Semi-empirical simulation of Zn/Cd binding site preference in the metal binding domains of mammalian metallothionein

Metallothionein, a two-domain protein, naturally binds sevengram atoms of divalent ions such as Zn and Cd. Four of the metals(Ml, M5, M6 and M7) are found in the -domain and three (M2,M3 and M4) in the ß-domain. Previous studies haveshown that metals in the -domain are more readily exchangeable,and the level of avidity is site specific. By semi-empiricalMNDO modified neglect of diatomic overlap calculations, we foundthe tendency of binding energy for Cd to be M3 > M2 >M4 in the ß-cluster and M5 > M7 > Ml, M6 inthe -cluster. Thus, the replacement of Zn by Cd can be expectedto follow the order M4 M2 M3 in the ß-domain andMS M7 M1 or M6 in the -domain. This is reflected by energydifferences computed with a series of simulated structures derivedfrom either X-ray crystallography or NMR coordinates.     

Effect of replacing helical glycine residues with alanines on reversible and irreversible stability and production of Aspergillus awamori glucoamylase

To decrease irreversible thermoinactivation of Aspergillus awamoriglucoamylase, five Gly residues causing helix flexibility werereplaced with Ala residues. Mutation of Gly57 did not affectthermostability. Mutation of Gly137 doubled it at pHs 3.5 and4.5 but barely changed it at pH 5.5. The Gly139Ala mutationdid not change thermostability at pH 3.5, improved it at pH4.5 and worsened it at pH 5.5. The Gly137/Gly139Ala/Ala mutationgave 1.5–2-fold increased thermostabilities at pHs 3.5–5.5.Mutations of Gly251 and Gly383 decreased it at all pHs. Gly137Alaand Gly137/Gly139Ala/Ala glucoamylases are the most stable yetproduced by mutation. Guanidine treatment at pH 4.5 decreasedthe reversible stabilities of Gly137Ala, Gly139Ala and Gly137/Gly139Ala/Alaglucoamylases at infinite dilution while not changing thoseof Gly251Ala and Gly 383Ala glucoamylases, which is, in general,opposite to what occurred with thermoinactivation. Mutationof Gly57 greatly improved the extracellular glucoamylase productionby yeast, that of Gly137 barely affected it and those of Gly139and of both Gly137 and Gly139 strongly impeded it. These observationssuggest that -helix rigidity can affect reversible and irreversibleglucoamylase stability differently, that the effects of multiplemutations within one -helix to improve stability are not alwaysadditive and that even single mutations can strongly affectextracellular enzyme production.     

Thermosensitive mutants of Aspergillus awamori glucoamylase by random mutagenesis: inactivation kinetics and structural interpretation

Abstract Seven thermosensitive glucoamylase mutants generated by randommutagenesis and expressed inSaccharomyces cerevisiae were sequencedand their inactivation kinetics were determined. Wild-type glucoamylaseexpressed in S.cerevisiae was more glycosylated and more stablethan the native Aspergillus niger enzyme. All mutants had lowerfree energies of inactivation than wild-type glucoamylase. Inthe Ala39 Val, Ala302 Val and Leu410 Phe mutants, small hydrophobicresidues were replaced by larger ones, showing that increasesin size and hydrophobicity of residues included in hydrophobicclusters were destabilizing. The Gly396 Ser and Gly407 Aspmutants had very flexible residues replaced by more rigid ones,and this probably induced changes in the backbone conformationthat destabilized the protein. The Prol28 Ser mutation changeda rigid residue in an a-helix to a more flexible one, and destabilizedthe protein by increasing the entropy of the unfolded state.The Ala residue in the Ala442 Thr mutation is in the highlyO-glycosylated region surrounded by hydrophilk residues, whereitmay be a hydrophobic anchor Unking the O-glycosylated arm tothe catalytic core. It was replaced by a residue that potentiallyis O-glycosylated. In five of the seven mutations, residuesthat were part of hydrophobic microdomains were changed, confirmingthe importance of the latter in protein stability and structure     

Mutations to alter Aspergillus awamori glucoamylase selectivity. IV. Combinations of Asn20->Cys/Ala27->Cys, Ser30->Pro, Gly137->Ala, 311314 Loop, Ser411->Ala and Ser436->Pro

Six previously constructed and nine newly constructed Aspergillusawamori glucoamylases with multiple mutations made by combiningexisting single mutations were tested for their ability to produceglucose from maltodextrins. Multiple mutations have cumulativeeffects on glucose yield, specific activity and thermostability.No general correlation between glucose yield and thermostabilitywas observed, although mutations that presumably impede unfoldingat high temperatures uniformly increase thermostability andgenerally increase glucose yield. Peak glucose yields decreasewith increasing temperature. The best combination of high glucoseyield, high specific activity and high thermostability occursin Asn20Cys/Ala27Cys/Ser30Pro/Gly137Ala glucoamylase.     

The use of alanine scanning mutagenesis to determine the role of the N-terminus of the regulatory chain in the heterotropic mechanism of Escherichia coli aspartate transcarbamoylase

The location of the first seven residues of the regulatory chainof Escherichia coli aspartate transcarbamoylase has been identifiedby X-ray crystallography to be near the binding site of theregulatory nucleotides. In order to determine the function ofthe N-terminus of the regulatory chain of aspartate transcarbamoylasein heterotropic regulation, alanine scanning mutagenesis wasused. Specifically, Thr2r, His3r, Asp4r, Asn5r, Lys6r and Leu7rwere each replaced with alanine. Analyses of these mutant enzymesindicate that none of these substitutions significantly alterthe catalytic properties of the enzyme. However, three of themutant enzymes, Asp4r Ala, Lys6r Ala and Leu7r Ala, exhibitednotable changes in their response to the regulatory nucleotides,while mutations at Thr2r, His3r and Asn5r exhibited only minorchanges in their heterotropic responses. For the Asp4r Alaenzyme, the responses to ATP and CTP were reduced 30 and 40%respectively, compared with the wild-type enzyme. For the Lys6r Ala enzyme, the response to ATP was reduced 70%, while theCTP response was reduced 50%. In the case of the Leu7r Alaenzyme, a 30 and 20% reduction in response to ATP and CTP respectively,was observed. The synergistk inhibition by UTP in the presenceof CTP for the Lys6r Ala enzyme was reduced 40% compared withthat of the wild type enzyme. For the Leu7r Ala enzyme, thesynergistic inhibition was abolished. In addition, UTP decreasedthe CTP binding affinity of the Leu7r Ala enzyme. Analysisof the kinetic data from these mutant enzymes suggests thatresidues Thr2r, His3r and Asn5r have little effect on the heterotropicmechanism, while residues Asp4r, Lys6r and Leu7r play a moresignificant role in the heterotropic response of the enzymetoward the nucleotides. Furthermore, residue Leu7r appears tobe directly involved in the mechanism for synergistic inhibitionof aspartate transcarbamoylase. In this study alanine scanningmutagenesis has provided a rapid method of identifying thoseresidues in the N-terminal region of the regulatory chain ofaspartate transcarbamoylase important for heterotropic regulation.     

Invariant amino acid replacement affects the dihydrofolate reductase function and its gene expression

Three different forms of dihydrofolate reductase (DHFR) fromEscherichia coli with amino acid replacements Thr35 Asp, Asn37 Ser and Arg57 His, and one form containing all three of thesechanges were obtained by oligonucleotide-directed mutagenesis.These amino acids are on the surface of the protein and twoof them (Thr35 and Arg57) are invariant for known sequencesof DHFR. Conversion of Asn37 Ser has no effect on the functionalactivity or the protein level in the cells. The Thr35 Asp replacementleads to a sharp decrease in the protein level, while the additionof a DHFR inhibitor, trimethoprim (Tmp), to the growth mediumincreases the level of DHFR in the ceus. There is a very smallquantity of DHFR with all three amino acid changes. The additionof Tmp to the growth medium also leads to an increase in themutant protein levels. The mutant with the Arg57 His replacementrenders the cells sensitive to Tmp, but the level of DHFR isthe same as for the wild-type protein. It is suggested thatthe invariant Thr35 is important for the stable conformationof DHFR whereas Arg57 is essential for protein activity. Variousstructural and functional aspects of these results are discussed.     

Identification and elimination by site-directed mutagenesis of thermolabile aspartyl bonds in Aspergillus awamori glucoamylase

Both Dative Aspergillus niger glucoamylase and wild-type Aspergillusawamori glucoamylase expressed in Saccharo-myces cerevisiae,which have identical primary structures, undergo hydrolysisat aspartyl bonds at low pH values and elevated temperatures.In native A.niger enzyme the Aspl26–Glyl27 bond was preferentiallycleaved at pH 3.5,while at pH 4.5 cleavage of the Asp257–Pro258and Asp293–Gly294 bonds was dominant. In wild-type A.awamoriglucoamylase, cleavage of the latter was dominant at both pH3.5 and 4.5. Site-directed mutations Aspl26Glu and Glyl27Alain wild-type enzyme decreased specific activities by 60 and30%, respectively, and increased irreversible thermoinactivationrates 3- to 4-fold at pH 4.5. Replacement of Asp257 with Gluand Asp293 with Glu or Gin decreased specific activities by20%, but greatly reduced cleavage of the Asp257–Pro258and Asp293–Gly294 bonds. The Asp257Glu mutant was producedvery slowly and was more thermostable than wild–type glucoamylaseat pH 4.5up to 70°C. Replacement of Asp293 with either Gluor Gln significantly raised protein production and slightlyincreased thermostability at pH 3.5 and 4.5, but not at pH5.     

Effect of amino acid deletions in the O-glycosylated region of Aspergillus awamori glucoamylase

Aspergillus awamori glucoamylase (GA) contains globular catalyticand starch-binding domains (residues 1–471 and 509–616,respectively). A heavily O-glycosylated sequence comprises twoparts. The first (residues 441–471) in the crystal structurewraps around an /-barrel formed by residues 1–440. Thesecond (residues 472–508) is an extended, semi-rigid linkerbetween the two domains. To investigate the functional roleof this linker, we made internal deletions to remove residues466–512 (GA1), 485–512 (GA2) and 466–483 (GA3).GA2 and GA3 were expressed in Saccharomyces cerevisiae culturesupernatants at 60 and 20% the wild-type level, respectively,while GA1 was almost undetectable. Western blots comparing extracellularand intracellular fractions indicated that the region deletedin GA3 was critical for secretion, while the region deletedin GA2 contributed to the production of a stable enzyme structure.The activities of purified GA2 and GA3 on soluble and insolublestarch were similar to those of wild-type GA, indicating thatfor soluble starch their deletions did not affect the catalyticdomain and for insoluble starch the linker does not coordinatethe activities of the catalytic and starch-binding domains.The deletions had a significant negative effect on GA2 and GA3thermos tabilities.     

Structural study of mutants of Escherichia coli ribonuclease HI with enhanced thermostability

Systematic replacement of the amino acid residues in Escherichiacoli ribonuclease HI with those in the thermophilic counterparthas revealed that two mutations, His62–Pro (H62P) andLys95Gly (K95G), increased the thermostability of the protein.These single-site mutant proteins, together with the mutantproteins His62Ala (H62A), Lys95Asn (K95N) and Lys95Ala (K95A),were crystallized and their structures were determined at 1.8Å resolution. The crystal structures of these mutant proteinsreveal that only the local structure around each mutation siteis essential for the increase in thermostability. For each mutantprotein, the stabilization mechanism is considered to be asfollows: (i) H62P is stabilized because of a decrease in theentropy of the unfolded state, without a change in the nativebackbone structure; (ii) K95G is stabilized since the straincaused by the left-handed backbone structure in the typical3:5 type loop is eliminated; and (iii) K95N is slightly stabilizedby a hydrogen bond formed between the side-chain N-atom of themutated aspargine residue and the main-chain carbonyl oxygenwithin the same residue.     

Inhibition of {beta}S-chain dependent polymerization by synergistic complementation of contact site perturbations of {alpha}-chain: application of semisynthetic chimeric {alpha}-chains

Mouse _1–30-horse _31–141 chimeric -chain, a semisyntheticsuper-inhibitory -chain, inhibits ß^S-chain dependent polymerizationbetter than both parent -chains. Although contact site sequencedifferences are absent in the _1–30 region of the chimericchain, the four sequence differences of the region _17-22 couldinduce perturbations of the side chains at ₁₆, ₂₀ and ₂₃, thethree contact sites of the region. A synergistic complementationof such contact site perturbation with that of horse _31–141probably results in the super-inhibitory activity of the chimeric-chain. The inhibitory contact site sequence differences, bythemselves, could also exhibit similar synergistic complementation.Accordingly, the polymerization inhibitory activity of Hb Le-Lamentin(LM) mutation [His20()Gln], a contact site sequence difference,engineered into human–horse chimeric -chain has been investigatedto map such a synergistic complementation. Gln20() has littleeffect on the O₂ affinity of HbS, but in human–horse chimeric-chain it reduces the O₂ affinity slightly. In the chimeric-chain, Gln20() increased sensitivity of the ßßcleft for the DPG influence, reflecting a cross-talk betweenthe ₁ß₁ interface and ßß cleft in this semisyntheticchimeric HbS. In the human -chain frame, the polymerizationinhibitory activity of Gln20() is higher compared with horse_1–30, but lower than mouse _1–30. Gln20() synergisticallycomplements the inhibitory propensity of horse _31–141.However, the inhibitory activity of LM–horse chimeric-chain is still lower than that of mouse–horse chimeric-chain. Therefore, perturbation of multiple contact sites inthe _1–30 region of the mouse–horse chimeric -chainand its linkage with the inhibitory propensity of horse _31–141has been now invoked to explain the super-inhibitory activityof the chimeric -chain. The `linkage-map' of contact sites canserve as a blueprint for designing synergistic complementationof multiple contact sites into -chains as a strategy for generatingsuper-inhibitory antisickling hemoglobins for gene therapy ofsickle cell disease.     

Effects of amino acid replacements around the reactive site of chicken ovomucoid domain 3 on the inhibitory activity toward chymotrypsin and trypsin

We have previously shown that replacing the P1-site residue(Ala) of chicken ovomucoid domain 3 (OMCHI3) with a Met or Lysresults in the acquisition of inhibitory activity toward chymotrypsinor trypsin, respectively. However, the inhibitory activitiesthus induced are not strong. In the present study, we introducedadditional amino acid replacements around the reactive siteto try to make the P1-site mutants more effective inhibitorsof chymotrypsin or trypsin. The amino acid replacement AspTyrat the P2' site of OMCHI3(P1Met) resulted in conversion to a35000-fold more effective inhibitor of chymotrypsin with aninhibitor constant (K_i) of 1.17x10^–11 M. The K_i valueof OMCHI3(P1Met, P2'Ala) indicated that the effect on the interactionwith chymotrypsin of removing a negative charge from the P2'site was greater than that of introducing an aromatic ring.Similarly, enhanced inhibition of trypsin was observed whenthe AspTyr replacement was introduced into the P2' site of OMCHI3(P1Lys).Two additional replacements, AspAla at the P4 site and ArgAlaat the P3' site, made the mutant a more effective inhibitorof trypsin with a K_i value of 1.44x10^–9 M. By contrast,ArgAla replacement at the P3' site of OMCHI3(P1Met, P2'Tyr)resulted in a greatly reduced inhibition of chymotrypsin, andAspAla replacement at the P4 site produced only a small changewhen compared with a natural variant of OMCHI3. These resultsclearly indicate that not only the P1-site residue but alsothe characteristics, particularly the electrostatic properties,of the amino acid residues around the reactive site of the proteaseinhibitor determine the strength of its interactions with proteases.Furthermore, amino acids with different characteristics arerequired around the reactive site for strong inhibition of chymotrypsinand trypsin.     

Replacing the ({beta}{alpha})-unit 8 of E.coli TIM with its chicken homologue leads to a stable and active hybrid enzyme

In order to investigate how structural modifications interferewith protein stability, we modified a (ß)-unit inE.coli triosephosphate isomerase (TIM), a typical (ß)-barrelprotein, assuming that the pseudosymmetrical ß-barrelcan be divided into eight successive loop/ß-strand/loop/-helixmotifs. We replaced the eighth (ß)-unit of E.coliTIM with the corresponding chicken (ß)-unit. The substitution,involving the replacement of 10 of the 23 residues of this (ß)-unit, was evaluated first by modelling, then experimentally.Modelling by bomology suggests how the amino add replacementsmight be accommodated in the hybrid E.coli/chicken TIM (ETCM8CHI).Both natural and hybrid recombinant TIMs, overproduced in E.coli,were purified to homogeneity and characterized as to their stabilityand kinetics. Our kinetic studies show that the modificationperformed here leads to an active enzyme. The stability studiesindicate that the stability of ETIM8CHI is comparable to thatof the wild type TIM.     

The T {leftrightharpoons} R structural transition of insulin; pathways suggested by targeted energy minimization

The transition of insulin between its crystallographically definedstates T and R is connected with considerable change even ofbackbone structure: the N-terminal B chain (residues B1 –B8) refolds from extended conformation in T into helical inR, and vice versa. Although hitherto observed only in hexamersthe transition of the monomer was adequate for developing andtesting the method of ‘targeted energy minimization’(TEM), capable of coping with conformational changes of suchextent at moderate computational expenditure. The simulationis performed in a predetermined number of steps consisting oftwo atomic displacements each, one by force in the directionof the target structure, the second by energy minimization releasingthe constraint caused in the first. The transition pathway isrepresented by the string of energy minimized transient structures.Due to the directedness of the algorithm the simulated pathwayfor R T is not the reversal of that for T R. It is, therefore,not pretended that the minimum energy pathway was identified.In the T R direction the N-terminal B chain first swivels whileremaining largely stretched and then winds up extending thepre-existing helix B9–B19. The A chain advances into thespace abandoned and withdraws from it in the R T simulation.In the latter the extended helix first kinks at B8/B9, and thenthe B1 B8 segment is unwound and stretched. The helical H-bondsof that segment are formed late in T R and are maintained duringalmost half of R T. The A_N helix is less stable and more involvedin the transitions than helix A_C. The two pathways seem plausiblefrom both the energetic and geometric points of view. Knowledgeof them will be of value to suggest mutations to test them byexperimental evidence.     

Study of cysteine residues in the {alpha} subunit of Escherichia coli tryptophan synthase. 2. Role in enzymatic function

To understand the functional roles of Cys residues in the subunitof tryptophan synthase from Escherichia coli, single mutantsof the subunit, in which each of the three Cys residues wassubstituted with Ser, Gly, Ala or Val, were constructed by site-directedmutagenesis. The effects of the substitutions on the functionof tryptophan synthase were investigated by activity measurements,calorimetric measurements of association with the ßsubunit and steadystate kinetic analysis of catalysis. Althoughthe three Cys residues are located away from the apparentlyimportant parts for enzymatic activity, substitutions at position81 by Ser, Ala or Val caused decreases in the intrinsic activityof the subunit. Furthermore, Cys81Ser and Cys81Val reducedstimulation activities in the and ß reactions dueto formation of a complex with the ß subunit. Thelower stimulation activities of the mutant proteins were notcorrelated with their abilities to associate with the ßsubunit but were correlated with decreases in k_cat. The presentresults suggest that position 81 plays an indirectly importantrole in the activity of the subunit itself and the mutual activationmechanism of the complex.     

An 8-fold {beta}{alpha} barrel protein with redundant folding possibilities

Protein sequences containing redundant segments of secondarystructure at both termini have the choice a priori of foldinginto several possible circularly permuted variants of the wild-typetertiary structure. To test this hypothesis the gene of phosphoribosylanthranilate isomerase from yeast, which is a single-domain8-fold ß barrel protein, was modified to produce a10-fold ß homologue in Escherichia coli. It containeda duplicate of the two C-terminal ß units of supersecondarystructure fused to its N-terminus. Most of the protein was recoveredfrom the insoluble fraction of disrupted cells by dissolutionin guanidinium chloride solutions and refolding. Pristine proteinwas purified from the soluble fraction. The purified (ß)₁₀proteins were enzymically almost fully active. Absorbance, fluorescenceand circular dichroism spectra as well as the reversible unfoldingbehaviour of both proteins were also very similar to the propertiesof the original (ß)₈ protein. Digestion with endopeptidasesconverted both the pristine and the refolded (ß)₁₀variant to the same large fragment that had the N-terminal sequenceand mol. wt of the wild-type ß)₈ protein. The datasuggest that the folding of the (ß)₁₀ variant is controlledthermodynamically both in vivo and in vitro.     

Analysis of structural determinants of the stability of thermolysinlike proteases by molecular modelling and site-directed mutagenesis

The thermolysin-like protease (TLP) produced by Bacillus stearothermophilusCU21 (TLP-ste) differs at 43 positions from the more thermallystable thermolysin (containing 316 residues in total). Of thesedifferences, 26 were analysed by studying the effect of replacingresidues in TLP-ste by the corresponding residues in thermolysin.Several stabilizing mutations were identified but, remarkably,considerable destabilizing mutational effects were also found.A Tyr-rich three residue insertion in TLP-ste (the only deletional/insertionaldifference between the two enzymes) appeared to make an importantcontribution to the stability of the enzyme. Mutations withlarge effects on stability were all localized in the ßpleatedN-terminal domain of TLP-ste, confirming observations that thisdomain has a lower intrinsic stability than the largely -helicalC-terminal domain. Rigidifying mutations such as Gly58 Alaand Ala69 Pro were among the most stabilizing ones. Apart fromthis observation, the analyses did not reveal general rulesfor stabilizing proteins. Instead, the results highlight theimportance of context in evaluating the stability effects ofmutations.     

The role of the sequence extensions in {beta}-crystallin assembly

The modular construction of the eye lens ß-crystallinsmakes them good candidates for protein engineering to ascertainthe rules of assembly of oligomers. X-ray studies have shownthat although the polypeptide chains of ßB2-crystallinand -crystallins fold to form similar N- and C-terminal domains,the conformation of the connecting peptides are such that the-crystallins are monomers and the ß-crystallin isa dimer. Unlike -crystallins, the numerous -crystallins haveextensions of variable sequence from the globular domains. Wehave tested the effect of removing the N- and C-terminal extensionsfrom rat ßB2-crystallin using a bacterial expressionsystem. Abundant proteins were produced in Escherichia coliusing the pET or pQE vectors. Full-length and truncated proteinswere purified and checked for refolding using circular dichroism.Sizing of the truncated proteins using gel filtration chroma-tographyshowed that the absence of either the N- or C-terminal extensiondoes not affect dimerization of ßB2-crystallin.     

Site-directed mutagenesis in hemoglobin: test of functional homology of the F9 amino acid residues of hemoglobin {alpha} and {beta} chains

The cysteine residue at F9(93) of the human hemoglobin (Hb A)ß chain, conserved in mammalian and avian hemoglobins,is located near the functionally important 1–ß2interface and C-terminal region of the ß chain and isreactive to sulfhydryl reagents. The functional roles of thisresidue are still unclear, although regulation of local bloodflow through allosteric S-nitrosylation of this residue is proposed.To clarify the role of this residue and its functional homologyto F9(88) of the chain, we measured oxygen equilibrium curves,UV-region derivative spectra, Soret-band absorption spectra,the number of titratable -SH groups with p-mercuribenzoate andthe rate of reaction of these groups with 4,4'-dipyridine disulfidefor three recombinant mutant Hbs with single amino acid substitutions:AlaCys at 88 (rHb A88C), CysAla at 93ß (rHb C93ßA)and CysThr at 93ß (rHb C93ßT). These Hbs showedincreased oxygen affinities and impaired allosteric effects.The spectral data indicated that the R to T transition upondeoxygenation was partially restricted in these Hbs. The numberof titratable -SH groups of liganded form was 3.2–3.5for rHb A88C compared with 2.2 for Hb A, whereas those for rHbC93ßA and rHb C93ßT were negligibly small. The reductionof rate of reaction with 4,4'-dipyridine disulfide upon deoxygenationin rHb A88C was smaller than that in Hb A. Our experimentaldata have shown that the residues at 88 and 93ß have definiteroles but they have no functional homology. Structure–functionrelationships in our mutant Hbs are discussed.     

The predicted secondary structure of the G-type glutamineamidotransferase is compatible with TEM-barrel topology

Glutamine amidotransferase (GAT) subunits or domains catalyzean important partial reaction in many complex biosynthetic reactions.The structure of one member of the F-type GATs is known, butthe structure of the unrelated G-type is still unknown. Becausemany protein sequences are available for anthranilate synthasecomponent II (product of the trpG gene), we have predicted itsaverage secondary structure by a joint prediction method [Niermannand Kirschner (1991a) Protein Engng, 4, 359–370]. Thepredicted eight ß-strands and seven -helices followan 8-fold cyclic repetition of a ß-strand-loop--helix-loopmodule with helix 7 missing. This pattern of secondary structuresuggests that the G-type GAT domain has an 8-fold ß-barreltopology, as found first in triose phosphate isomerase (TIM-barrel).This model is supported by the location of known catalyticallyessential residues in loops between (ß-strands and-helices. Evidence from published sequencing and mutationalstudies on selected members of the GAT superfamily (carbamoylphosphate, imidazoleglycerol phosphate, GMP and CTP synthases)support both the secondary structure prediction and the TIM-barreltopology.     

Family of G protein {alpha} chains: amphipathic analysis and predicted structure of functional domains

The G proteins transduce hormonal and other signals into regulationof enzymes such as adenylyl cyclase and retinal cGMP phosphodiesterase.Each G protein contains an subunit that binds and hydrolyzesguanine nucleotides and interacts with ß subunitsand specific receptor and effector proteins. Amphipathic andsecondary structure analysis of the primary sequences of fivedifferent chains (bovine _s, _t1 and _t2, mouse _i, and rat _o)predicted the secondary structure of a composite chain (_avg).The chains contain four short regions of sequence homologousto regions in the GDP binding domain of bacterial elongationfactor Tu (EF-Tu). Similarities between the predicted secondarystructures of these regions in _avg and the known secondary structureof EF-Tu allowed us to construct a three-dimensional model ofthe GDP binding domain of _avg. Identification of the GDP bindingdomain of _avg defined three additional domains in the compositepolypeptide. The first includes the amino terminal 41 residuesof _avg, with a predicted am phipathic helical structure; thisdomain may control binding of the chains to the ßcomplex. The second domain, containing predicted ßstrands and helices, several of which are strongly amphipathic,probably contains sequences responsible for interaction of chains with effector enzymes. The predicted structure of thethird domain, containing the carhoxy terminal 100 amino acids,is predominantly ß sheet with an amphipathic helixat the carboxy terminus. We propose that this domain is reponsiblefor receptor binding. Our model should help direct further experimentsinto the structure and function of the G protein chain.