HIV-1 protease function and structure studies with the simplicial neighborhood analysis of protein packing method

The Simplicial Neighborhood Analysis of Protein Packing (SNAPP) method was used to predict the effect of mutagenesis on the enzymatic activity of the HIV-1 protease (HIVP). SNAPP relies on a four-body statistical scoring function derived from the analysis of spatially nearest neighbor residue compositional preferences in a diverse and representative subset of protein structures from the Protein Data Bank. The method was applied to the analysis of HIVP mutants with residue substitutions in the hydrophobic core as well as at the interface between the two protease monomers. Both wild-type and tethered structures were employed in the calculations. We obtained a strong correlation, with R(2) as high as 0.96, between DeltaSNAPP score (i.e., the difference in SNAPP scores between wild-type and mutant proteins) and the protease catalytic activity for tethered structures. However, a weaker but significant correlation was obtained for nontethered structures. Our analysis identified residues both in the hydrophobic core and at the dimeric interface that are very important for the protease function. This study demonstrates a potential utility of the SNAPP method for rational design of mutagenesis studies and protein engineering.     

Evaluation of the relative stability of liganded versus ligand-free protein conformations using Simplicial Neighborhood Analysis of Protein Packing (SNAPP) method

Many proteins change their conformation upon ligand binding. For instance, bacterial periplasmic binding proteins (bPBPs), which transport nutrients into the cytoplasm, generally consist of two globular domains connected by strands, forming a hinge. During ligand binding, hinge motion changes the conformation from the open to the closed form. Both forms can be crystallized without a ligand, suggesting that the energy difference between them is small. We applied Simplicial Neighborhood Analysis of Protein Packing (SNAPP) as a method to evaluate the relative stability of open and closed forms in bPBPs. Using united residue representation of amino acids, SNAPP performs Delaunay tessellation of the protein, producing an aggregate of space-filling, irregular tetrahedra with nearest neighbor residues at the vertices. The SNAPP statistical scoring function is derived from log-likelihood scores for all possible quadruplet compositions of amino acids found in a representative subset of the Protein Data Bank, and the sum of the scores for a given protein provides the total SNAPP score. Results of scoring for bPBPs suggest that in most cases, the unliganded form is more stable than the liganded form, and this conclusion is corroborated by similar observations of other proteins undergoing conformation changes upon binding their ligands. The results of these studies suggest that the SNAPP method can be used to predict the relative stability of accessible protein conformations. Furthermore, the SNAPP method allows delineation of the role of individual residues in protein stabilization, thereby providing new testable hypotheses for rational site-directed mutagenesis in the context of protein engineering.     

Examining protein surface structure in highly conserved sequence variants with mass spectrometry

A simple mass spectrometry-based method capable of examining protein structure called SNAPP (selective noncovalent adduct protein probing) is used to evaluate the structural consequences of point mutations in naturally occurring sequence variants from different species. SNAPP monitors changes in the attachment of noncovalent adducts to proteins as a function of structural state. Mutations that lead to perturbations to the electrostatic surface structure of a protein affect noncovalent attachment and are easily observed with SNAPP. Mutations that do not alter the tertiary structure or electrostatic surface structure yield similar results by SNAPP. For example, bovine, porcine, and human insulin all have very similar backbone structures and no basic or acidic residue mutations, and the SNAPP distributions for all three proteins are very similar. In contrast, four variants of cytochrome c (cytc) have varying degrees of sequence homology, which are reflected in the observed SNAPP distributions. Bovine and pigeon cytc have several basic or acidic residue substitutions relative to horse cytc, but the SNAPP distributions for all three proteins are similar. This suggests that these mutations do not significantly influence the protein surface structure. On the other hand, yeast cytc has the least sequence homology and exhibits a unique, though related, SNAPP distribution. Even greater differences are observed for lysozyme. Hen and human lysozyme have identical tertiary structures but significant variations in the locations of numerous basic and acidic residues. The SNAPP distributions are quite distinct for the two forms of lysozyme, suggesting significant differences in the surface structures. In summary, SNAPP experiments are relatively easy to perform, require minimal sample consumption, and provide a facile route for comparison of protein surface structure between highly homologous proteins.     

Fluorous effect in proteins: de novo design and characterization of a four-alpha-helix bundle protein containing hexafluoroleucine

Several studies have demonstrated that proteins incorporating fluorinated analogues of hydrophobic amino acids such as leucine and valine into their hydrophobic cores exhibit increased stability toward thermal denaturation and unfolding by guanidinium chloride. However, estimates for the increase in the thermodynamic stability of a protein (DeltaDeltaG(unfold)) afforded by the substitution of a hydrophobic amino acid with its fluorinated analogue vary quite significantly. To address this, we have designed a peptide that adopts an antiparallel four-helix bundle structure in which the hydrophobic core is packed with leucine, and investigated the effects of substituting the central two layers of the core with L-5,5,5,5',5',5'-hexafluoroleucine (hFLeu). We find that DeltaDeltaG(unfold) is increased by 0.3 kcal/mol per hFLeu residue. This is in good agreement with the predicted increase in DeltaDeltaG(unfold) of 0.4 kcal/mol per residue arising from the increased hydrophobicity of the hFLeu side chain, which we determined experimentally from partitioning measurements on hFLeu and leucine. The increased stability of this fluorinated protein may therefore be ascribed to simple hydrophobic effects, rather than specific "fluorous" interactions between the hFLeu residues.     

Free energies for coarse-grained proteins by integrating multibody statistical contact potentials with entropies from elastic network models

We propose a novel method of calculation of free energy for coarse grained models of proteins by combining our newly developed multibody potentials with entropies computed from elastic network models of proteins. Multi-body potentials have been of much interest recently because they take into account three dimensional interactions related to residue packing and capture the cooperativity of these interactions in protein structures. Combining four-body non-sequential, four-body sequential and pairwise short range potentials with optimized weights for each term, our coarse-grained potential improved recognition of native structure among misfolded decoys, outperforming all other contact potentials for CASP8 decoy sets and performance comparable to the fully atomic empirical DFIRE potentials. By combing statistical contact potentials with entropies from elastic network models of the same structures we can compute free energy changes and improve coarse-grained modeling of protein structure and dynamics. The consideration of protein flexibility and dynamics should improve protein structure prediction and refinement of computational models. This work is the first to combine coarse-grained multibody potentials with an entropic model that takes into account contributions of the entire structure, investigating native-like decoy selection.     

Multibody coarse-grained potentials for native structure recognition and quality assessment of protein models

Multibody potentials have been of much interest recently because they take into account three dimensional interactions related to residue packing and capture the cooperativity of these interactions in protein structures. Our goal was to combine long range multibody potentials and short range potentials to improve recognition of native structure among misfolded decoys. We optimized the weights for four-body nonsequential, four-body sequential, and short range potentials to obtain optimal model ranking results for threading and have compared these data against results obtained with other potentials (26 different coarse-grained potentials from the Potentials 'R'Us web server have been used). Our optimized multibody potentials outperform all other contact potentials in the recognition of the native structure among decoys, both for models from homology template-based modeling and from template-free modeling in CASP8 decoy sets. We have compared the results obtained for this optimized coarse-grained potentials, where each residue is represented by a single point, with results obtained by using the DFIRE potential, which takes into account atomic level information of proteins. We found that for all proteins larger than 80 amino acids our optimized coarse-grained potentials yield results comparable to those obtained with the atomic DFIRE potential.     

Stability Changes upon Mutation of Solvent- accessible Residues in Proteins Evaluated by Database-derived Potentials

The stability changes in peptides and proteins caused by the substitution of a single amino acid, which can be measured experimentally by the change in folding free energy, are evaluated here using effective potentials derived from known protein structures. The analysis is focused on mutations of residues that are accessible to the solvent. These represent in total 106 mutations, introduced at different sites in barnase, bacteriophage T4 lysozyme and chymotrypsin inhibitor 2, and in a synthetic helical peptide. Assuming that the mutations do not modify the backbone structure, the changes in folding free energies are computed using various types of database-derived potentials and are compared with the measured ones. Distance-dependent residue – residue potentials are found to be inadequate for estimating the stability changes caused by these mutations, as they are dominated by hydrophobic interactions, which do not play an essential role at the protein surface. On the contrary, the potentials based on backbone torsion angle propensities yield quite good results. Indeed, for a subset of 96 out of the 106 mutations, the computed and measured changes in folding free energy correlate with a linear correlation coefficient of 0.87. Moreover, the ten mutations that are excluded from the correlation either seem to cause modifications of the backbone structure or to involve strong hydrophobic interactions, which are atypical for solvent-accessible residues. We find furthermore that raising the ionic strength of the solvent used for measuring the changes in folding free energies improves the correlation, as it tends to mask the electrostatic interactions. When adding to these 106 mutations 44 mutations performed in staphylococcal nuclease and chemotactic protein, which were first discarded because some of them were suspected to affect the backbone conformation or the denatured state, the correlation between measured and computed folding free energy changes remains quite good: the correlation coefficient is 0.86 for 135 out of the 150 mutations. The success of the backbone torsion potentials in predicting stability changes indicates that the approximations made for deriving these potentials are adequate. It suggests moreover that the local interactions along the chain dominate at the protein surface.     

Effect of site-directed point mutations on protein misfolding: A simulation study

A Monte Carlo simulation based sequence design method is proposed to investigate the role of site-directed point mutations in protein misfolding. Site-directed point mutations are incorporated in the designed sequences of selected proteins. While most mutated sequences correctly fold to their native conformation, some of them stabilize in other nonnative conformations and thus misfold/unfold. The results suggest that a critical number of hydrophobic amino acid residues must be present in the core of the correctly folded proteins, whereas proteins misfold/unfold if this number of hydrophobic residues falls below the critical limit. A protein can accommodate only a particular number of hydrophobic residues at the surface, provided a large number of hydrophilic residues are present at the surface and critical hydrophobicity of the core is preserved. Some surface sites are observed to be equally sensitive toward site-directed point mutations as the core sites. Point mutations with highly polar and charged amino acids increases the misfold/unfold propensity of proteins. Substitution of natural amino acids at sites with different number of nonbonded contacts suggests that both amino acid identity and its respective site-specificity determine the stability of a protein. A clash-match method is developed to calculate the number of matching and clashing interactions in the mutated protein sequences. While misfolded/unfolded sequences have a higher number of clashing and a lower number of matching interactions, the correctly folded sequences have a lower number of clashing and a higher number of matching interactions. These results are valid for different SCOP classes of proteins.     

Hydrogen-exchange stabilities of RNase T1 and variants with buried and solvent-exposed Ala --> Gly mutations in the helix

Hydrogen-exchange rates were measured for RNase T1 and three variants with Ala --> Gly substitutions at a solvent-exposed (residue 21) and a buried (residue 23) position in the helix: A21G, G23A, and A21G + G23A. These results were used to measure the stabilities of the proteins. The hydrogen-exchange stabilities (DeltaG(HX)) for the most stable residues in each variant agree with the equilibrium conformational stability measured by urea denaturation (DeltaG(U)), if the effects of D(2)O and proline isomerization are included [Huyghues-Despointes, B. M. P., Scholtz, J. M., and Pace, C. N. (1999) Nat. Struct. Biol. 6, 210-212]. These residues also show similar changes in DeltaG(HX) upon Ala --> Gly mutations (DeltaDeltaG(HX)) as compared to equilibrium measurements (DeltaDeltaG(U)), indicating that the most stable residues are exchanging from the globally unfolded ensemble. Alanine is stabilizing compared to glycine by 1 kcal/mol at a solvent-exposed site 21 as seen by other methods for the RNase T1 protein and peptide helix [Myers, J. K., Pace, C. N., and Scholtz, J. M. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 3833-2837], while it is destabilizing at the buried site 23 by the same amount. For the A21G variant, only local NMR chemical shift perturbations are observed compared to RNase T1. For the G23A variant, large chemical shift changes are seen throughout the sequence, although X-ray crystal structures of the variant and RNase T1 are nearly superimposable. Ala --> Gly mutations in the helix of RNase T1 at both helical positions alter the native-state hydrogen-exchange stabilities of residues throughout the sequence.     

Protein chemistry at membrane interfaces: non-additivity of electrostatic and hydrophobic interactions

Non-specific binding of proteins and peptides to charged membrane interfaces depends upon the combined contributions of hydrophobic (DeltaG(HPhi)) and electrostatic (DeltaG(ES)) free energies. If these are simply additive, then the observed free energy of binding (DeltaG(obs)) will be given by DeltaG(obs)=DeltaG(HPhi)+DeltaG(ES), where DeltaG(HPhi)=-sigma(NP)A(NP) and DeltaG(ES)=zFphi. In these expressions, A(NP) is the non-polar accessible area, sigma(NP) the non-polar solvation parameter, z the formal peptide valence, F the Faraday constant, and phi the membrane surface potential. But several lines of evidence suggest that hydrophobic and electrostatic binding free energies of proteins at membrane interfaces, such as those associated with cell signaling, are not simply additive. In order to explore this issue systematically, we have determined the interfacial partitioning free energies of variants of indolicidin, a cationic proline-rich antimicrobial peptide. The synthesized variants of the 13 residue peptide covered a wide range of hydrophobic free energies, which allowed us to examine the effect of hydrophobicity on electrostatic binding to membranes formed from mixtures of neutral and anionic lipids. Although DeltaG(obs) was always a linear function of DeltaG(HPhi), the slope depended upon anionic lipid content: the slope was 1.0 for pure, zwitterionic phosphocholine bilayers and 0.3 for pure phosphoglycerol membranes. DeltaG(obs) also varied linearly with surface potential, but the slope was smaller than the expected value, zF. As observed by others, this suggests an effective peptide valence z(eff) that is smaller than the formal valence z. Because of our systematic approach, we were able to establish a useful rule-of-thumb: z(eff) is reduced relative to z by about 20 % for each 3 kcal mol(-1) (1 kcal=4.184 kJ) favorable increase in DeltaG(HPhi). For neutral phosphocholine interfaces, we found that DeltaG(obs) could be predicted with remarkable accuracy using the Wimley-White experiment-based interfacial hydrophobicity scale.     

Four-body scoring function for mutagenesis

MOTIVATION: There is a need for an efficient and accurate computational method to identify the effects of single- and multiple-residue mutations on the stability and reactivity of proteins. Such a method should ideally be consistent and yet applicable in a widespread manner, i.e. it should be applied to various proteins under the same parameter settings, and have good predictive power for all of them. RESULTS: We develop a Delaunay tessellation-based four-body scoring function to predict the effects of single- and multiple-residue mutations on the stability and reactivity of proteins. We test our scoring function on sets of single-point mutations used by several previous studies. We also assemble a new, diverse set of 237 single- and multiple-residue mutations, from over 24 different publications. The four-body scoring function correctly predicted the changes to the stability of 169 out of 210 mutants (80.5%), and the changes to the reactivity of 17 out of 27 mutants (63%). For the mutants that had the changes in stability/reactivity quantified (using reaction rates, temperatures, etc.), an average Spearman rank correlation coefficient of 0.67 was achieved with the four-body scores. We also develop an efficient method for screening huge numbers of mutants of a protein, called combinatorial mutagenesis. In one study, 64 million mutants of a cold-shock nucleus binding domain protein 1CSQ, with six of its residues being changed to all possible (20) amino acids, were screened within a few hours on a PC, and all five stabilizing mutants reported were correctly identified as stabilizing by combinatorial mutagenesis.     

Statistical significance of hierarchical multi-body potentials based on Delaunay tessellation and their application in sequence-structure alignment.

Statistical potentials based on pairwise interactions between C alpha atoms are commonly used in protein threading/fold-recognition attempts. Inclusion of higher order interaction is a possible means of improving the specificity of these potentials. Delaunay tessellation of the C alpha-atom representation of protein structure has been suggested as a means of defining multi-body interactions. A large number of parameters are required to define all four-body interactions of 20 amino acid types (20(4) = 160,000). Assuming that residue order within a four-body contact is irrelevant reduces this to a manageable 8,855 parameters, using a nonredundant dataset of 608 protein structures. Three lines of evidence support the significance and utility of the four-body potential for sequence-structure matching. First, compared to the four-body model, all lower-order interaction models (three-body, two-body, one-body) are found statistically inadequate to explain the frequency distribution of residue contacts. Second, coherent patterns of interaction are seen in a graphic presentation of the four-body potential. Many patterns have plausible biophysical explanations and are consistent across sets of residues sharing certain properties (e.g., size, hydrophobicity, or charge). Third, the utility of the multi-body potential is tested on a test set of 12 same-length pairs of proteins of known structure for two protocols: Sequence-recognizes-structure, where a query sequence is threaded (without gap) through the native and a non-native structure; and structure-recognizes-sequence, where a query structure is threaded by its native and another non-native sequence. Using cross-validated training, protein sequences correctly recognized their native structure in all 24 cases. Conversely, structures recognized the native sequence in 23 of 24 cases. Further, the score differences between correct and decoy structures increased significantly using the three- or four-body potential compared to potentials of lower order.     

High-performance liquid chromatographic chiral separation of beta2-homoamino acids

Reversed-phase high-performance liquid chromatographic methods were developed for the separation of enantiomers of eleven unnatural beta(2)-homoamino acids on chiral stationary phases containing macrocyclic glycopeptides (teicoplanin-containing Chirobiotic T and T2) or the macrocyclic peptide teicoplanin aglycone (Chirobiotic TAG) as chiral selectors. The effects of the organic modifier, the mobile phase composition, temperature, and the structures of the analytes on the separations were investigated. Separations were carried out at constant mobile phase compositions in temperature range 7-45 degrees C and the changes in enthalpy, Delta(DeltaH(o)), entropy, Delta(DeltaS(o)), and free energy, Delta(DeltaG(o)), were calculated. The -Delta(DeltaG(o)) values obtained on the three columns indicated that Chirobiotic TAG, without sugar units, may promote the interactions of the enantiomers of beta(2)-homoamino acids with branched alkyl or aryl side-chains, whereas for beta(2)-homoamino acids with alkyl side-chains Chirobiotic T and T2 seem to be more favorable. The elution sequence was determined in some cases and was observed to be R < S.     

Four-body contact potentials derived from two protein datasets to discriminate native structures from decoys

Two-body inter-residue contact potentials for proteins have often been extracted and extensively used for threading. Here, we have developed a new scheme to derive four-body contact potentials as a way to consider protein interactions in a more cooperative model. We use several datasets of protein native structures to demonstrate that around 500 chains are sufficient to provide a good estimate of these four-body contact potentials by obtaining convergent threading results. We also have deliberately chosen two sets of protein native structures differing in resolution, one with all chains' resolution better than 1.5 A and the other with 94.2% of the structures having a resolution worse than 1.5 A to investigate whether potentials from well-refined protein datasets perform better in threading. However, potentials from well-refined proteins did not generate statistically significant better threading results. Our four-body contact potentials can discriminate well between native structures and partially unfolded or deliberately misfolded structures. Compared with another set of four-body contact potentials derived by using a Delaunay tessellation algorithm, our four-body contact potentials appear to offer a better characterization of the interactions between backbones and side chains and provide better threading results, somewhat complementary to those found using other potentials.     

Contribution of intra- and intermolecular hydrogen bonds to the conformational stability of human lysozyme(,).

In globular proteins, there are intermolecular hydrogen bonds between protein and water molecules, and between water molecules, which are bound with the proteins, in addition to intramolecular hydrogen bonds. To estimate the contribution of these hydrogen bonds to the conformational stability of a protein, the thermodynamic parameters for denaturation and the crystal structures of five Thr to Val and five Thr to Ala mutant human lysozymes were determined. The denaturation Gibbs energy (DeltaG) of Thr to Val and Thr to Ala mutant proteins was changed from 4.0 to -5.6 kJ/mol and from 1.6 to -6.3 kJ/mol, respectively, compared with that of the wild-type protein. The contribution of hydrogen bonds to the stability (DeltaDeltaG(HB)) of the Thr and other mutant human lysozymes previously reported was extracted from the observed stability changes (DeltaDeltaG) with correction for changes in hydrophobicity and side chain conformational entropy between the wild-type and mutant structures. The estimation of the DeltaDeltaG(HB) values of all mutant proteins after removal of hydrogen bonds, including protein-water hydrogen bonds, indicates a favorable contribution of the intra- and intermolecular hydrogen bonds to the protein stability. The net contribution of an intramolecular hydrogen bond (DeltaG(HB[pp])), an intermolecular one between protein and ordered water molecules (DeltaG(HB[pw])), and an intermolecular one between ordered water molecules (DeltaG(HB[ww])) could be estimated to be 8. 5, 5.2, and 5.0 kJ/mol, respectively, for a 3 A long hydrogen bond. This result shows the different contributions to protein stability of intra- and intermolecular hydrogen bonds. The entropic cost due to the introduction of a water molecule (DeltaG(H)()2(O)) could be also estimated to be about 8 kJ/mol.     

Propagation of a single destabilizing mutation throughout the Escherichia coli ribonuclease HI native state

A point mutation (I53A) in the core of Escherichia coli RNase H* is known to destabilize both the native conformation (DeltaG(UN)) and the kinetic intermediate (DeltaG(UI)) by 2 kcal/mole. Here, we have used native-state hydrogen deuterium exchange to ask how this destabilization is propagated throughout the molecule. Stability parameters were obtained for individual residues in I53A and compared with those from the wild-type protein. A destabilization of 2 kcal/mole was observed in residues in the core but was not detected in the periphery of the molecule. These results are consistent with the localized destabilization of the core observed in the early intermediate of the kinetic folding pathway, supporting the resemblance of this kinetic intermediate to the partially unfolded form detected in the native state at equilibrium. A thermodynamic cycle also shows no interaction between Ile 53 and a residue in the periphery. There is, however, an increase in the number of denaturant-independent exchange events in the periphery of I53A, showing that effects of the point mutation are communicated to regions outside the core, although perhaps not through changes in stability. In sum, this work shows that localized regions within a protein can be destabilized independently. Furthermore, it implies a correspondence between the kinetic intermediate and the equilibrium PUF, as the magnitude and localization of the destabilization are the same in both.     

Comprehensive mutagenesis of HIV-1 protease: a computational geometry approach

A computational geometry technique based on Delaunay tessellation of protein structure, represented by C(alpha) atoms, is used to study effects of single residue mutations on sequence-structure compatibility in HIV-1 protease. Profiles of residue scores derived from the four-body statistical potential are constructed for all 1881 mutants of the HIV-1 protease monomer and compared with the profile of the wild-type protein. The profiles for an isolated monomer of HIV-1 protease and the identical monomer in a dimeric state with an inhibitor are analyzed to elucidate changes to structural stability. Protease residues shown to undergo the greatest impact are those forming the dimer interface and flap region, as well as those known to be involved in inhibitor binding.     

An approach for the assessment of the order of disruption of the elements of protein structure upon protein unfolding: A study of carbonic anhydrase B

An experimental approach named μ-analysis has been developed in order to elucidate the sequence of the loss of ordered structure by elements of a protein during the denaturation of the molecule. This approach is applicable for the analysis of proteins that fold (unfold) in a multistep process that involve the formation (destruction) of a range of intermediate states. The concept of the approach consists in systematic analysis of mutagenized forms of the protein with point substitutions of hydrophobic amino-acid residues and additional cysteine bridges. Importantly, the substitutions of the amino-acid residues must be localized to the same structural elements of the protein. Point substitutions of hydrophobic amino-acid residues mainly provide information on the structural elements of the protein that are disrupted at the final stages of protein denaturation. The addition of cysteine bridges to the surface of the protein molecule allows investigation of structural elements of the protein that are the first to unfold upon protein denaturation. Calorimetric studies of non-equilibrium melting of bovine carbonic anhydrase B yielded information on the rate constants of the unfolding of ten mutant forms of the protein. The analysis of the effects of mutations on the rates of different stages of protein unfolding allowed for elucidation of the order of disruption of structural elements of carbonic anhydrase B upon thermal denaturation.