Achieving stability and conformational specificity in designed proteins via binary patterning

We have developed a method to determine the optimal binary pattern (arrangement of hydrophobic and polar amino acids) of a target protein fold prior to amino acid sequence selection in protein design studies. A solvent accessible surface is generated for a target fold using its backbone coordinates and "generic" side-chains, which are constructs whose size and shape are similar to an average amino acid. Each position is classified as hydrophobic or polar according to the solvent exposure of its generic side-chain. The method was tested by analyzing a set of proteins in the Protein Data Bank and by experimentally constructing and analyzing a set of engrailed homeodomain variants whose binary patterns were systematically varied. Selection of the optimal binary pattern results in a designed protein that is monomeric, well-folded, and hyperthermophilic. Homeodomain variants with fewer hydrophobic residues are destabilized, while additional hydrophobic residues induce aggregation. Binary patterning, in conjunction with a force field that models folded state energies, appears sufficient to satisfy two basic goals of protein design: stability and conformational specificity.     

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.     

Predicting surface exposure of amino acids from protein sequence

The amino acid residues on a protein surface play a key role in interaction with other molecules, determined many physical properties, and constrain the structure of the folded protein. A database of monomeric protein crystal structures was used to teach computer-simulated neural networks rules for predicting surface exposure from local sequence. These trained networks are able to correctly predict surface exposure for 72% of residues in a testing set using a binary model, (buried/exposed) and for 54% of residues using a ternary model (buried/intermediate/exposed). In the ternary model, only 11% of the exposed residues are predicted as buried and only 5% of the buried residues are predicted as exposed. Also, since the networks are able to predict exposure with a quantitative confidence estimate, it is possible to assign exposure for over half of the residues in a binary model with greater than 80% accuracy. Even more accurate predictions are obtained by making a consensus prediction of exposure for a homologous family. The effect of the local environment of an amino acid on its accessibility, though smaller than expected, is significant and accounts for the higher success rate of prediction than obtained with previously used criteria. In the absence of a three-dimensional structure, the ability to predict surface accessibility of amino acids directly from the sequence is a valuable tool in choosing sites of chemical modification or specific mutations and in studies of molecular interaction.     

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.     

Topography of the prostaglandin endoperoxide H2 synthase-2 in membranes

The topology of association of the monotopic protein cyclooxygenase-2 (COX-2) with membranes has been examined using EPR spectroscopy of spin-labeled recombinant human COX-2. Twenty-four mutants, each containing a single free cysteine substituted for an amino acid in the COX-2 membrane binding domain were expressed using the baculovirus system and purified, then conjugated with a nitroxide spin label and reconstituted into liposomes. Determining the relative accessibility of the nitroxide-tagged amino acid side chains for the solubilized COX-2 mutants, or COX-2 reconstituted into liposomes to nonpolar (oxygen) and polar (NiEDDA or CrOx) paramagnetic reagents allowed us to map the topology of COX-2 interaction with the lipid bilayer. When spin-labeled COX-2 was reconstituted into liposomes, EPR power saturation curves showed that side chains for all but two of the 24 mutants tested had limited accessibility to both polar and nonpolar paramagnetic relaxation agents, indicating that COX-2 associates primarily with the interfacial membrane region near the glycerol backbone and phospholipid head groups. Two amino acids, Phe(66) and Leu(67), were readily accessible to the non-polar relaxation agent oxygen, and thus likely inserted into the hydrophobic core of the lipid bilayer. However these residues are co-linear with amino acids in the interfacial region, so their extension into the hydrophobic core must be relatively shallow. EPR and structural data suggest that membrane interaction of COX-2 is also aided by partitioning of 4 aromatic amino acids, Phe(59), Phe(66), Tyr(76), and Phe(84) to the interfacial region, and by the electrostatic interactions of two basic amino acids, Arg(62) and Lys(64), with the phospholipid head groups.     

Analysis of accessible surface of residues in proteins

We analyzed the total, hydrophobic, and hydrophilic accessible surfaces (ASAs) of residues from a nonredundant bank of 587 3D structure proteins. In an extended fold, residues are classified into three families with respect to their hydrophobicity balance. As expected, residues lose part of their solvent-accessible surface with folding but the three groups remain. The decrease of accessibility is more pronounced for hydrophobic than hydrophilic residues. Amazingly, Lysine is the residue with the largest hydrophobic accessible surface in folded structures. Our analysis points out a clear difference between the mean (other studies) and median (this study) ASA values of hydrophobic residues, which should be taken into consideration for future investigations on a protein-accessible surface, in order to improve predictions requiring ASA values. The different secondary structures correspond to different accessibility of residues. Random coils, turns, and beta-structures (outside beta-sheets) are the most accessible folds, with an average of 30% accessibility. The helical residues are about 20% accessible, and the difference between the hydrophobic and the hydrophilic residues illustrates the amphipathy of many helices. Residues from beta-sheets are the most inaccessible to solvent (10% accessible). Hence, beta-sheets are the most appropriate structures to shield the hydrophobic parts of residues from water. We also show that there is an equal balance between the hydrophobic and the hydrophilic accessible surfaces of the 3D protein surfaces irrespective of the protein size. This results in a patchwork surface of hydrophobic and hydrophilic areas, which could be important for protein interactions and/or activity.     

Comparison of the structures and stabilities of coiled‐coil proteins containing hexafluoroleucine and t‐butylalanine provides insight into the stabilizing effects of highly fluorinated amino acid side‐chains

Highly fluorinated analogs of hydrophobic amino acids are well known to increase the stability of proteins toward thermal unfolding and chemical denaturation, but there is very little data on the structural consequences of fluorination. We have determined the structures and folding energies of three variants of a de novo designed 4‐helix bundle protein whose hydrophobic cores contain either hexafluoroleucine (hFLeu) or t‐butylalanine (tBAla). Although the buried hydrophobic surface area is the same for all three proteins, the incorporation of tBAla causes a rearrangement of the core packing, resulting in the formation of a destabilizing hydrophobic cavity at the center of the protein. In contrast, incorporation of hFLeu, causes no changes in core packing with respect to the structure of the nonfluorinated parent protein which contains only leucine in the core. These results support the idea that fluorinated residues are especially effective at stabilizing proteins because they closely mimic the shape of the natural residues they replace while increasing buried hydrophobic surface area.     

Development of indole chemistry to label tryptophan residues in protein for determination of tryptophan surface accessibility

Solvent accessibility can be used to evaluate protein structural models, identify binding sites, and characterize protein conformational changes. The differential modification of amino acids at specific sites enables the accessible surface residues to be identified by mass spectrometry. Tryptophan residues within proteins can be differentially labeled with halocompounds by a photochemical reaction. In this study, tryptophan residues of carbonic anhydrase are reacted with chloroform, 2,2,2-trichloroethanol (TCE), 2,2,2-trichloroacetate (TCA), or 3-bromo-1-propanol (BP) under UV irradiation at 280 nm. The light-driven reactions with chloroform, TCE, TCA, and BP attach a formyl, hydroxyethanone, carboxylic acid, and propanol group, respectively, onto the indole ring of tryptophan. Trypsin and chymotrypsin digests of the modified carbonic anhydrase are used to map accessible tryptophan residues using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Tryptophan reactivity is determined by identifying peptides with tryptophan residues modified with the appropriate label. The reactivity is calculated from the frequency that the modification is identified and a semiquantitative measure of the amount of products formed. Both of these measures of tryptophan reactivity correlate significantly with the accessible surface area of tryptophan residues in carbonic anhydrase determined from the X-ray crystal structure. Therefore the photochemical reaction of halocompounds with tryptophan residues in carbonic anhydrase indicates the degree of solvent accessibility of these residues.     

The apolar surface area of amino acids and its empirical correlation with hydrophobic free energy

A linear equation is presented which accounts quantitatively for the free energy of transfer of amino acids from water to apolar solvent as a function of accessible surface area and partial charges of the constituents of amino acids. Starting from these parameters the apolar surface area is defined and correlated with the measured free transfer energy. The resulting linear correlation makes it possible to calculate more precisely the "hydrophobic" contribution of both apolar and polar groups including uncharged side chains of arginine, lysine, glutamic and aspartic acids, and histidine, respectively, to protein stabilization.     

Quantifying the effect of burial of amino acid residues on protein stability

The average contribution of individual residue to folding stability and its dependence on buried accessible surface area (ASA) are obtained by two different approaches. One is based on experimental mutation data, and the other uses a new knowledge-based atom-atom potential of mean force. We show that the contribution of a residue has a significant correlation with buried ASA and the regression slopes of 20 amino acid residues (called the buriability) are all positive (pro-burial). The buriability parameter provides a quantitative measure of the driving force for the burial of a residue. The large buriability gap observed between hydrophobic and hydrophilic residues is responsible for the burial of hydrophobic residues in soluble proteins. Possible factors that contribute to the buriability gap are discussed.     

Stability scale and atomic solvation parameters extracted from 1023 mutation experiments

The stability scale of 20 amino acid residues is derived from a database of 1023 mutation experiments on 35 proteins. The resulting scale of hydrophobic residues has an excellent correlation with the octanol-to-water transfer free energy corrected with an additional Flory-Huggins molar-volume term (correlation coefficient r = 0.95, slope = 1.05, and a near zero intercept). Thus, hydrophobic contribution to folding stability is characterized remarkably well by transfer experiments. However, no corresponding correlation is found for hydrophilic residues. Both the hydrophilic portion and the entire scale, however, correlate strongly with average burial accessible surface (r = 0.76 and 0.97, respectively). Such a strong correlation leads to a near uniform value of the atomic solvation parameters for atoms C, S, O/N, O(-0.5), and N(+0.5,1). All are in the range of 12-28 cal x mol(-1) A(-2), close to the original estimate of hydrophobic contribution of 25-30 cal x mol(-1) A(-2) to folding stability. Without any adjustable parameters, the new stability scale and new atomic solvation parameters yielded an accurate prediction of protein-protein binding free energy for a separate database of 21 protein-protein complexes (r = 0.80 and slope = 1.06, and r = 0.83 and slope = 0.93, respectively).     

Solution nonideality related to solute molecular characteristics of amino acids.

By measuring the freezing-point depression for dilute, aqueous solutions of all water-soluble amino acids, we test the hypothesis that nonideality in aqueous solutions is due to solute-induced water structuring near hydrophobic surfaces and solute-induced water destructuring in the dipolar electric fields generated by the solute. Nonideality is expressed with a single solute/solvent interaction parameter I, calculated from experimental measure of delta T. A related parameter, I(n), gives a method of directly relating solute characteristics to solute-induced water structuring or destructuring. I(n)-values correlate directly with hydrophobic surface area and inversely with dipolar strength. By comparing the nonideality of amino acids with progressively larger hydrophobic side chains, structuring is shown to increase with hydrophobic surface area at a rate of one perturbed water molecule per 8.8 square angstroms, implying monolayer coverage. Destructuring is attributed to dielectric realignment as described by the Debye-Hückel theory, but with a constant separation of charges in the amino-carboxyl dipole. By using dimers and trimers of glycine and alanine, this destructuring is shown to increase with increasing dipole strength using increased separation of fixed dipolar charges. The capacity to predict nonideal solution behavior on the basis of amino acid characteristics will permit prediction of free energy of transfer to water, which may help predict the energetics of folding and unfolding of proteins based on the characteristics of constituent amino acids.     

Local flexibility facilitates oxidization of buried methionine residues

In proteins, all amino acid residues are susceptible to oxidation by various reactive oxygen species (ROS), with methionine and cysteine residues being particularly sensitive to oxidation. Methionine oxidation is known to lead to destabilization and inactivation of proteins, and oxidatively modified proteins can accumulate during aging, oxidative stress, and in various age-related diseases. Although the efficiency of a given methionine oxidation can depend on its solvent accessibility (evaluated from a protein structure as the accessible surface area of the corresponding methionine residue), many experimental results on oxidation rate and oxidation sites cannot be unequivocally explained by the methionine solvent accessible surface area alone. In order to explore other possible mechanisms, we analyzed a set of seventy-one oxidized methionines contained in thirty-one proteins by various bioinformatics tools. In which, 41% of the methionines are exposed, 15% are buried but with various degree of flexibility, and the rest 44% are buried and structured. Buried but highly flexible methionines can be oxidized. Buried and less flexible methionines can acquire additional local structural flexibility from flanking regions to facilitate the oxidation. Oxidation of buried and structured methionine can also be promoted by the oxidation of neighboring methionine that is more exposed and/or flexible. Our data are consistent with the hypothesis that protein structural flexibility represents another important factor favoring the oxidation process.     

Thermodynamic stability of histone H3 is a necessary but not sufficient driving force for its evolutionary conservation

Determining the forces that conserve amino acid positions in proteins across species is a fundamental pursuit of molecular evolution. Evolutionary conservation is driven by either a protein's function or its thermodynamic stability. Highly conserved histone proteins offer a platform to evaluate these driving forces. While the conservation of histone H3 and H4 "tail" domains and surface residues are driven by functional importance, the driving force behind the conservation of buried histone residues has not been examined. Using a computational approach, we determined the thermodynamically preferred amino acids at each buried position in H3 and H4. In agreement with what is normally observed in proteins, we find a significant correlation between thermodynamic stability and evolutionary conservation in the buried residues in H4. In striking contrast, we find that thermodynamic stability of buried H3 residues does not correlate with evolutionary conservation. Given that these H3 residues are not post-translationally modified and only regulate H3-H3 and H3-H4 stabilizing interactions, our data imply an unknown function responsible for driving conservation of these buried H3 residues.     

Position-dependence of stabilizing polar interactions of asparagine in transmembrane helical bundles

Recent studies with model peptides and statistical analyses of the crystal structures of membrane proteins have shown that buried polar interactions contribute significantly to the stabilization of the three-dimensional structures of membrane proteins. Here, we probe how the location of these polar groups along the transmembrane helices affect their free energies of interaction. Asn residues were placed singly and in pairs at three positions within a model transmembrane helix, which had previously been shown to support the formation of trimers in micelles. The model helix was designed to form a transmembrane coiled coil, with Val side chains at the "a" positions of the heptad repeat. Variants of this peptide were prepared in which an Asn residue was introduced at one or more of the "a" positions, and their free energies of association were determined by analytical ultracentrifugation. When placed near the middle of the transmembrane helix, the formation of trimers was stabilized by at least -2.0 kcal/mol per Asn side chain. When the Asn was placed at the interface between the hydrophobic and polar regions of the peptide, the substitution was neither stabilizing nor destabilizing (0.0 +/- 0.5 kcal/mol of monomer). Finally, it has previously been shown that a Val-for-Asn mutation in a water-soluble coiled coil destabilizes the structure by approximately 1.5 kcal/mol of monomer [Acharya, A., et al. (2002) Biochemistry 41, 14122-14131]. Thus, the headgroup region of a micelle appears to have a conformational impact intermediate between that of bulk water and the apolar region of micelle. A similarly large dependence on the location of the polar residues was found in a statistical survey of helical transmembrane proteins. The tendency of different types of residues to be buried in the interiors versus being exposed to lipids was analyzed. Asn and Gln show a very strong tendency to be buried when they are located near the middle of a transmembrane helix. However, when placed near the ends of transmembrane helices, they show little preference for the surface versus the interior of the protein. These data show that Asn side chains within the apolar region of the transmembrane helix provide a significantly larger driving force for association than Asn residues near the apolar/polar interface. Thus, although polar interactions are able to strongly stabilize the folding of membrane proteins, the energetics of association depend on their location within the hydrophobic region of a transmembrane helix.     

Analysis of tryptophan surface accessibility in proteins by MALDI-TOF mass spectrometry

Surface accessible amino acids can play an important role in proteins. They can participate in enzyme's active center structure or in specific intermolecular interactions. Thus, the information about selected amino acids' surface accessibility can contribute to the understanding of protein structure and function. In this paper, we present a simple method for surface accessibility mapping of tryptophan side chains by their chemical modification and identification by MALDI-TOF mass spectrometry. The reaction with 2-hydroxy-5-nitrobenzyl bromide, a common and highly specific covalent modification of tryptophan, seems to be very useful for this purpose. The method was tested on four model proteins with known spatial structure. In the native proteins (1) only surface accessible tryptophan side chains were found to react with the modification agent and (2) no buried one was found to react at lower reagent concentrations. These results indicate that the described method can be a potent tool for identification of surface-located tryptophan side chain in a protein of unknown conformation.     

Local hydrophobicity stabilizes secondary structures in proteins

The probability of occurrence of helix and β-sheet residues in 47 globular proteins was determined as a function of local hydrophobicity, which was defined by the sum of the Nozaki-Tanford transfer free energies at two nearest-neighbors on both sides of the amino acid sequence. In general, hydrophilic amino acids favor neither helix nor β-sheet formations when neighbor residues are also hydrophilic but favor helix formation at higher local hydrophobicity. On the other hand, some hydrophobic amino acids such as Met, Leu, and Ile favor helix formation when neighbor residues are hydrophilic. None of the hydrophobic amino acids favor β-sheet formation with hydrophilic neighbors, but most of them strongly favor β-sheet formation at high local hydrophobicity. When the average of 20 amino acids is taken, both helix and β-sheet residue probabilities are higher at higher local hydrophobicity, although the increase is steeper for β-sheets. Therefore, β-sheet formation is more influenced by local hydrophobicity than helix formation. Generally, helices are nearer the surface and tend to have hydrophilic and hydrophobic faces at opposite sides. The tendency of alternating regions of hydrophilic and hydrophobic residues in a helical sequence was revealed by calculating the correlation of the Nozaki-Tanford values. Such amphipathic helices may be important in protein–protein and protein–lipid interactions and in forming hydrophilic channels in the membrane. The choice of 30 nonhomologous proteins as the data set did not alter the above results.     

Interior and surface of monomeric proteins

The solvent-accessible surface area (As) of 46 monomeric proteins is calculated using atomic co-ordinates from high-resolution and well-refined crystal structures. The As of these proteins can be determined to within 1 to 2% and that of their individual residues to within 10 to 20%. The As values of proteins are correlated with their molecular weight (Mr) in the range 4000 to 35,000: the power law As = 6.3 M0.73 predicts protein As values to within 4% on average. The average water-accessible surface is found to be 57% non-polar, 24% polar and 19% charged, with 5% root-mean-square variations. The molecular surface buried inside the protein is 58% non-polar, 39% polar and 4% charged. The buried surface contains more uncharged polar groups (mostly peptides) than the surface that remains accessible, but many fewer charged groups. On average, 15% of residues in small proteins and 32% in larger ones may be classed as "buried residues", having less than 5% of their surface accessible to the solvent. The accessibilities of most other residues are evenly distributed in the range 5 to 50%. Although the fraction of buried residues increases with molecular weight, the amino acid compositions of the protein interior and surface show no systematic variation with molecular weight, except for small proteins that are often very rich in buried cysteines. From amino acid compositions of protein surfaces and interiors we calculate an effective coefficient of partition for each type of residue, and derive an implied set of transfer free energy values. This is compared with other sets of partition coefficients derived directly from experimental data. The extent to which groups of residues (charged, polar and non-polar) are buried within proteins correlates well with their hydrophobicity derived from amino acid transfer experiments. Within these three groups, the correlation is low.     

A Dynamic Hydrophobic Core and Surface Salt Bridges Thermostabilize a Designed Three-Helix Bundle

Thermostable proteins are advantageous in industrial applications, as pharmaceuticals or biosensors, and as templates for directed evolution. As protein-design methodologies improve, bioengineers are able to design proteins to perform a desired function. Although many rationally designed proteins end up being thermostable, how to intentionally design de novo, thermostable proteins is less clear. UVF is a de novo-designed protein based on the backbone structure of the Engrailed homeodomain (EnHD) and is highly thermostable (T_m > 99°C vs. 52°C for EnHD). Although most proteins generally have polar amino acids on their surfaces and hydrophobic amino acids buried in their cores, protein engineers followed this rule exactly when designing UVF. To investigate the contributions of the fully hydrophobic core versus the fully polar surface to UVF’s thermostability, we built two hybrid, chimeric proteins combining the sets of buried and surface residues from UVF and EnHD. Here, we determined a structural, dynamic, and thermodynamic explanation for UVF’s thermostability by performing 4 μs of all-atom, explicit-solvent molecular dynamics simulations at 25 and 100°C, Tanford-Kirkwood solvent accessibility Monte Carlo electrostatic calculations, and a thermodynamic analysis of 40 temperature runs by the weighted-histogram analysis method of heavy-atom, structure-based models of UVF, EnHD, and both chimeric proteins. Our models showed that UVF was highly dynamic because of its fully hydrophobic core, leading to a smaller loss of entropy upon folding. The charged residues on its surface made favorable electrostatic interactions that contributed enthalpically to its thermostability. In the chimeric proteins, both the hydrophobic core and charged surface independently imparted thermostability.