simple fragment threading test. They vary from -0.4 - -0.5 kcal/mole per residue for phi/psi/chi1 propensities to a range of -0.15 - -0.6 kcal/mole per residue for each of the other 3 terms. The combined energy function, containing no interactions between atoms more than 4 residues apart, identifies the correct structural fragment for randomly selected 15 mers over 40% of the time, after searching through 232,000 alternative conformations. For 14 out of 20 sets of all-atom Rosetta decoys analyzed, the native structure has a combined score lower than any of the 1700-1900 decoy conformations. The ability of this energy function to detect energetically important details of local structure is demonstrated by its power to distinguish high-resolution crystal structures from NMR solution structures.     

High-resolution solution structure of reduced French bean plastocyanin and comparison with the crystal structure of poplar plastocyanin

The three-dimensional solution structure of reduced (CuI) plastocyanin from French bean leaves has been determined by distance geometry and restrained molecular dynamics methods using constraints obtained from 1H n.m.r. (nuclear magnetic resonance) spectroscopy. A total of 1244 experimental constraints were used, including 1120 distance constraints, 103 dihedral angle constraints and 21 hydrogen bond constraints. Stereospecific assignments were made for 26 methylene groups and the methyls of 11 valines. Additional constraints on copper co-ordination were included in the restrained dynamics calculations. The structures are well defined with average atomic root-mean-square deviations from the mean of 0.45 A for all backbone heavy atoms and 1.08 A for side-chain heavy atoms. French bean plastocyanin adopts a beta-sandwich structure in solution that is similar to the X-ray structure of reduced poplar plastocyanin; the average atomic root-mean-square difference between 16 n.m.r. structures and the X-ray structure is 0.76 A for all backbone heavy atoms. The conformations of the side-chains that constitute the hydrophobic core of French bean plastocyanin are very well defined. Of 47 conserved residues that populate a single chi 1 angle in solution, 43 have the same rotamer in the X-ray structure. Many surface side-chains adopt highly preferred conformations in solution, although the 3J alpha beta coupling constants often indicate some degree of conformational averaging. Some surface side-chains are disordered in both the solution and crystal structures of plastocyanin. There is a striking correlation between measures of side-chain disorder in solution and side-chain temperature factors in the X-ray structure. Side-chains that form a distinctive acidic surface region, believed to be important in binding other electron transfer proteins, appear to be disordered. Fifty backbone amide protons form hydrogen bonds to carbonyls in more than 60% of the n.m.r. structures; 45 of these amide protons exchange slowly with solvent deuterons. Ten hydrogen bonds are formed between side-chain and backbone atoms, eight of which are correlated with decreased proton exchange. Of the 60 hydrogen bonds formed in French bean plastocyanin, 56 occur in the X-ray structure of the poplar protein; two of the missing hydrogen bonds are absent as a result of mutations. It appears that molecular dynamics refinement of highly constrained n.m.r. structures allows accurate prediction of the pattern of hydrogen bonding.     

Side-chain conformational entropy in protein unfolded states

The largest force disfavoring the folding of a protein is the loss of conformational entropy. A large contribution to this entropy loss is due to the side-chains, which are restricted, although not immobilized, in the folded protein. In order to accurately estimate the loss of side-chain conformational entropy that occurs upon folding it is necessary to have accurate estimates of the amount of entropy possessed by side-chains in the ensemble of unfolded states. A new scale of side-chain conformational entropies is presented here. This scale was derived from Monte Carlo computer simulations of small peptide models. It is demonstrated that the entropies are independent of host peptide length. This new scale has the advantage over previous scales of being more precise with low standard errors. Better estimates are obtained for long (e.g., Arg and Lys) and rare (e.g., Trp and Met) side-chains. Excellent agreement with previous side-chain entropy scales is achieved, indicating that further advancements in accuracy are likely to be small at best. Strikingly, longer side-chains are found to possess a smaller fraction of the theoretical maximum entropy available than short side-chains. This indicates that rotations about torsions after chi(2) are significantly affected by side-chain interactions with the polypeptide backbone. This finding invalidates previous assumptions about side-chain-backbone interactions. Proteins 2000;40:443-450.     

Optimization of linear disorder predictors yields tight association between crystallographic disorder and hydrophobicity

X-ray crystallographic protein structures often contain disordered regions that are observed as missing electron density. Diffraction data may give little or no direct evidence as to the specific nature of disordered regions. We have developed a weighted window-based disorder predictor optimized using crystallographic data. Performance of a predictor is strongly influenced by chain termini. Optimized score adjustment values for amino- and carboxy-terminal positions demonstrate a simple, monotonic relationship between disorder and residue distance from termini. This optimized disorder predictor performs similarly to DISOPRED2 on crystallographically disordered regions. Data-optimized residue disorder propensities show strong linear correlation with experimentally determined amino acid transfer energies between water and hydrogen-bonding organic solvents, which primarily reflect residue hydrophobicity (exemplified by the Nozaki-Tanford hydrophobicity scale). Disorder propensities do not correlate as well with transfer energies between water and apolar solvents, which primarily reflect a different hydropathic property: residue hydrophilicity (also reflected by the Kyte-Doolittle hydropathy scale). Our results suggest that while hydrophobic side-chain interactions are primarily involved in determining stability of the folded conformation, hydrogen bonding, and similar polar interactions are primarily involved in conformational and interaction specificity.     

Protein structure, stability and solubility in water and other solvents

Proteins carry out the most difficult tasks in living cells. They do so by interacting specifically with other molecules. This requires that they fold to a unique, globular conformation that is only marginally more stable than the large ensemble of unfolded states. The folded state is stabilized mainly by the burial and tight packing of over 80% of the peptide groups and non-polar side chains. If life as we know it is to exist in a solvent other than water, the folded state must be stable and soluble in the new solvent. Our analysis suggests that proteins will be unstable in most polar solvents such as ethanol, extremely stable in non-polar solvents such as cyclohexane, and even more stable in a vacuum. Our solubility studies suggest that protein solubility will be markedly lower in polar solvents such as ethanol and that proteins will be essentially insoluble in non-polar solvents such as cyclohexane. For these and other reasons it seems unlikely that the life we know could exist in any solvent system other than water.     

Thermodynamic consequences of burial of polar and non-polar amino acid residues in the protein interior

Effects of amino acid substitutions at four fully buried sites of the ubiquitin molecule on the thermodynamic parameters (enthalpy, Gibbs energy) of unfolding were evaluated experimentally using differential scanning calorimetry. The same set of substitutions has been incorporated at each of four sites. These substitutions have been designed to perturb packing (van der Waals) interactions, hydration, and/or hydrogen bonding. From the analysis of the thermodynamic parameters for these ubiquitin variants we conclude that: (i) packing of non-polar groups in the protein interior is favorable and is largely defined by a favorable enthalpy of van der Waals interactions. The removal of one methylene group from the protein interior will destabilize a protein by approximately 5 kJ/mol, and will decrease the enthalpy of a protein by 12 kJ/mol. (ii) Burial of polar groups in the non-polar interior of a protein is highly destabilizing, and the degree of destabilization depends on the relative polarity of this group. For example, burial of Thr side-chain in the non-polar interior will be less destabilizing than burial of Asn side-chain. This decrease in stability is defined by a large enthalpy of dehydration of polar groups upon burial. (iii) The destabilizing effect of dehydration of polar groups upon burial can be compensated if these buried polar groups form hydrogen bonding. The enthalpy of this hydrogen bonding will compensate for the unfavorable dehydration energy and as a result the effect will be energetically neutral or even slightly stabilizing.     

Assignment of the 1H nuclear magnetic resonance spectrum of the proteinase inhibitor IIA from bull seminal plasma by two-dimensional nuclear magnetic resonance at 500 MHz

The assignment of the 1H nuclear magnetic resonance (n.m.r.) spectrum of the protease inhibitor IIA from bull seminal plasma is described and documented. The assignments are based entirely on the amino acid sequence and on two-dimensional n.m.r. experiments at 500 MHz. Individual assignments were obtained at 18 degrees C and 45 degrees C for the backbone protons of all 57 amino acid residues, with the single exception of the N-terminal pyroglutamate amide proton. The amino acid side-chain resonance assignments are complete, with the exception of 17 long side-chains, i.e. Pro13, Met43 and all the Glu, Gln, Lys and Arg, where only one or two resonances of C beta H2 and in some cases C gamma H2 could be identified. The sequential assignments showed that the order of the two C-terminal residues in the previously established primary structure had to be changed; this was then confirmed by chemical methods. The chemical shifts for the assigned resonances at 18 degrees C and 45 degrees C are listed for an aqueous solution at pH 4.9. A preliminary characterization of the polypeptide secondary structure was obtained from the observed patterns of sequential connectivities.     

Amino acid side-chain partition energies and distribution of residues in soluble proteins

Energies required to transfer amino acid side chains from water to less polar environments were calculated from results of several studies and compared with several statistical analyses of residue distributions in soluble proteins. An analysis that divides proteins into layers parallel with their surfaces is more informative than those that simply classify residues as exposed or buried. Most residues appear to be distributed as a function of the distance from the protein-water interface in a manner consistent with partition energies calculated from partitioning of amino acids between water and octanol phases and from solubilities of amino acids in water, ethanol, and methanol. Lys, Arg, Tyr, and Trp residues tend to concentrate near the water-protein interface where their apolar side-chain components are more buried than their polar side-chain components. Residue distributions calculated in this manner do not correlate well with side-chain solvation energies calculated from vapor pressures of side-chain analogs over a water phase. Results of statistical studies that classify residues as exposed to solvent or buried inside the protein interior appear to depend on the method used to classify residues. Data from some of these studies correlate better with solvation energies, but other data correlate better with partition energies. Most other statistical methods that have been used to evaluate effects of water on residue distributions yield results that correlate better with partition energies than with solvation energies.     

Partitioning of four modern volatile general anesthetics into solvents that model buried amino acid side-chains.

Partitioning of four modern inhalational anesthetics (halothane, isoflurane, enflurane, and sevoflurane) between the gas phase and nine organic solvents that model different amino acid side-chains and lipid membrane domains was performed in an effort to define which microenvironments present in proteins and lipid bilayers might be favored. Compared to a purely aliphatic environment (hexane), the presence of an aromatic-, alcohol-, thiol- or sulfide group on the solvent improved anesthetic partitioning, by factors of 1.3-5.2 for halothane, 1.7-5.6 for isoflurane, 1.7-7.6 for enflurane, and 1.5-7.3 for sevoflurane. The most favorable solvent for halothane partitioning was ethyl methyl sulfide, a model for methionine. Enflurane and isoflurane partitioned most extensively into methanol, a model for serine, and sevoflurane into ethanol, a model for threonine. Isoflurane also partitioned favorably into ethyl methyl sulfide. The results suggest that volatile general anesthetics interact better with partly polar groups, which are present on amino acids frequently found buried in the hydrophobic core of proteins, compared to purely aliphatic side-chains. Furthermore, if an anesthetic molecule was located in a saturated region of a phospholipid bilayer membrane, there would be an energetically favorable driving force for it to move into several higher dielectric microenvironments present on membrane proteins. The results provide evidence that proteins rather than lipids are the likely targets of volatile general anesthetics in biological membranes.     

A simple method for displaying the hydropathic character of a protein

A computer program that progressively evaluates the hydrophilicity and hydrophobicity of a protein along its amino acid sequence has been devised. For this purpose, a hydropathy scale has been composed wherein the hydrophilic and hydrophobic properties of each of the 20 amino acid side-chains is taken into consideration. The scale is based on an amalgam of experimental observations derived from the literature. The program uses a moving-segment approach that continuously determines the average hydropathy within a segment of predetermined length as it advances through the sequence. The consecutive scores are plotted from the amino to the carboxy terminus. At the same time, a midpoint line is printed that corresponds to the grand average of the hydropathy of the amino acid compositions found in most of the sequenced proteins. In the case of soluble, globular proteins there is a remarkable correspondence between the interior portions of their sequence and the regions appearing on the hydrophobic side of the midpoint line, as well as the exterior portions and the regions on the hydrophilic side. The correlation was demonstrated by comparisons between the plotted values and known structures determined by crystallography. In the case of membrane-bound proteins, the portions of their sequences that are located within the lipid bilayer are also clearly delineated by large uninterrupted areas on the hydrophobic side of the midpoint line. As such, the membrane-spanning segments of these proteins can be identified by this procedure. Although the method is not unique and embodies principles that have long been appreciated, its simplicity and its graphic nature make it a very useful tool for the evaluation of protein structures.     

Periodicity in alpha-helix lengths and C-capping preferences.

We surveyed 299 high resolution, non-homologous protein crystal structures for alpha-helix lengths and capping preferences. We find that helices show a preference to have close to an integral number of turns. Helices can be usefully subdivided into either "favoured length" with 6, 7, 10, 11, 13, 14, 17, 18, 21, 22, 24, 25, 28, 29 or 31 residues, or "disfavoured length" with 8, 9, 12, 15, 16, 19, 20, 23, 26, 27 or 30 residues. Favoured length helices have their N and C-caps on the same side of the helix so they can lie on the protein surface. There is no significant difference in amino acid preferences at the N terminus between favoured and disfavoured length helices. At the C terminus, favoured length helices prefer non-polar side-chains at C4 and polar amino acid residues at C2, while disfavoured length helices prefer non-polar amino acid residues at C2. There are strong periodic trends in the likelihood of terminating a helix with a Schellman or alphaL C-capping motif. These can be rationalised by the preference for a non-polar side-chain at C3 with these motifs, favouring placing C3 on the buried side of the helix. We suggest that algorithms aiming to predict helices or C-capping in proteins should include a weight for the helix length.     

Quantitative IR spectrophotometry of peptide compounds in water (H2O) solutions. II. Amide absorption bands of polypeptides and fibrous proteins in alpha-, beta-, and random coil conformations.

Infrared spectra of poly(D,L-alanine), poly(L-glutamic acid), poly(L-lysine), silk fibroin, and tropomyosin have been registered for various conformations of the polypeptide chain. Assuming additivity of the main- and side-chain absorption, spectral parameters of amide I and II absorption bands corresponding to alpha-, beta-, and random coil conformations have been derived. The amide I band parameters for H2O and D2O have been compared.     

Structure of actinidin,after refinement at 1.7 Å resolution

The structure of the sulphydryl protease, actinidin, after refinement at 1.7 Å resolution, is described. The positions of most of the 1666 atoms have been determined with an accuracy better than 0.1 Å; only two residues (219 and 220) and the side-chain of a third (87) cannot be seen. In addition, the model contains 272 solvent molecules, all taken as water, except one which may be an ammonium ion. Atomic B values give a good indication of the mobility of different parts of the structure. Actinidin has a double domain structure, with one domain mostly helical in its secondary structure, and the other domain built around a twisted β-sheet. The geometry of hydrogen bonds in helices, β-structure and turns has been analysed. All are significantly non-linear, with the angle N-?…O ~160 °. Carbonyl groups are tilted outwards from the axis of each helix, the tilting apparently unaffected by whether or not additional hydrogen bonds are made (e.g. to water or side-chain atoms). Each domain is folded round a substantial core of non-polar side-chains, but the interface between domains is mostly polar. Interactions across this interface involve a network of eight buried water molecules, the buried carboxyl and amino groups of Glu35, Glu50, Lys181 and Lys17, other polar side-chains and a few hydrophobic groups. One other internal charged side-chain, that of Glu52, is adjacent to a buried solvent molecule, probably an ammonium ion. Other side-chain environments are described. One proline residue has a cis configuration. The sulphydryl group is oxidized, probably to SO₂^?, with one oxygen atom clearly visible but the other somewhat less certain. The active site geometry is otherwise compatible with the mechanism proposed by Drenth et al. (1975,1976) for papain. The positions of the 272 solvent molecules are described. The best-ordered water molecules are those that are internal (total of 17), in surface pockets, or in the intermolecular contact regions. These generally form three or four hydrogen bonds, two to proton acceptors and one or two to proton donors. Other water molecules make water bridges on the surface, sometimes covering the exposed edges of non-polar groups. Intermolecular contacts involve few protein atoms, but many water molecules.     

Automated extraction of backbone deuteration levels from amide H/2H mass spectrometry experiments

A Fourier deconvolution method has been developed to explicitly determine the amount of backbone amide deuterium incorporated into protein regions or segments by hydrogen/deuterium (H/D) exchange with high-resolution mass spectrometry. Determination and analysis of the level and number of backbone amide exchanging in solution provide more information about the solvent accessibility of the protein than do previous centroid methods, which only calculate the average deuterons exchanged. After exchange, a protein is digested into peptides as a way of determining the exchange within a local area of the protein. The mass of a peptide upon deuteration is a sum of the natural isotope abundance, fast exchanging side-chain hydrogens (present in MALDI-TOF H/2H data) and backbone amide exchange. Removal of the components of the isotopic distribution due to the natural isotope abundances and the fast exchanging side-chains allows for a precise quantification of the levels of backbone amide exchange, as is shown by an example from protein kinase A. The deconvoluted results are affected by overlapping peptides or inconsistent mass envelopes, and evaluation procedures for these cases are discussed. Finally, a method for determining the back exchange corrected populations is presented, and its effect on the data is discussed under various circumstances.