Evaluation of atomic level mean force potentials via inverse folding and inverse refinement of protein structures: atomic burial position and pairwise non-bonded interactions

Two atomic level knowledge-based mean force interaction potentials(KBPs), a centrosymmetric burial position term and a long-rangepairwise term, were developed. These were tested by comparingmultiple configurations of three structurally unrelated proteinsand were found successfully to (i) discriminate native stateproteins from grossly misfolded structures in inverse foldingtests, (ii) rank identify, using the KBP energy/r.m.s.d. correlation,native from progressively less native-like (compact and dilated)structures generated via molecular dynamics sampling, providingan energy gradient sloping from partially unfolded structurestowards near-native states in inverse refinement tests, (iii)smooth the overall potential energy surface in the region ofdilated non-native structures by countering local minima ofthe in vacuo molecular mechanical potential and (iv) serve asa local minimum detector during simulated temperature quenchingstudies. These atomic KBPs discriminated native from non-nativestructures with greater overall sensitivity than did eithera residue-based pairwise interaction potential or an effectivesolvation potential based on atomic contact volume occupancy.The KBPs presented here are immediately useful as a tool forselecting ‘good refinement candidates’ from an arbitrarycollection of protein configurations and may play a role indynamic computational protein refinement.     

Variable gap penalty for protein sequence-structure alignment

The penalty for inserting gaps into an alignment between two protein sequences is a major determinant of the alignment accuracy. Here, we present an algorithm for finding a globally optimal alignment by dynamic programming that can use a variable gap penalty (VGP) function of any form. We also describe a specific function that depends on the structural context of an insertion or deletion. It penalizes gaps that are introduced within regions of regular secondary structure, buried regions, straight segments and also between two spatially distant residues. The parameters of the penalty function were optimized on a set of 240 sequence pairs of known structure, spanning the sequence identity range of 20-40%. We then tested the algorithm on another set of 238 sequence pairs of known structures. The use of the VGP function increases the number of correctly aligned residues from 81.0 to 84.5% in comparison with the optimized affine gap penalty function; this difference is statistically significant according to Student's t-test. We estimate that the new algorithm allows us to produce comparative models with an additional approximately 7 million accurately modeled residues in the approximately 1.1 million proteins that are detectably related to a known structure.     

Experimental verification of the `stability profile of mutant protein' (SPMP) data using mutant human lysozymes

The stability profile of mutant protein (SPMP) (Ota,M., Kanaya,S.and Nishikawa,K., 1995, J. Mol. Biol., 248, 733–738) estimatesthe changes in conformational stability due to single aminoacid substitutions using a pseudo-energy potential developedfor evaluating structure–sequence compatibility in thestructure prediction method, the 3D–1D compatibility evaluation.Nine mutant human lysozymes expected to significantly increasein stability from SPMP were constructed, in order to experimentallyverify the reliability of SPMP. The thermodynamic parametersfor denaturation and crystal structures of these mutant proteinswere determined. One mutant protein was stabilized as expected,compared with the wild-type protein. However, the others werenot stabilized even though the structural changes were subtle,indicating that SPMP overestimates the increase in stabilityor underestimates negative effects due to substitution. Thestability changes in the other mutant human lysozymes previouslyreported were also analyzed by SPMP. The correlation of thestability changes between the experiment and prediction dependedon the types of substitution: there were some correlations forproline mutants and cavity-creating mutants, but no correlationfor mutants related to side-chain hydrogen bonds. The presentresults may indicate some additional factors that should beconsidered in the calculation of SPMP, suggesting that SPMPcan be refined further.     

Search for the most stable folds of protein chains: II. Computation of stable architectures of {beta}-proteins using a self-consistent molecular field theory

In a preceding paper we presented a novel approach to computationof 3-D folds of protein chains from their amino acid sequences.This approach is a physically correct generalization of the‘threading’ methods. It is based on a self-consistentmolecular field theory and on a physical theory of protein foldingpatterns, which make it possible to examine all the varietyof ‘potentially stable’ folding patterns and allthe variety of the chain conformations within each of them andto determine the thermodynamically stable structure. In thispaper, we apply this approach to single out stable folding patternsand conformations for the chains of ß-sandwich proteinsand show that the similarity of the calculated and observedstructures is usually rather close.     

The influence of Glu44 and Glu56 of cytochrome b5 on the protein structure and interaction with cytochrome c

The gene encoding trypsin-solubilized bovine liver microsomalcytochrome b₅ (82 residues in length) has been mutated, in whichthe codons of Glu44 and Glu56 were changed to those of Ala.The mutated genes were expressed in Escherichia coli successfullyand three mutant proteins (E44A, E56A and E44/56A) were obtained.The UV-visible, CD and ¹H NMR spectra of proteins have beenstudied. The results show that the mutagenesis at surface residuesdoes not alter the secondary and tertiary structures of cytochromeb₅ significantly. The interactions between recombinant cytochromeb₅ and its mutants with cytochrome c were studied by using opticaldifference spectra. The results demonstrated that both Glu44and Glu56 of cytochrome b₅ participate in the formation of acomplex between cytochrome b₅ and cytochrome c.     

Amino acid changes in the repressor of bacteriophage lambda due to temperature-sensitive mutations in its cI gene and the structure of a highly temperature-sensitive mutant repressor

The mutant cIts genes from seven different cIts phages carryingtsU50, tsU9, tsU46, ts1, tsU51, tsI-22 and ts2 mutations werecloned in plasmid. The positions of these mutations and theresulting changes of amino acids in the repressor were determinedby DNA sequencing. The first four mutations mapping in the N-terminaldomain show the following changes: I21S, G53S, A62T and V73A,respectively. Of the three remaining mutations mapping in theC-terminal domain, cItsI-22 and cIts2 show N207T and K224E substitutionsrespectively, while the mutant cItsU51 gene carries F141I andP153L substitutions. Among these ts repressors, CIts2 havingthe charge-reversal change K224E was overexpressed from tacpromoter in a plasmid and purified, and its structure and functionwere studied. Operator-binding studies suggest that the ts2repressor is somewhat defective in monomer–dimer equilibriumand/or cooperativity even at permissive temperatures and losesits operator-binding ability very rapidly above 25°C. Comparativestudies of fluorescence and CD spectra, sulfhydryl group reactivityand elution behaviour in size-exclusion HPLC of both wild-typeand ts2-mutant repressors at permissive and non-permissive temperaturessuggest that the C-terminal domain of the ts2 repressor carryinga K224E substitution has a structure that does not favor tetramerformation at non-permissive temperatures.