Alignment of protein sequences using secondary structure: a modified dynamic programming method

A method for comparison of protein sequences based on theirprimary and secondary structure is described. Protein sequencesare annotated with predicted secondary structures (using a modifiedChou and Fasman method). Two lettered code sequences are generated(Xx, where X is the amino acid and x is its annotated secondarystructure). Sequences are compared with a dynamic programmingmethod (STRALIGN) that includes a similarity matrix for boththe amino acids and secondary structures. The similarity valuefor each paired two-lettered code is a linear combination ofsimilarity values for the paired amino acids and their annotatedsecondary structures. The method has been applied to eight globinproteins (28 pairs) for which the X-ray structure is known.For protein pairs with high primary sequence similarity (>45%),STRALIGN alignment is identical to that obtained by a dynamicprogramming method using only primary sequence information.However, alignment of protein pairs with lower primary sequencesimilarity improves significantly with the addition of secondarystructure annotation. Alignment of the pair with the least primarysequence similarity of 16% was improved from 0 to 37% ‘correct’alignment using this method. In addition, STRALIGN was successfullyapplied to seven pairs of distantly related cytochrome c proteins,and three pairs of distantly related picornavirus proteins.     

Effects of introduced aspartic and glutamic acid residues on the Pi substrate specificity, pH dependence and stability of carboxypeptidase Y

Carboxypeptidase Y is a serine carboxypeptidase isolated fromSaccharomyces cerevisiae with a preference for Cterminal hydrophobicamino acid residues. In order to alter the inherent substratespecificity of CPD-Y into one for basic amino acid residuesin P'₁, we have introduced Asp and/or Glu residues at a numberof selected positions within the Si binding site. Hie effectsof these substitutions on the substrate specificity, pH dependenceand protein stability have been evaluated. The results presentedhere demonstrate that it is possible to obtain significant changesin the substrate preference by introducing charged amino acidsinto the framework provided by an enzyme with a quite differentspecificity. The introduced acidic amino acid residues providea marked pH dependence of the (k_{cat/Km)FA-A-R-OH/(kcatm})_FA-A-R-OHratio. The change in stability upon introduction of Asp/Gluresidues can be correlated to the difference in the mean buriedsurfac surface area between the substituted and the substitutingamino acid. Thus, the effects of acidic amino acid residueson the protein stability depend upon whether the introducedamino acid protrudes from the solvent accessible surface asdefined by the surrounding residues in the wild type enzymeor is submerged below.     

Analysis of the catalyic mechanism of a fungal lipase using computer-aided design and structural mutants

Both an active enzyme conformation and stabilization of tetrahedraltransition states are essential for the catalysis of ester bondhydrolysis by lipases. X-ray structural data and results fromsite-directed mutagenesis experiments with proteases have beenused as a basis for predictions of amino acid residues likelyto have key functions in lipases. The gene encoding a lipasefrom Rhizopus oryzae was cloned and expressed in Escherichiacoli. Site-directed mutagenesis of this gene was used to testthe validity of computer-aided predictions of the functionalroles of specific amino acids in this enzyme. Examination ofthe kinetic constants of the Rhizopus oryzae lipase variantsallowed us to identify amino acid residues which are directlyinvolved in the catalytic reaction or which stabilize the activegeometry of the enzyme. The combination of these results withmolecular mechanics simulations, based on a homology-derivedstructural model, provided new information about structure-functionrelationships. The interpretation of the data is consistentwith results obtained with other hydrolases, such as proteases.     

On the role of conserved proline residues in the structure and function of Clostridium pasteurianum 2[4Fe-4S] ferredoxin

The widespread occurrence of Pro residues adjacent to Cys ligandsin the sequences of [4Fe-4S] electron transfer proteins hasnot yet found a functional basis. The two such Pro of Clostridiumpasteurianum 2[4Fe-4S] ferredoxin have been probed by site-directedmutagenesis. Any one of them, but not both simultaneously, canbe substituted without impairing the proper folding of the protein.The reduction potentials of the ferredoxin variants fall ina narrow range of <20 mV above the potential of the nativeprotein. The biological activities with C.pasteurianum hydrogenaseand pyruvate-ferredoxin oxidoreductase do not change significantly,except when Lys replaces Pro. In these cases, the data suggestthat the two clusters of 2[4Fe-4S] ferredoxin may not alwaysbe equivalent in the interaction with the redox partners. Destabilizationof the structure has been observed as the consequence of theProl9 or Pro48 substitutions. Using 2-D NMR, this effect hasbeen associated with perturbations of both the hydrogen bondnetwork and one amino acid side chain around the [4Fe-4S] clusters.Thus, the conserved Pro found in the binding motif of [4Fe-4S]clusters in proteins strongly stabilizes the active site butdoes not play an essential role in the mechanism of electrontransfer.     

Conversion of human 15-lipoxygenase to an efficient 12-lipoxygenase: the side-chain geometry of amino acids 417 and 418 determine positional specificity

Positional specificity determinants of human 15-lipoxygenasewere examined by site-directed mutagenesis and by kinetic analysisof the wild-type and variant enzymes. By comparing conserveddifferences among sequences of 12- and 15-lipoxygenases, a smallregion responsible for functional differences between 12- and15-lipoxygenases has been identified. Furthermore, the replacementof only two amino acids in 15-lipoxygenase (at 417 and 418 inthe primary sequence) by those found in certain 12-lipoxygenasesresults in an enzyme that has activity similar to 12-lipoxygenase.An examination of the activity of nine variants of lipoxygenasedemonstrated that the amino acid side-chain bulk and geometryof residues 417 and 418 are the key components of the positionalspecificity determinant of 15-lipoxygenase. Overexpression ofa variant (containing valines at positions 417 and 418) thatperforms predominantly 12-lipoxygenation was achieved in a baculovirus-insectcell culture system. This variant was purified to >90% homogeneityand its kinetics were compared with the wild-type 15-lipoxygenase.The variant enzyme has no change in its apparent K_M for arachidonicacid and a minor(3-fold) change in its V_max. For linoleic acid,the variant has no change in its K_M and a 10-fold reductionin its V_max, as expected for an enzyme performing predominantly12-lipoxygenation. The results are consistent with a model inwhich two amino acids of 15-lipoxygenase (isoleucine 417 andmethionine 418) constitute a structural element which contributesto the regiospecificity of the enzyme. Replacement of theseamino acids with those found in certain 12-lipoxygenases resultsin an enzyme which can bind arachidonic acid in a catalyticregister that prefers 12-lipoxygenation.     

A new method for random mutagenesis of complete genes: enzymatic generation of mutant libraries in vitro

A new efficient in vitro mutagenesis method for the generationof complete random mutant libraries, containing all possiblesingle base substitution mutations in a cloned gene is described.The method is based on controlled use of polymerases. Four populationsof DNA molecules are first generated by primer elongation sothat they terminate randomly, but always just before a knowntype of base (before A, C, G or T respectively). Each of thefour populations is then mutagenized in a separate misincorporationreaction, where the correct base can now be omitted. The regenerationof wild-type sequences can thus be efficiently avoided. Also,the misincorporating nucleotide concentrations can be optimizedto give the three possible single mutations in close to equalratio. The mutagenesis can be precisely localized within a predeterminedtarget region of any size, and vector sequences remain intact.We have mutagenized the DNA coding for the -fragment of Escherichiacoli ß-galactosidase, and identified 176 differentbase substitution mutations by sequencing. The present methodgives mutant yields of 40–60%, when the mutants containabout one amino acid change per protein molecule. All typesof base substitution mutations can be generated and deletionsare rare. The efficiency of this method permits the use of relativelyelaborate screening systems to isolate mutants of either structuralgenes or regulatory regions.     

The {alpha}/{beta} hydrolase fold

We have identified a new protein fold—the /ßhydrolase fold—that is common to several hydrolytic enzymesof widely differing phylogenetic origin and catalytic function.The core of each enzyme is similar: an /ß sheet, notbarrel, of eight ß-sheets connected by -helices. Theseenzymes have diverged from a common ancestor so as to preservethe arrangement of the catalytic residues, not the binding site.They all have a catalytic triad, the elements of which are borneon loops which are the best-conserved structural features inthe fold. Only the histidine in the nucleophile-histidine-acidcatalytic triad is completely conserved, with the nucleophileand acid loops accommodating more than one type of amino acid.The unique topological and sequence arrangement of the triadresidues produces a catalytic triad which is, in a sense, amirror-image of the serine protease catalytic triad. There arenow four groups of enzymes which contain catalytic triads andwhich are related by convergent evolution towards a stable,useful active site: the eukaryotic serine proteases, the cysteineproteases, subtilisins and the /ß hydrolase fold enzymes.     

The hierarchy of mutations influencing the folding of antibody domains in Escherichia coli

In a systematic study of the periplasmic folding of antibodyfragments in Escherichia coli, we have analysed the expressionof an aggregation-prone and previously non-functional anti-phosphorylcholineantibody, T15, as a model system and converted it to a functionalmolecule. Introduction of heavy chain framework mutations previouslyfound to improve the folding of a related antibody led to improvedfolding of T15 fragments and improved physiology of the hostE.coli cells. Manipulation of the complementarity determiningregions (CDR) of the framework-mutated forms of T15 furtherimproved folding and bacterial host physiology, but no improvementwas seen in the wild type, suggesting the existence of a hierarchyin sequence positions leading to aggregation. Rational mutagenesisof the T15 light chain led to the production of functional T15fragments for the first time, with increased levels of functionalprotein produced from V_H manipulated constructs. We proposethat a hierarchical analysis of the primary amino acid sequence,as we have described, provides guidelines on how correctly folding,functional antibodies might be achieved and will allow furtherdelineation of the decisive structural factors and pathwaysfavouring protein aggregation.     

Simultaneous and multivariate alignment of protein sequences: correspondence between physicochemical profiles and structurally conserved regions (SCR)

A general protein sequence alignment methodology for detectinga priori unknown common structural and functional regions isdescribed. The method proposed in this paper is based on twobasic requirements for a meaningful alignment. First, each sequenceor segment of a sequence is characterized by a multivariatephysicochemical profile. Second, the alignment is performedby considering all the sequences simultaneously, and the algorithmdetects those regions that form a set of similar profiles. Inorder to test the structural meaning of the alignment obtainedfrom the sequences, quantitative comparisons are performed withstructurally conserved regions (SCR) determined from the X-raystructures of three serine proteases. Results suggest that thelimits of the SCR may be predicted from the similarities betweenthe physicochemical profiles of the sequences. The proceduresare not completely automated. The final step requires a visualscreening of alternative pathways in order to determine an optimalalignment.     

Potential of genetic algorithms in protein folding and protein engineering simulations

Genetic algorithms are very efficient search mechanisms whichmutate, recombine and select amongst tentative solutions toa problem until a near optimal one is achieved. We introducethem as a new tool to study proteins. The identification andmotivation for different fitness functions is discussed. Theevolution of the zinc finger sequence motif from a random startis modelled. User specified changes of the repressor structurewere simulated and critical sites and exchanges for mutagenesisidentified. Vast conformational spaces are efficiently searchedas illustrated by the ab initio folding of a model protein ofa four ß strand bundle. The genetic algorithm simulationwhich mimicked important folding constraints as overall hydrophobicpackaging and a propensity of the betaphilic residues for transpositions achieved a unique fold. Cooperativity in the ßstrand regions and a length of 3–5 for the interconnectingloops was critical. Specific interaction sites were considerablyless effective in driving the fold.     

Does a backwardly read protein sequence have a unique native state?

Amino acid sequences of native proteins are generally not palindromic.Nevertheless, the protein molecule obtained as a result of readingthe sequence backwards, i.e. a retro-protein, obviously hasthe same amino acid composition and the same hydrophobicityprofile as the native sequence. The important questions whicharise in the context of retro-proteins are: does a retro-proteinfold to a well defined native-like structure as natural proteinsdo and, if the answer is positive, does a retro-protein foldto a structure similar to the native conformation of the originalprotein? In this work, the fold of retro-protein A, originatedfrom the retro-sequence of the B domain of Staphylococcal proteinA, was studied. As a result of lattice model simulations, itis conjectured that the retro-protein A also forms a three-helixbundle structure in solution. It is also predicted that thetopology of the retro-protein A three-helix bundle is that ofthe native protein A, rather than that corresponding to themirror image of native protein A. Secondary structure elementsin the retro-protein do not exactly match their counterpartsin the original protein structure; however, the amino acid sidechain contact pattern of the hydrophobic core is partly conserved.     

The thermostability of DNA-binding protein HU from bacilli

The primary and tertiary structures of DNA-binding protein HUfrom Bacillus stearothermophilus are already known. The primarystructure has been previously determined for HU from the closelyrelated B.globigü and the determinations of the sequencesfrom B.caldolyticus and B.subtilis are described here. Thesebacteria have optimum growth temperatures of > 70C (B.caldolyticus),65C (B.stearothermophilus), 37C (B.subtilis) and 30C (B.globigü).in vitro measurements from circular dichroic spectra describedhere give T_m values reflecting these growth temperatures, of68, 64, 43 and 41C respectively. We discuss here the relativethermostability of the four proteins in terms of the amino aciddifferences between the sequences and the three-dimensionalmodel of the B.stearothermophilus HU. The current model forthe interaction of the protein with DNA is only discussed interms of its relevance with regard to thermostability.     

Evolutionary divergence and conservation of trypsin

The trypsin sequences currently available in the data bankshave been collected and aligned using first the amino acid sequencehomology and, subsequently, the superposed crystal structuresof trypsins from the cow, the bacterium Streptomyces griseusand the fungus Fusarium oxysporum. The phylogenetic tree constructedaccording to this multiple alignment is consistent with a continuousevolutionary divergence of trypsin from a common ancestor ofboth prokaryotes and eukaryotes. Comparison of crystal structuresreveals a strict conservation of secondary structure. Similarly,in the alignment of all the sequences, insertions and deletionsoccur only in regions corresponding to loops between the secondarystructure elements in the known crystal structures. The conservedresidues cluster around the active site. Almost all conservedresidues can be associated with one of the basic functionalfeatures of the protein: zymogen activation, catalysis and substratespecificity. In contrast, the residues of the hydrophobic coreof the protein and the calcium ion binding sites are generallynot conserved. The conserved features of trypsin and the natureof the conservation are discussed In detail     

Structure of a 16 kDa integral membrane protein that has identity to the putative proton channel of the vacuolar H+-ATPase

A 16 kDa protein has been isolated in a homogeneous form asthe major component of a paracrystalline paired membrane structureclosely resembling the gap junction. The primary structure ofthis protein from arthropod and vertebrate species has beendetermined by protein and cDNA sequencing. The amino acid sequencesare highly conserved and virtually identical to the amino acidsequence of the proteolipid subunit of the vacuolar H⁺-ATPases.The disposition of the protein in the membrane has been studiedusing proteases and the N,N'-dicyclohexylcarbodiimide reactivesite identified. These data, together with secondary structurepredictions, suggest that the 16 kDa protein is for the mostpart buried in the membrane, arranged in a bundle of four hydrophobicß-helices. Using computer graphics, a model has beenconstructed based on this arrangement and on the electron microscopicimages of the paracrystalline arrays     

Mutational analysis supports a structural model for the cell cycle protein kinase p34

Structural models for the eukaryotic cell cycle control proteinp34 from human, S.pombe and S.cerevisiae have been derived fromthe crystallographic coordinates of the cAMP-dependent proteinkinase (cAPK) catalytic subunit (active conformation) and comparedwith the structure of Inactive CDK2 apoenzyme. Differences betweenthe p34 and cAPK catalytic sites provide a possible explanationfor their different substrate specificities. The p34 modelslocalize Tyrl5 and Thrl4 close to the sites of catalysis andsubstrate recognition where their phosphorylatlon could inhibitp34 kinase activity either by blocking MgATP or substrate binding.The conserved sequences PSTAIRE and LYLIFEFL are both closeto the catalytic site and accessible on the protein surfaceavailable to mediate interactions with other proteins. It ispredicted that p34 has an active-site cleft composed almostentirely of sequences common to all protein kinases and sequencesunique to the p34 protein family. Genetic and biochemical analysesof p34 have shown that it interacts extensively with a numberof other proteins. The model allows the relative dispositionof these sites of mutation to each other and to the sites ofcatalysis and substrate recognition to be appreciated. Surfaceregions on p34 that are important for function have been identified.These sites identify residues that may interact with p13^SUCL,cydin, plO7^WEEL and p80^cdc25     

Effects of amino acid substitutions in the hydrophobic core of {alpha}-lactalbumin on the stability of the molten globule state

Five mutant –lactalbumins, with one or two amino acidsubstitution(s) in the B helix, were engineered to examine therelation between the stability of the molten globule state andthe hydrophobicity of these amino acids. The mutation sites(Thr29, Ala30 and Thr33) have been chosen on the basis of comparisonof the amino acid sequences of goat, bovine and gunea pig –lactalbumin,in which the guinea pig protein shows a remarkably more stablemolten globule than the other proteins. The recombinant proteinswere expressed Escherichia coli and then purified and refoldedefficiently to produce the active proteins. The stability ofthe molten globule state of these engineered proteins has beeninvestigated by urea–induced unfolding transition underan acidic condition (pH 2.0), where the molten globule stateis stable in the absence of urea. The results show that themolten globule state is stabilized by the amino acid substitutionswhich raise the hydrophobicity of the residues, suggesting thatthe hydrophobic core in a globular protein plays an importantrole in the stability of the molten globule state. The changein stabilization free energy of the molten globule state causedby each amino acid substitution has been evaluated, and molecularmechanisms of stabilization of the molten globule state arediscussed.     

Lipid modification of proteins and their membrane transport

An effective method for artificial attachment of lipid anchorsto water-soluble proteins has been developed. To this end, aprotein molecule is modified in a system of reversed micellesby a water-insoluble reagent, e.g. fatty acid chloride. Fattyacylated proteins acquire an ability to translocate across lipidmembranes and penetrate intact cells. This principle of impartingtransmembrane properties to water-soluble proteins makes itpossible to realize in vivo a direct transport of antibodiesacross the hemato-encephalic barrier into the brain and to developa method for virus suppression by fatty acylated anti-viralantibodies capable of penetrating infected cells. The effectof a drastic increase in the biological activity of exogenousprotein factors, e.g. Staphylococcus aureus enterotoxin A, asa result of their artificial fatty acylation has been discovered.The above-mentioned phenomena are discussed in relation to thein vivo data, indicating that post-translational modificationof proteins by fatty acids and phospholipids is very widespreadin nature and evidently plays an important role in protein transportand sorting. In this connection, lipid modification of proteinsis regarded as a possible general step of protein transportin vivo.     

Structure prediction of the EcoRV DNA methyltransferase based on mutant profiling, secondary structure analysis, comparison with known structures of methyltransferases and isolation of catalytically inactive single mutants

The EcoRV DNA methyltransferase (M·EcoRV) is an -adeninemethyltransferase. We have used two different programs to predictthe secondary structure of M·EcoRV. The resulting consensusprediction was tested by a mutant profiling analysis. 29 neutralmutations of M·EcoRV were generated by five cycles ofrandom mutagenesis and selection for active variants to increasethe reliability of the prediction and to get a secondary structureprediction for some ambiguously predicted regions. The predictedconsensus secondary structure elements could be aligned to thecommon topology of the structures of the catalytic domains ofM·HhaI and M·TaqI. In a complementary approachwe have isolated nine catalytically inactive single mutants.Five of these mutants contain an amino acid exchange withinthe catalytic domain of M·EcoRV (Val20-Ala, Lys81Arg,Cys192Arg, Asp193Gly, Trp231Arg). The Trp231Arg mutant bindsDNA similarly to wild-type M·EcoRV, but is catalyticallyinactive. Hence this mutant behaves like a bona fide activesite mutant. According to the structure prediction, Trp231 islocated in a loop at the putative active site of M·EcoRV.The other inactive mutants were insoluble. They contain aminoacid exchanges within the conserved amino acid motifs X, IIIor IV in M·EcoRV confirming the importance of these regions.