Folding of chains with random and edited sequences: similarities and differences

We have investigated the process of protein folding by Monte-Carlosimulation of folding occurring in a simple 3D lattice modelof a protein globule. We have found the range of ‘optimal’temperatures where the native fold is achieved by the Monte-Carloprocess much faster than that by exhaustive sorting of all thechain folds. The ‘optimal’ temperatures are essentiallythe same for different random and lsquo;edited’ sequences(for the latter, the native fold energy is separated by a considerablegap from the energies of other low-energy folds; for randomsequences, this gap is negligible). At the ‘optimal’temperatures, the ‘edited’ chains attain their nativefold faster than the random ones. However, the essence is thatthe native folds of ‘edited’ chains are thermodynamicallystable at temperatures optimal for fast folding, while the nativefolds of random chains are unstable at the temperatures optimalfor fast folding; also, at low temperatures where the nativefolds of random chains are stable, folding kinetics is veryslow. Consequently, stable native folds are formed slowly byrandom sequences and rapidly by the ‘edited’ ones     

Proline versus charge concept for protein stabilization against proteolytic attack

The virtue of the so-called ‘proline concept’ andthe ‘charge concept’ for stabilizing protease-susceptibleregions of a protein structure was compared on bovine pancreaticribonuclease A. Alanine 20 and serine 21, both of whichare located in a loop that is susceptible to the unspecificproteases subtilisin Carlsberg, subtilisin BPN', proteinaseK and elastase, were replaced with proline or lysine by site-directedmutagenesis. The rate constant of proteolysis was decreasedby up to three orders of magnitude for the proline mutants dependingon the site of the mutation and the protease used. In contrast,substitution by lysine increased the proteolytic resistanceby only one order of magnitude characterizing the ‘prolineconcept’ as superior to the ‘charge concept’.Although the four applied proteases are considered to be unspecific,the degree of stabilization of the ribonuclease molecule variedconsiderably, indicating the impact of individual differencesin their substrate specificity on the proteolytic resistanceand degradation pathway of the target protein. Received May 12, 2003; revised October 23, 2003; accepted October 30, 2003     

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.     

Identifying the mechanism of protein loop closure: a molecular dynamics simulation of the Bacillus stearothermophilus LDH loopin solution

The ‘loop’ involving residues 98–110 in Bacillusstearothermophilus lactate dehydrogenase (BSLDH) is of greatinterest as substrate-induced ‘loop’ closure isthought to berate-limiting and essential in catalyzing the reactionand in determining substrate specificity. Consequently, we haveexplored the mechanism underlying ‘loop’ openingin BSLDH through a molecular dynamics simulation at high temperature(1000 K) in the presence of explicit solvent, starting fromthe X-ray structure of BSLDH complexed with the co-enzyme NAD+and oxamate at 2.5 Å. During the simulation, a significantconformational change occurred, as evidenced by sharp dihedralangle transitions, hydrogen bond breaking and formation andlarge root mean square deviations from the starting structure;these changes define the criteria for ‘loop’ opening.The mechanical elements responsible for ‘loop’ opening,i.e. ‘loop’ hinges and flap, are defined througha combination of order parameters, dihedral angle changes andtheir correlations and the dynamical cross-correlation map ofatomic displacements for the ‘loop’ residues. Theresults indicate that the ‘loop’ consists of twoflexible hinge regions on either side of a relatively rigidthree-residue segment that undergoes a significant spatial displacementduring ‘loop’opening. ‘Loop’ openingis made possible through an array of correlated dihedral anglechanges and intra-& ‘loop’ rearrangements ofhydrogen-bond interactions. The presentfindings are comparedto previous work related to ‘loop’ opening and site-directedmutagenesis experiments.     

Search for the most stable folds of protein chains: I. Application of a self-consistent molecular field theory to a problem of protein three-dimensional structure prediction

We present a general approach to the prediction of 3-D foldsof protein chains from their amino acid sequences. The approachis based on the use of the self-consistent molecular field theoryfor long-range interactions, the use of 1-D statistical mechanicsfor short-range interactions and on the discovery that thereis and should only be a relatively small discrete set of foldingpatterns. This makes it possible to examine the full varietyof ‘potentially stable’ folds and to determine thethermodynamically stable structure. In this paper, we give thegeneral theoretical background of the approach. The encouragingresults of the application of this approach to ß-domainsare described in another paper.     

Role of the tyrosine corner motif in the stability of neocarzinostatin

Although the immunoglobulin-like ß-sandwich fold hasno specifically conserved function, some common structural featureshave been observed, in particular a structural motif, the tyrosinecorner. Such a motif was described in neocarzinostatin (NCS),a bacterial protein the structure of which is very similar tothat of the immunoglobulin domain. Compared with the other ß-sheetproteins, the NCS ‘tyrosine corner’ presents non-standardstructural features. To investigate the role of this motif inthe NCS structure and stability, we studied the properties ofa mutant where the H bond interaction had been eliminated byreplacing the tyrosine with a phenylalanine. This mutation costs4.0 kcal/mol showing that the NCS ‘tyrosine corner’is involved in protein stability as in the other Greek key proteins.This destabilization is accompanied by remote structural effects,including modification of the binding properties, suggestingan increase in the internal flexibility of the protein. Witha view to using this protein for drug targeting, these resultsalong with those obtained previously allow us to define clearlythe limitations of the modifications that can be performed onthis scaffold. Received December 3, 2002; revised June 6, 2003; accepted September 3, 2003.     

Structural background of cyclodextrin-protein interactions

Cyclodextrins are cyclic oligosaccharides with the shape ofa hollow truncated cone. Their exterior is hydrophilic and theircavity is hydrophobic, which gives cyclodextrins the abilityto accommodate hydrophobic molecules/moieties in the cavity.This special molecular arrangement accounts for the varietyof beneficial effects cyclodextrins have on proteins, whichis widely used in pharmacological applications. We have studiedthe interaction between ß-cyclodextrin and four non-carbohydrate-bindingmodel proteins: ubiquitin, chymotrypsin inhibitor 2 (CI2), S6and insulin SerB9Asp by NMR spectroscopy at varying structuraldetail. We demonstrate that the interaction of ß-cyclodextrinand our model proteins takes place at specific sites on theprotein surface, and that solvent accessibility of those sitesis a necessary but not compelling condition for the occurrenceof an interaction. If this behaviour can be generalized, itmight explain the wide range of different effects of cyclodextrinson different proteins: aggregation suppression (if residuesresponsible for aggregation are highly solvent accessible),protection against degradation (if point of attack of a proteaseis sterically ‘masked’ by cyclodextrin), alterationof function (if residues involved in function are ‘masked’by cyclodextrin). The exact effect of cyclodextrins on a givenprotein will always be related to the particular structure ofthis protein. Received May 30, 2003; revised October 27, 2003; accepted October 30, 2003     

In search of the ideal protein sequence

The inverse of a folding problem is to find the ideal sequencethat folds into a particular protein structure. This problemhas been addressed using the topology fingerprintbased threadingalgorithm, capable of calculating a score (energy) of an arbitrarysequence-structure pair. At first, the search is conducted byunconstrained minimization of the energy in sequence space.It is shown that using energy as the only design criterion leadsto spurious solutions with incorrect amino acid composition.The problem lies in the general features of the protein energysurface as a function of both structure and sequence. The proposedsolution is to design the sequence by maximizing the differencebetween its energy in the desired structure and in other knownprotein structures. Depending on the size of the database ofstructures ‘to avoid’, sequences bearing significantsimilarity to the native sequence of the target protein areobtained using this procedure.     

Knowledge based modelling of homologous proteins, part I: three-dimensional frameworks derived from the simultaneous superposition of multiple structures

An approach is described for modelling the three-dimensionalstructure of a protein from the tertiary structures of severalhomologous proteins that have been determined by X-ray analysis.A method is developed for the simultaneous superposition ofseveral protein molecules and for the calculation of an ‘averagestructure’ or ‘framework’. Investigation ofthe convergence properties of this method, in the case of bothweighted and unweighted least squares, demonstrates that bothgive a unique answer and the latter is robust for an homologousfamily of proteins. Multi-dimensional scaling is used to subgroupthe proteins with respect to structural homology. The frameworkcalculated on the basis of the family of homologous proteins,or of an appropriate subgroup, is used to align fragments ofthe known protein structures of high sequence homology withthe unknown. This alignment provides a basis for model buildingthe tertiary structure. Different techniques for using the frameworkto model the mainchain of various globins and an immunoglobulindomain in the structurally conserved regions are in vestigated.     

When awaiting 'Bio' Champollion: dynamic programming regularization of the protein secondary structure predictions

Predictions of protein secondary structure using current methodsare often unrealistic, i.e. the predicted -helices or ß-strandsare too short. To improve the realism, various heuristic ‘filtering’or ‘smoothing’ methods are used. They are more orless intuitive and are based on ad hoc corrections. We presenta regularization method to obtain a realistic secondary structurefrom predicted propensities. It is based on the known dynamicprogramming algorithm and is quite objective. It can be usedwith any prediction method which yields propensities. The regularizedpredictions conserve well the overall prediction accuracy andimprove the ‘protein-likeness’ of the prediction.     

Geometric and energy parameters in lysine-retinal chromophores

The parameters used in the computer program ECEPP (EmpiricalConformational Energy Program for Peptides) have been expandedto cover some key elements in retinal-containing proteins. Theseelements are ‘all-trans retinal lysine with unprotonatedimine’, ‘all-trans retinal lysine with protonatedimine’, ‘13-cis retinal lysine with unprotonatedimine’ and ‘13-cis retinal lysine with protonatedimine’ respectively. The geometric parameters of thesefour new ‘amino acid residues’ were derived by optimizingtheir molecular structures with the AMI Hamiltonian includedin MOPAC (Molecular Orbital PACkage), and their partial atomiccharges were determined with a CNDO/2 (Complete Neglect of DifferentialOverlap) calculation. The parameters for nonbonded interactionsand torsional potentials were obtained from the existing ECEPPparameters through a logical extension. The augmented ECEPPsystem thus obtained can be employed to investigate the conformationof bacteriorhodopsin and its proton-pumping mechanism from anenergetic point of view. The computer modeling study on bacteriorhodopsinand other seven-helix membrane proteins, e.g. serotonin receptorand dopamine receptor, is under way in the Upjohn Laboratories.     

Vicinal disulfide turns

The formation of a disulfide bond between adjacent cysteineresidues is accompanied by the formation of a tight turn ofthe protein backbone. In nearly 90% of the structures analyzeda type VIII turn was found. The peptide bond between the twocysteines is in a distorted trans conformation, the omega torsionangle ranges from 159 to –133°, with an average valueof 171°. The constrained nature of the vicinal disulfideturn and the pronounced difference observed between the oxidizedand reduced states, suggests that vicinal disulfides may beemployed as a ‘redox-activated’ conformational switch. Received December 16, 2002; revised June 30, 2003; accepted July 30, 2003.     

A backbone-reversed all-{beta} polypeptide (retro-CspA) folds and assembles into amyloid nanofibres

The backbone-reversed or ‘retro’, form of a modelall-ß-sheet protein, Escherichia coli CspA, was producedfrom a synthetic gene in E.coli in fusion with an N-terminalaffinity tag. Following purification under denaturing conditionsand dialysis-based removal of urea, the protein was found tofold into a soluble, poorly structured multimer. Upon concentration,this state readily transformed into amyloid nanofibres. CongoRed-binding amorphous forms were also observed. Since a ß-sheet-formingsequence is expected to retain high ß-sheet-formingpropensity even after backbone reversal and given the fact thatfolding of retro-CspA occurs only to a poorly structured form,we conclude that the increase effected in protein concentrationmay be responsible for the formation of intermolecular ß-sheets,facilitating the bleeding away of the protein’s conformationalequilibrium into aggregates that generate well-formed fibres.Since every molecule in these fibres contains a peptide tagfor binding Ni²⁺, the fibres may provide a template for depositionof nickel to generate novel materials. Received April 1, 2003; revised October 27, 2003; accepted October 30, 2003     

On the choice of reference mutant states in the application of the double-mutant cycle method

Pairwise interactions in proteins can be detected and, in certaincircumstances, their strength measured by applying the methodof double-mutant cycles. Such cycles comprise wild type protein,two single mutants and the corresponding double mutant The analysisof double-mutant cycles is most straightforward when the mutationsare to alanine since interactions are mostly removed withoutnew interactions being formed. Here, ‘not-to-alanine’double-mutant cycles are analysed. It is shown that a ‘not-to-alanine’double-mutant cycle can be decomposed into four double-mutantcycles with mutations only to alanine. The coupling energy correspondingto the ‘not-to-alanine’ double-mutant cycle is expressedas a function of the coupling energies of these four cycles.     

The role of heat-shock and chaperone proteins in protein folding: possible molecular mechanisms

Recently some heat-shock proteins have been linked to functionsof ‘chaperoning’ protein folding in vivo. Here currentexperimental evidence is reviewed and possible requirementsfor such an activity are discussed. It is proposed that onemode of chaperone action is to actively unfold misfolded orbadly aggregated proteins to a conformation from whkh they couldrefold spontaneously; that improperly folded proteins are recognizedby excessive stretches of solvent-exposed backbone, rather thanby exposed hydrophobic patches; and that the molecular mechanismfor unfolding is either repeated binding and dissociation (‘plucking’)or translocation of the protein backbone through a binding cleft(‘threading’), allowing the threaded chain to refoldspontaneously. The observed hydrolysis of ATP would providethe energy for active unfolding. These hypotheses can be appliedto both monomeric folding and oligomeric assembly and are sufficientlydetailed to be open to directed experimental verification.     

Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus: functional and structural consequences of mutation at the S4 substrate binding pocket

Serine endoproteases such as trypsins and subtilisins are knownto have an extended substrate binding region that interactswith residues P6 to P3' of a substrate. In order to investigatethe structural and functional effects of replacing residuesat the S4 substrate binding pocket, the serine protease fromthe alkalophilic Bacillus strain PB92, which shows homologywith the subtilisins, was mutated at positions 102 and 126–128.Substitution of Val102 by Trp results in a 12–fold increasein activity towards succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide(sAAPFpNA). An X-ray structure analysis of the V102W mutantshows that the Trp side chain occupies a hydrophobic pocketat the surface of the molecule leaving a narrow crevice forthe P4 residue of a substrate. Better binding of sAAPFpNA bythe mutant compared with the wild type protein as indicatedby the kinetic data might be due to the hydrophobic interactionof Ala P4 of the substrate with the introduced Trp102 side chain.The observed difference in binding of sAAPFpNA by protease PB92and thermitase, both of which possess a Trp at position 102,is probably related to the amino acid substitutions at positions105 and 126 (in the protease PB92 numbering).Kinetic data forthe variants obtained by random mutation of residues Serl26,Prol27 and Serl28 reveal that the activity towards sAAPFpNAincreases when a hydrophobic residue is introduced at position126. An X-ray diffraction analysis was carried out for the threeprotease PB92 mutants which have residues Serl26-Prol27-Serl28replaced by Met-Ala-Gly(‘MAG’ mutant), Phe-Gln-Ser(‘FQS’ mutant) and Asn-Ser-Ala (‘NSA’mutant). Met 126 and Phel26 in the crystal structures of thecorresponding mutants are fixed in the same hydrophobic environmentas Trp102 in the V102W mutant.In contrast, Asnl26 in the ‘NSA’mutant is completely disordered in both crystal forms for whichthe structure has been determined. According to our kineticmeasurements none of the mutants with Met, Phe, Leu or Val atposition 126 binds sAAPFpNA better than the wild type enzyme.Resultsof the site-directed mutagenesis at position 127 imply thatpossible interaction of this residue with a substrate has almostno effect on activity towards sAAPFpNA and casein.     

Are database-derived potentials valid for scoring both forward and inverted protein folding?

Database-derived potentials, compiled from frequencies of sequenceand structure features, are often used for scoring the compatibilityof protein sequences and conformations. It is often believedthat these scores correspond to differences in free energy with,in addition, a term containing the partition function of thesystem. Since this function does not depend on the conformation,the potentials are considered to be valid for scoring the compatibilityof different conformations with a given sequence (‘forwardfolding’), but not of sequences with a given structure(‘inverted folding’). This interpretation is questionedhere. It is argued that when many body-effects, which dominatefrequencies compiled from the protein database, are correctedfor, the potentials approximate a physically meaningful freeenergy difference from which the partition function term cancelsout It is the difference between the free energy of a givensequence in a specific conformation and that of the same sequencein a denatured-like state. Two examples of denatured-like statesare discussed. Depending on the considered state, the free energydifference reduces to the commonly used scoring scheme, or containsadditional terms that depend on the sequence. In both cases,all the terms can be derived from sequence-structure frequenciesin the database. Such free energy difference, commonly definedas the folding free energy, is a measure of protein stabilityand can be used for scoring both forward and inverted proteinfolding. The implications for the use of knowledge-based potentialsin protein structure prediction are described. Finally, thedifficulty of designing tests that could validate the proposedapproach, and the inherent limitations of such tests, are discussed     

Parameter relation rows: a query system for protein structure function relationships

In the course of molecular modeling or mutant prediction oneoften wants quick answers to questions such as: ‘Are thereany residues in a beta-strand that point into an internal cavity,and are highly mutable?;’ ‘Are there large polarresidues in a helix that make a contact with a hydrophobic residuein a sheet, and don't make the maximal number of hydrogen bonds?’or ‘Which hydrophobic residues are in a helix with a largehydrophobic moment, and make a contact with a co-factor, butat the same time still have a large accessible surface?’.I describe here a method to get answers to these kinds of questionsin a very quick and easy manner. The method described is partlybased on the principles used in the design of relational databases,and its mode of operation is similar to the query methods usedin a relational database environment. Although designed foraiding in molecular modeling, its applicability is much moregeneral. The method has been implemented as part of a largemolecular modeling package which copes with the numerous problemsin systematic handling of protein structures, e.g. residue numbering.This also implies that many normal tools such as graphical analyses,I/O facilities, etc. are available on-line.