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.     

Functional roles and subsite locations of Leu177, Trp178 and Asn182 of Aspergillus awamori glucoamylase determined by site-directed mutagenesis

Fungal glucoamylases contain four conserved regions. One regionfrom the Aspergillus niger enzyme contains three key carboxylicacid residues, the general acid catalytic group, Glu179, alongwith Asp176 and Glu180. Three site-directed mutations, Leu177– His, Trp178 – Arg and Asn182 – Ala, wereconstructed near these acidic groups to reveal the functionof other conserved residues in this region. Leu177 and Trp178are strictly conserved among fungal glucoamylases, while anamide, predominantly Asn, always occurs at position 182. Substitutionsof Leu177 or Trp178 cause significant decreases in k_cat withthe substrates tested. Similar increases in activation energiesobtained with Leu177 – His with both -(1,4)- and -(1,6)-linkedsubstrates indicate Leu177 is located in subsite 1. K_M valuesobtained with the Trp178 – Arg mutation increase for an-(1,6)-linked substrate, but not for -(1,4)-linked substrates.Calculated differences in activation energy between substratesindicate Trp178 interacts specifically with subsite 2. The Asn182 Ala mutation did not change k_cat or K_M values, indicating thatAsn182 is not crucial for activity. These results support amechanism for glucoamylase catalytic activity consisting ofa fast substrate binding step followed by a conformational changeat subsite 1 to stabilize the transition state complex.     

Specificity determinants of rat tissue kallikrein probed by site-directed mutagenesis

Site-specific mutagenesis was employed to study structure-functionrelationships at the substrate binding site of rat tissue kallikrein.Four kallikrein mutants, the Pro219 deletion (P219del), the34–38 loop Tyr-Tyr-Phe-Gly to Ile-Asn mutation [YYFG(34–38)IN],the Trp215Gly exchange (W215G) and the double mutant with Tyr99Hisand Trp215Gly exchange (Y99H:W215G) were created by site-directedmutagenesis to probe their function in substrate binding. Themutant proteins were expressed in Esclzerichia coli at highlevels and analyzed by Western blot. These mutant enzymes werepurified to apparent homogeneity. Each migrated as a singleband on SDS-PAGE, with slightly lower molecular mass (36 kDa)than that of the native enzyme, (38 kDa) because of their lackof glycosylation. The recombinant kallikreins are immunologicallyidentical to the native enzyme, displaying parallelism withthe native enzyme in a direct radioimmunoassay for rat tissuekallikrein. Kinetic analyses of K_m and k_cat using fluorogenicpeptide substrates support the hypothesis that the Tyr99–Trp215interaction is a major determinant for hydrophobic P2 specificity.The results suggest an important role for the 34–38 loopin hydrophobic P3 affinity and further show that Pro219 is essentialto substrate binding and efficient catalysis of tissue kallikrein.     

Increasing the thermostability of a neutral protease by replacing positively charged amino acids in the N-terminal turn of {alpha}-helices

The 247–260 and 289–299 -helices of Bacillus subtilisneutral protease have a lysine in their N-terminal turn. Theselysines were replaced by Ser or Asp in order to improve electrostaticinteractions with the -helix dipole. After replacing Lys bySer at positions 249 or 290, the thermostability of the enzymewas increased by 0.3 and 1.0°C, respectively. The Asp249and Asp290 mutants exhibited a stabilization of 0.6 and 1.2°C,respectively. The results show the feasibility of stabilizingenzymes by introducing favourable residues at the end of -helices.     

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.     

Direct energy transfer to study the 3D structure of non-native proteins: AGH complex in molten globule state of apomyoglobin

The direct energy transfer technique was modified and appliedto probe the relative localization of apomyoglobin A-, G- andH-helixes, which are partly protected from deuterium exchangein the equilibrium molten globule state and in the molten globule-likekinetic intermediate. The non-radiative transfer of tryptophanelectronic energy to 3-nitrotyrosine was studied in differentconformational states of apomyoglobin (native, molten globule,unfolded) and interpreted in terms of average distances betweengroups of the protein chain. The experimental data show thatthe distance between the middle of A-helix and the N-terminusof G-helix as well as the distance between the middle of theA-helix and the C-terminus of the H-helix in the molten globulestate are close to those in the native state. This is a strongargument in favor of similarity of the overall architectureof the molten globule and native states.     

Synthesis, purification and initial structural characterization of octarellin, a de novo polypeptide modelled on the {alpha}/{beta}-barrel Proteins

We have attempted to construct an artificial polypeptide thatfolds like the eight-stranded parallel ß-barrel structures.Our approach consists of repeating eight times a unit peptidedesigned to adopt a ‘ß-strand/-helix’pattern. A first ‘test’ sequence for this structuralunit was deduced from a series of parameters defined after ananalysis of three natural /ß-barrel proteins and includingprincipally the lengths of the secondary structure elements,the /ß packing and the fitting on average Garnierprofiles. The gene encoding this structural unit was synthesized,cloned and expressed in Escherichia coli either as a monomeror as direct repeats of 2–12 units. Preliminary structuralcharacterization of the 7-, 8- and 9-fold unit polypeptidesby circular dichroism measurements indicates the presence ofthe predicted amount of -helix in the three proteins. Furtheranalysis by urea-gradient gel electrophoresis demonstrates that,in the conditions tested, only the 8-fold unit polypeptide formsa compact structure through a cooperative and rapid two-statefolding transition involving long-range molecular interactions.     

Modelling of the disulphide-swapped isomer of human insulin-like growth factor-1: implications for receptor binding

Insulin-like growth factor-1 (IGF-1) is a serum protein whichunexpectedly folds to yield two stable tertiary structures withdifferent disulphide connectivities; native IGF-1 [18–61,6–48,47–52]and IGF-1 swap [18–61,6–47, 48–52]. Here we demonstratein detail the biological properties of recombinant human nativeIGF-1 and IGF-1 swap secreted from Saccharomyces cerevisiae.IGF-1 swap had a ~30 fold loss in affinity for the IGF-1 receptoroverexpressed on BHK cells compared with native IGF-1.The parallelincrease in dose required to induce negative cooperativity togetherwith the parallel loss in mitogenicity in NIH 3T3 cells impliesthat disruption of the IGF-1 receptor binding interaction ratherthan restriction of a post-binding conformational change isresponsible for the reduction in biological activity of IGF-1swap. Interestingly, the affinity of IGF-1 swap for the insulinreceptor was ~200 fold lower than that of native IGF-1 indicatingthat the binding surface complementary to the insulin receptor(or the ability to attain it) is disturbed to a greater extentthan that to the IGF-1 receptor. A 1.0 ns high-temperature moleculardynamics study of the local energy landscape of IGF-1 swap resultedin uncoiling of the first A-region -helix and a rearrangementin the relative orientation of the A- and B-regions. The modelof IGF-1 swap is structurally homologous to the NMR structureof insulin swap and CD spectra consistent with the model arepresented. However, in the model of IGF-1 swap the C-regionhas filled the space where the first A-region -helix has uncoiledand this may be hindering interaction of Val44 with the secondinsulin receptor binding pocket.     

Investigation of the solution structure of chymotrypsin inhibitor 2 using molecular dynamics: comparison to X-ray crystallographic and NMR data

The native solution structure and dynamics of chymotrypsin inhibitor2 (CI2) have been studied using a long (5.3 ns) molecular dynamics(MD) simulation without any imposed restraints. The majorityof the experimentally observed spin–spin coupling constants,short– and long–range nuclear Overhauser effect(NOE) cross peaks and the amide hydrogen exchange behavior werereproduced by the MD simulation. This good correspondence suggeststhat the major structural features of the protein during thesimulation are representative of the true protein structurein solution. Two water molecules formed hydrogen bond bridgesbetween ß₂ and ß₃, in agreement with X–raycrystallographic data and a recent reassessment of the solutionstructure using time averaged NMR restraints during MD refinement.The active–site loop of the protein displayed the greateststructural changes and the highest mobility. When this loopregion was excluded, the average C r.m.s. deviation of the simulatedsolution structures from the crystal structure was 1.5 from0.5 to 5 3 ns. There is structural heterogeneity in particularregions of the NMR–derived solution structures, whichcould be a result of imprecision or true internal motion. Astudy of the distribution of mobility through the protein allowsus to distinguish between these two alternatives. In particular,deviations in the active–site loop appear to be a resultof heightened mobility, which is also supported by good correspondencebetween calculated and experimental S² N–H order parameters.On the other hand, other ill–defined regions of the NMR-derivedstructures are well defined in the simulation and are probablythe result of a lack of structural restraints (i.e. NOEs), asopposed to reflecting the true mobility.     

Stabilization of lysozyme against irreversible inactivation by alterations of the Asp-Gly sequences

Site-directed mutagenesis was performed at Asp-Gly (48–49,66–67, 101–102) and Asn-Gly (103–104) sequencesof hen egg-white lysozyme to protect the enzyme against irreversiblethermoinactivation. Because the lysozyme inactivation was causedby the accumulation of multiple chemical reactions, includingthe isomerization of the Asp-Gly sequence and the deamidationof Asn [Tomizawa et al.(1994) Biochemistry, 33, 13032–13037],the suppression of these reactions by the substitution of Glyto Ala, or the introduction of a sequence of human-type lysozyme,was attempted and the mutants (where each or all labile sequenceswere replaced) were prepared. The substitution resulted in thereversible destabilization from 1 to 2 kcal/mol per substitution.The destabilization was caused by the introduction of ß-carbonto the constrained position that had conformational angles withinthe allowed range for the Gly residue. Despite the decreasein the reversible conformational stability, the mutants hadmore resistance to irreversible inactivation at pH 4 and 100°C.In particular, the rate of irreversible inactivation of themutant, which was replaced at four chemically labile sequences,was the latest and corresponded to 18 kcal/mol of the reversibleconformational stability. Therefore, replacement of the chemicallylabile sequence was found to be more effective at protectingenzymes against irreversible thermoinactivation than at strengtheningreversible conformational stability.     

The 3.0 A crystal structure of xylose isomerase from Streptomyces olivochromogenes

The crystal structure of xylose isomerase [E.C. 5.3.1.5 [EC] ] fromStreptomyces olivochromogenes has been determined to 3.0 Åresolution. The crystals belong to space group P22₁2₁ with unitcell parameters a = 98.7, b = 93.9, c = 87.7. The asymmetricunit contains half of a tetrameric molecule of 222 symmetry.The two-fold axis relating the two molecules in the asymmetricunit is close to where a crystallographic two-fold would beif the space group were 1222. This causes the diffraction patternto have strong 1222 pseudo-symmetry, so all data were collectedin this pseudo-space group. Since the sequence of this enzymehas not been reported, a polyalanine backbone has been fittedto the electron density. Xylose isomerase has two domains: theN-terminal domain is an eight-stranded /ß barrel of299 residues. The C-terminal domain is a large loop of 50 residueswhich is involved in inter-molecular contacts. Comparison ofxylose isomerase with the archetypical /ß barrel protein,triose phosphate isomerase, reveals that the proteins overlapbest when the third (ß) strand of xylose isomeraseis superimposed on the first (ß) strand of triosephosphate isomerase. This same overlap has also been found betweenthe muconate lactonising enzyme and triose phosphate isomerase[Goldman et al. (1987) J. Mol. Biol., in press].     

Studies of the pore-forming domain of a voltage-gated potassium channel protein

Recent mutagenesis studies nave identified a stretch of aminoacid residues which form the ion-selective pore of the voltage-gatedpotassium channel. It has been suggested that this sequenceof amino acids forms a ß-barrel structure making upthe structure of the ion-selective pore [Hartman,H.A., Kirsch,G.E.,DreweJ.A., Taglialatela.M., Joho.R.H. and Brown,A.M. (1991)Science, 251, 942–944; YeUen.G., Jurman,M.E., Abramson,T.and MacKinnon,R. (1991) Science, 251, 939–942; Yool,AJ.and Schwarz.T.L. (1991) Nature, 349, 700–704]. We havesynthesized a polypeptide corresponding to this amino add sequence(residues 431–449 of the ShA potassium channel from Drosophila).A tetrameric version of this sequence was also synthesized byUnking together four of these peptldes onto a branching lysinecore. Fourier transform infrared (FT-LR) and circular dichroism(CD) spectroscopy have been used to investigate the structureof these peptides after their reconstitution into lyso phos-phatidylcholinemicelles and lipid bilayers composed of dimyristoyl phosphatidyfcholineand dimyristoyl phosphatidyl-glycerol. The spectroscopic studiesshow that these peptides are predominantly a-helical in theselipid environments. When Incorporated into planar lipid bilayersboth peptides induce ion channel activity. Molecular modellingstudies based upon the propensity of these peptides to forman -helical secondary structure in a hydrophobfc environmentare described. These results are discussed in the light of recentmutagenesis and binding studies of the Drosophila Shaker potassiumion channel protein     

Protien side-chain conformational entropy derived from fusion data-comparison with other empirical scales

The loss of conformational entropy of protein side-chains isa major effect in the energetics of folding. The simplest approachis to enumerate the number of freely rotatable bonds. Recently,two scales of side-chain conformational entropy have been proposedbased on the definition of entropy as the Boltzmann samplingover all accessible states (S –Rp_iInp_i, where p_i is theprobability of being in a rotameric state). In one scale, derivedonly for aliphatic and aromatic side-chains, the values of p_iwere obtained from Monte Carlo simulations. In the other scale,the observed frequencies of different rotameric states in adatabase of protein crystal structures yielded an estimate forp_i. Here an empirical estimation of the fusion entropy of theside-chains is used to derive a third scale. The fusion entropyis obtained as a sum of empirically derived contributions fromcomponent hydrocarbon and functional groups. There is a goodagreement between the fusion scale and the other two scales.This suggests that the magnitude of conformational entropy isbeing correctly established     

Trp59 to Tyr substitution enhances the catalytic activity of RNase T1 and of the Tyr to Trp variants in positions 24, 42 and 45

Using point mutated overproducing strains of E.coli, ribonucleaseT1 was prepared with the single substitutions Tyr24Trp, Tyr42Trp,Tyr45Trp or Trp59Tyr and the corresponding double substitutionsTyr24Trp/Trp59Tyr, Tyr42Trp/Trp59Tyr and Tyr45Trp/Trp59Tyr.Steady state kinetics of the transesterification reaction forthe two dinucleoside monophosphate substrates guanylyl-3', 5'-cytidineand guanylyl-3', 5'-adenosine indicate that the tryptophan canbe introduced in different positions within the ribonucleaseT1 molecule without abolishing enzymatic activity. The Trp59Tyrexchange even enhances catalysis of the cleavage reaction (k_cat/K_m)relative to the wild type enzyme and similar effects are foundwith single tyrosine to tryptophan substitutions. For the pHdependencies of the guanylyl-3', 5'-cytidine transesterificationreaction of wild type ribonuclease T1 and of the variants, typicallybell-shaped curves are observed with a plateau in the rangepH 4.5–7.0. Their shapes and slopes indicate that theenzymes are comparable in their macroscopic pK_a, values. AtpH 7.5, the variant Tyr45Trp/Trp59Tyr shows a more than 3-foldhigher transesterification activity for guanylyl-3', 5'-adenosineand a 2-fold increase for guanylyl-3', 5'-cytidine comparedto the wild type enzyme, i.e. this variant catalyses the transesterificationof the substrate guanylyl-3', 5'-adenosine with the same orbetter efficiency as guanylyl-3', 5'-cytidine.     

Spectroscopic investigation of structure in octarellin (a de novo protein designed to adopt the {alpha}/{beta}-barred packing)

We present here a spectroscopic structural characterizationof octarellin, a recently reported de novo protein modelledon /ß-barrel proteins [K. Go raj, A.Renard and J.A.Martial(1990) Protein Engng, 3, 259–266]. Infrared and Ramanspectra analyses of octarellin‘s secondary structure revealthe expected percentage of -helices (30%) and a higher ß-sheetcontent (40%) than predicted from the design. When the Ramanspectra obtained with octarellin and native triosephosphateisomerase (a natural /ß-barrel) are compared, similarpercentages of secondary structures are found. Thermal denaturationof octarellin monitored by CD confirms that its secondary structuresare quite stable, whereas its native-like tertiary fold is not.Tyrosine residues, predicted to be partially hidden from solvent,are actually exposed as revealed by Raman and UV absorptionspectra. We conclude that the attempted /ß-barrelconformation in octarellin may be loosely packed. The criteriaused to design octarellin are discussed and improvements suggested.     

Stability, activity and flexibility in {alpha}-lactalbumin

-Lactalbumins and the type-c lysozymes are homologues with similarfolds that differ in function and stability. To determine ifthe lower stability of -lactalbumin results from specific substitutionsrequired for its adaptation to a new function, the effects oflysozyme-based and other substitutions on thermal stabilitywere determined. Unblocking the upper cleft in -lactalbuminby replacing Tyr103 with Ala, perturbs stability and structurebut Pro, which also generates an open cleft, is compatible withnormal structure and activity. These effects appear to reflectalternative enthalpic and entropic forms of structural stabilizationby Tyr and Pro. Of 23 mutations, only three, which involve substitutionsfor residues in flexible substructures adjacent to the functionalsite, increase stability. Two are lysozyme-based substitutionsfor Leu110, a component of a region with alternative helix andloop conformations, and one is Asn for Lys114, a residue whosemicroenvironment changes when -lactalbumin interacts with itstarget enzyme. While all substitutions for Leu110 perturb activity,a Lys114 to Asn mutation increases T_m by more than 10°Cand reduces activity, but two other destabilizing substitutionsdo not affect activity. It is proposed that increased stabilityand reduced activity in Lys114Asn result from reduced flexibilityin the functional site of -lactalbumin.     

Structure-function relationships in the catalytic and starch binding domains of glucoamylase

Sixteen primary sequences from five sub-families of fungal,yeast and bacterial glucoamylases were related to structuralinformation from the model of the catalytic domain of Aspergillusawamori var. X100 glucoamylase obtained by protein crystallography.This domain is composed of thirteen -belices, with five conservedregions defining the active site. Interactions between methyl-maltoside and active site residues were modelled, and the importanceof these residues on the catalytic action of different glucoamylaseswas shown by their presence in each primary sequence. The overallstructure of the starch binding domain of some fungal glucoamylaseswas determined based on homology to the Cterminal domains ofBacillus cyclodextrin glucosyltransferases. Crystallographyindicated that this domain contains 6–8 ß-strandsand homology allowed the attribution of a disulfide bridge inthe glucoamylase starch binding domain. Glucoamylase residuesThr525, Asn530 and Trp560, homologous to Bacillus stearothermophiluscyclodextrin glucosyltransferase residues binding to maltosein the Cterminal domain, could be involved in raw-starch binding.The structure and length of the linker region between the catalyticand starch binding domains in fungal glucoamylases can varysubstantially, a further indication of the functional independenceof the two domains.     

Molecular model-building of amylin and {alpha}-calcitonin gene-related polypeptide hormones using a combination of knowledge sources

Amylin is the major component of the amyloid found in the pancreasesof noninsulin-dependent diabetics (type 2 diabetes). It is a37 amino acid polypeptide and has been shown to have 46% sequenceidentity with the neuropeptide -calcitonin gene-related peptide(-CGRP). Both amylin and -CGRP are known to be potent inhibitorsof glycogen synthesis in stripped rat soleus muscle. Secondarystructure prediction and tertiary structure model-building showthe two polypeptides to have an -helix/ß-strand motifsimilar to that observed in the insulin B-chain. The resultshave been supported by CD spectroscopy, although there is nosequence similarity between insulin and amylin/-CGRP. Aggregationstates have been predicted based on the dimeric and hexamericarrangements seen in porcine insulin. Rat and hamster amylinhave a changed sequence motif in the ß-strand whichresults in lack of amyloid formation and type 2 diabetes. This,we propose, is caused by disruption of hydrogen bonding whichprevents the formation of the dimer.     

Roles of catalytic residues in {alpha}-amylases as evidenced by the structures of the product-complexed mutants of a maltotetraose-forming amylase

The crystal structures of the four product-complexed singlemutants of the catalytic residues of Pseudomonas stutzeri maltotetraose-forming-amylase, E219G, D193N, D193G and D294N, have been determined.Possible roles of the catalytic residues Glu219, Asp193 andAsp294 have been discussed by comparing the structures amongthe previously determined complexed mutant E219Q and the presentmutant enzymes. The results suggested that Asp193 predominantlyworks as the base catalyst (nucleophile), whose side chain atomlies in close proximity to the C1-atom of Glc4, being involvedin the intermediate formation in the hydrolysis reaction. WhileAsp294 works for tightly binding the substrate to give a twistedand a deformed conformation of the glucose ring at position–1 (Glc4). The hydrogen bond between the side chain atomof Glu219 and the O1-atom of Glc4, that implies the possibilityof interaction via hydrogen, consistently present throughoutthese analyses, supports the generally accepted role of thisresidue as the acid catalyst (proton donor).