Effect of mutations on the dimer stability and the pH optimum of the human foamy virus protease

To explore the role of residues being close to the catalytic aspartates in the higher pH optimum and in the lower dimer stability of human foamy virus (HFV) protease (PR) in comparison with human immunodeficiency virus type 1 (HIV-1) protease, single (Q8R, H22L, S25T, T28D) and double (Q8R-T28D, H22L-T28D) mutants were created based on sequence alignments and on the molecular model of HFV PR. The wild-type and mutant enzymes were expressed in fusion with maltose binding protein in Escherichia coli and the fusion proteins were purified by affinity chromatography. Specificity constant of most mutants was lower, but the value of Q8R-T28D double mutant enzyme was higher than that of the wild-type HFV PR. Furthermore, urea denaturation at two pH values and pH optimum values showed an increased stability and pH optimum for most mutants. These results suggest that the mutated residues may not be responsible for the higher pH optimum of HFV PR, but they may contribute to the lower dimer stability as compared with that of HIV-1 PR.     

Stability effects associated with the introduction of a partial and a complete Ca2+-binding site into human lysozyme

Two mutants of human lysozyme were synthesized. Mutant A92D,in which Ala92 was substituted by Asp, contains a partial Ca²⁺-bindingsite and mutant M4, in which Ala83, Gm86, Asn88 and Ala92 werereplaced by Lys, Asp, Asp and Asp respectively, contains thecomplete Ca -binding site of bovine a-lactalbumin. The Ca²⁺-bindingconstants of wild type human lysozyme and of mutants A92D andM4, measured at 25C and pH 7.5, were 2(1) x 102 M"1, 8(2)x l^M"1 and 9(0.5) x 10* M"1 respectively. Information gatheredfrom mkrocalorimetrk and CD spectro-scopic measurements indicatesthat the conformational changes of the M4 mutant lysozyme, inducedby Ca²⁺ binding, are smaller than those observed for bovinea-lactalbumin and for the Ca²⁺-binding equine lysozyme. At pH4.5, the thermostability of both the apo and Ca²⁺ forms of theA92D human was decreased in comparison with that of native humanlysozyme. In particular, within the apo form of this mutantan a-helix-containing sequence was destabilized. In contrast,at the same pH the thermostability of the apo and Ca²⁺ formsof the M4 mutant lysozyme was increased. The e-ammonium groupof the Lys83 side chain is assumed to be responsible for thestabilization of the apo form of this mutant.     

Improvement of the refolding yield and solubility of hen egg-white lysozyme by altering the Met residue attached to its N-terminus to Ser

When hen egg-white lysozyme was produced in Escherichia coli, it possessed an extra methionine residue at the N-terminus (Met(-1)- lysozyme). The Met(-1)-lysozyme showed a decreased refolding yield and solubility compared with the native hen egg-white lysozyme, as the methionine is a hydrophobic amino acid. A Met(-2)Pro(-1) or Met(-2)Ser(- 1) sequence was introduced at the N-terminus of hen egg-white lysozyme. The methionine residue in these hen egg-white lysozymes was completely removed by methionine aminopeptidase, as expected, since the penultimate residue was proline or serine. From the analyses of solubility, stability and refolding yield, it was found that an extra Ser residue attached to the N-terminus of hen egg-white lysozyme (Ser(- 1)-lysozyme) showed closer characteristics to the native hen egg-white lysozyme than did Met(-1) or an extra Pro residue attached to the N- terminus of hen egg-white lysozyme (Pro(-1)-lysozyme). Moreover, the tertiary conformation of Ser(-1)-lysozyme examined by NMR spectroscopy and its activity were almost identical with those of native hen egg- white lysozyme.      

Are the parameters of various stabilization factors estimated from mutant human lysozymes compatible with other proteins?

The various factors which contribute to protein stability havebeen extensively examined using mutant proteins, but the samekinds of substitutions have given different results dependingon the substitution sites. Recently, the contributions of somestabilization factors have been quantitatively derived as parametersby a unique equation, considering the conformational changesdue to the mutations using mutant human lysozymes [Funahashiet al. (1999) Protein Eng. 12, 841–850]. To evaluate theseparameters estimated from the mutant human lysozymes, stability–structuredatasets for the mutant T4 lysozymes were selected. The stabilitiesfor the mutant T4 lysozymes could be roughly estimated usingthese parameters. Notable differences between the estimatedand experimental stabilities were caused by the uncertaintyin part of the structures due to some Arg and Lys residues fluctuatingon the surface of the T4 lysozyme. Excluding these atoms fromthe estimation gave a good correlation between the estimatedand experimental stabilities. These results suggest that theparameters of the various stabilization factors derived fromthe mutant human lysozymes are compatible with the mutant T4lysozymes, although they should be improved with respect tosome points using more information.     

Reduction of membrane protein hydrophobicity by site-directed mutagenesis: introduction of multiple polar residues in helix D of bacteriorhodopsin

Introduction of polar and charged residues on the lipid-exposed face of transmembrane proteins using site-directed mutagenesis represents a novel approach to render membrane proteins more soluble in aqueous solution. We have sequentially introduced as many as five polar and charged amino acids onto the lipid-exposed face of helix D of bacteriorhodopsin from Halobacterium salinarium. The most polar mutant (Q4D) has four glutamine residues at positions 113, 116, 120 and 124 and an aspartate at position 117. In combination with wild-type residues Gln105, Thr107, Thr121 and Thr128, the Q4D mutant has a nearly uninterrupted stripe of polar residues on the surface of helix D. All of the mutants refold, bind retinal and the resulting pigments exhibit light- and dark-adapted UV and visible spectroscopic properties that are similar to the wild-type pigment, indicating that the secondary, tertiary and active site structures are similar to the wild-type protein. These results demonstrate that micelle-solubilized bacteriorhodopsin can tolerate multiple non-conservative substitution of amino acids that face the non-polar portion of the lipid bilayer in vivo, thus lending credence to the notion of partial or complete solubilization of integral membrane proteins by site-directed mutagenesis.      

Use of site-directed mutagenesis to obtain isomorphous heavy-atom derivatives for protein crystallography: cysteine-containing mutants of phage T4 lysozyme

Five different cysteine-containing mutants of the lysozyme frombacteriopbage T4 were used to explore the feasibility of usingsite-directed mutagenesis to generate isomorphous heavy-atomderivatives for protein crystallography. Cysteines 54 and 97,present in wild-type lysozyme, can be readily reacted with mercuricion to produce an excellent isomorphous heavy-atom derivative.Mutants with an additional cysteine at position 86,146,153 or157, or with Cys 97 replaced by Val, were engineered by site-directedmutagenesis. The mutant lysozyme Thr 157 - Cys reacts with mercuricchloride to give an excellent new derivatve although Cys 157is only -60% substituted with the heavy atom. The cysteine atposition 146 is largely buried but reacts readily with mercuricchloride. In this case the isomorphism is poor and the resultantderivative is of marginal quality. Cys 153 reacts rapidly withmercuric ion but the derivative crystals do not diffract. Themutant Pro 86 - Cys does not yield a particularly good heavy-atomderivative. This is due in part to a loss of isomorphism associatedwith the mutation. In addition, Cys 86 shows very little reactivitytowards mercurials even though it is fully exposed to solvent.The mutation Cys 97 Val was used to explore the possibilityof creating an independent derivative by deleting a heavy-atomsite already present in wild-type lysozyme. In all cases thatwere tested, the quality of the heavy-atom derivative was improvedby using as an isomorphous pair mercury-substituted mutant versusnon-substituted mutant rather than mercury-substituted mutantversus (non-substituted) wild-type lysozyme. Unexpectedly, thecysteines that are most exposed to solvent and most mobile areleast reactive toward mercuric chloride. The cysteines thatprovide the best heavy-atom sites are those that are locatedin surface crevices and are only partly exposed to solvent.     

Relationship between local structure and stability in hen egg white lysozyme mutant with alanine substituted for glycine

We prepared five mutant lysozymes in which glycines whose dihedralangles are located in the region of the left-handed helix, Gly49,Gly67, Gly71, Gly102 and Gly117, were mutated to an alanineresidue. From analyses of their thermal stabilities using differentialscanning calorimetry, most of them were more destabilized thanthe native lysozyme, except for the G102A mutant, which hasa stability similar to that of the native lysozyme at pH 2.7.As for the destabilized mutant lysozymes, their X-ray crystallographicanalyses showed that their global structures did not changebut that the local structures changed slightly. By examiningthe dihedral angles at the mutation sites based on X-ray crystallographicresults, it was found that the dihedral angles at these mutationsites tended to adopt favorable values in a Ramachandran plotand that the extent and direction of their shifts from the originalvalue had similar tendencies. Therefore, the change in dihedralangles may be the cause of the slight local structural changesaround the mutation site. On the other hand, regarding the mutationof G102A, the global structure was almost identical with thatof the native structure but the local structure was drasticallychanged. Therefore, it was suggested that the drastic localconformational change might be effective in releasing the unfavorableinteraction of the native state at the mutation site.     

Lysozyme requires fluctuation of the active site for the manifestation of activity

Mutations around His15 which lie far away from the active site,stimulated glycol chitin activity of lysozyme at physiologicaltemperature. Del-Argl4Hisl5 lysozyme, a mutant lysozyme whoseArgl4 and Hisl5 were deleted together, and has the highest activityamong these mutant lysozymes, had a similar binding abilityto a trimer of N-acetyl-glucosamine, a substrate analogue, relativeto native lysozyme. This suggests that the increased activitywas due to an increased k_cat in the catalysis reaction. TheH-D exchange rate of the N-1 proton in the Trp63 which is locatedin the active site cleft, was enhanced in the Del-Argl4Hisl5lysozyme, while 2-D proton NMR analysis revealed no conformationalchange around Trp63. We conclude that some sort of fluctuationat the active site might be required for the manifestation ofactivity. This theory is supported by the finding that the Del-Argl4Hisl5lysozyme showed a shift in temperature dependency of activityto lower temperatures compared with that of native lysozyme.     

The role of net charge on the renaturation of reduced lysozyme by the sulfhydryl-disulfide interchange reaction

Reduced and acetylated lysozymes are basic proteins. When theiramino groups were variously acetylated and then renatured bysulfhydryl-disulfide (SH-SS) interchange reaction at pH 8.0,the final folding yield decreased as the number of positivecharges decreased. The final folding yield of native and Ac1lysozyme, with one positive charge eliminated, was less sensitiveto increasing protein concentration than that of Ac2 lysozyme,where two positive charges had been eliminated. The final foldingyield of reduced Ac2 lysozyme increased in the presence of 1M urea, which reduced the aggregation of unfolded lysozyme.Thus, the aggregation of unfolded lysozymes, which leads toa decrease in the final folding yield, was found to be heavilydependent on their net charges. Moreover, the final foldingyield of reduced lysozyme was shown to be increased by use ofcystamine as an oxidizing reagent in comparison with 2-hydroxyethyldisulfide or dithiodiglycolic acid. This may support the ideathat the final folding yield is influenced by electrostaticinteraction between unfolded lysozymes in the early stage ofrenaturation. In contrast, the concentration dependency of thefinal folding yield of Ac1 lysozyme was different from thoseof carboxymethylated His15 and Asp106 lysozymes whose positivenet charges were similar to that of Ac1 lysozyme. On the basisof the observations, it is suggested that the formation of theaggregates in the renaturation process might also be affectedby the structure of the unfolded state of lysozyme in solution.     

Expression of a synthetic gene encoding canine milk lysozyme in Escherichia coli and characterization of the expressed protein

A high-expression plasmid of the canine milk lysozyme, whichbelongs to the family of calcium-binding lysozymes, was constructedin order to study its physico-chemical properties. Because thecDNA sequence of the protein has not yet been determined, a400 base-pair gene encoding canine milk lysozyme was first designedon the basis of the known amino acid sequence. The gene wasconstructed by an enzymatic assembly of 21 chemically synthesizedoligonucleotides and inserted into an Escherichia coli expressionvector by stepwise ligation. The expression plasmid thus constructedwas transformed into BL21(DE3)/pLysS cells. The gene productaccumulated as inclusion bodies in an insoluble fraction. Recombinantcanine milk lysozyme was obtained by purification and refoldingof the product and showed the same characteristics in termsof bacteriolytic activity and far- and near-UV circular dichroismspectra as the authentic protein. The NMR spectra of refoldedlysozyme were also characteristic of a native globular protein.It was concluded that recombinant canine milk lysozyme was foldedinto the correct native structure. Moreover, the thermal unfoldingprofiles of the refolded recombinant lysozyme showed a stableequilibrium intermediate, indicating that the molten globulestate of this protein was extraordinarily stable. This expressionsystem of canine milk lysozyme will enable biophysical and structuralstudies of this protein to be extended.     

CTAB辅助溶菌酶复性过程动力学

研究了溶菌酶在十六烷基三甲基溴化铵（CTAB）溶液中的复性过程,通过测定溶液表面张力与酶活力的变化证实变性溶菌酶首先与CTAB形成复合物,进而在氧化-还原剂作用下开始复性并与CTAB发生解离.非还原型SDS-PAGE分析结果表明,复性反应的主要产物有三类：具有天然结构的溶菌酶单体、溶菌酶多聚体及溶菌酶单体与CTAB形成的无活性复合物.不同产物的含量取决于溶液中CTAB与溶菌酶的摩尔比.当变性溶菌酶浓度为0.1～0.4mg•ml^-1,CTAB与溶菌酶摩尔比为5～20时,采用“变性-复性”二态复性模型分析了CTAB辅助溶菌酶复性过程的宏观动力学.结果表明,随CTAB与溶菌酶摩尔比的增大,变性反应的速率常数显著增大,而折叠反应的速率常数先增大后减小,CTAB与溶菌酶的摩尔比为10时,复性率最高.而蛋白质浓度提高则导致折叠反应速率常数减小,变性反应速率常数增大,复性率缓慢下降.     

分子伴侣GroEL促进溶菌酶复性动力学

建立了溶菌酶复性的表观动力学模型 ,研究了分子伴侣GroEL促进变性溶菌酶复性的动力学行为 ,包括酶浓度、三磷酸腺苷 (ATP)浓度、GroEL与酶的摩尔比对复性率和复性速率常数的影响 .酶浓度对复性速率常数的影响比对复性率的影响显著 ;酶的复性率和复性速率常数随ATP浓度的增大而提高 ;在蛋白质浓度一定的情况下 ,存在适宜的GroEL和ATP浓度 ,使复性率和复性速率常数最大     

利用体积排阻色谱法进行蛋白质折叠

以溶菌酶为模拟体系对体积排阻色谱法进行蛋白质折叠过程实验研究 .圆二色性光谱法分析结果证实了复性溶菌酶与天然溶菌酶的二级结构一致性 ;复性溶菌酶与天然溶菌酶色谱保留体积的差异揭示出折叠过程中无活性蛋白质聚集体的存在及其向复性蛋白质转化的机制 ;不同初始浓度的复性实验证实了蛋白质聚集体的存在及其与变性蛋白质初始浓度的关系 ;采用短色谱柱的折叠分离实验结果表明蛋白质折叠是一个快速过程 ;不同尿素浓度下的折叠分离实验结果表明尿素在SEC法中具有非常重要的作用 .与稀释复性法的对比实验表明 :体积排阻色谱法具有稀释倍数小、复性产品活性收率高、复性蛋白质浓度高等优点 .     

高浓度变性-还原溶菌酶的流加复性动力学特性

研究了高浓度变性-还原溶菌酶的流加复性过程动力学特性,重点考察了影响复性速率和复性收率的主要因素,包括盐酸胍浓度、酶浓度和氧化还原剂浓度.结果表明,与直接稀释复性相比,流加操作可有效降低肽链分子间聚集,提高蛋白质的复性收率;在流加复性条件下,随着酶浓度的提高,复性速率和复性收率均有所下降,但适当提高复性液中盐酸胍浓度仍可获得高浓度蛋白质的高复性收率.另外,随着变性酶浓度的提高,需要适当提高复性液中氧化型谷胱甘肽浓度,以加快溶菌酶分子内二硫键的形成,提高复性反应速度.     

Tolerance of point substitution of methionine for isoleucine in hen egg white lysozyme

X-ray structure determination of proteins by using the multiple-wavelengthanomalous dispersion method targeting selenomethionine is nowwidely employed. Isoleucine was examined for the second choiceof the `safe' substitution of methionine next to leucine. Weperformed a systematic mutational study of the substitutionsof methionine for isoleucine. All mutated lysozymes were lessstable than the wild-type by about 1 kcal/mol and it is suggestedthat this instability was caused by the change in residual hydrophobicityfrom isoleucine to methionine. The X-ray structures of all mutantlysozymes were very similar to that of the wild-type. In addition,both the accessible surface areas and the conformation of theside chain of methionine in all mutant lysozymes were similarto those of the side chain at the respective isoleucine in thewild-type. Therefore, it is suggested that the mutation fromisoleucine to methionine in a protein can be considered as a`safe' substitution.     

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

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

The spontaneous gating activity of OmpC porin is affected by mutations of a putative hydrogen bond network or of a salt bridge between the L3 loop and the barrel

Porins are trimeric channel-forming proteins of the outer membrane of Escherichia coli. Each subunit contains 16 beta-strands forming a transmembrane beta-barrel whose pore is constricted by the third extracellular loop (L3). We investigated the effects of site-directed mutations at two critical regions of the OmpC porin: (i) the D315A mutation targets a key component of a putative hydrogen bond network linking the L3 loop to the adjacent barrel wall and (ii) the D118Q, R174Q and R92Q mutations target putative salt bridges at the root of the L3 loop. We purified the outer membrane fractions obtained from each mutant and reconstituted them in liposomes suitable for electrophysiology. Patch clamp experiments showed that the frequency of spontaneous transitions between open and closed states is increased in the D315A, D118Q and R92Q mutants but unchanged in the R174Q mutant. These transitions are not driven by transmembrane voltage changes and represent the thermal oscillations between functionally distinct conformations. The asymmetric voltage-dependent inactivation of the channels is not affected by the mutations, however, suggesting different molecular mechanisms for the spontaneous and voltage- dependent gating processes. We propose that the positioning or flexibility of the L3 loop across the pore, as governed by the putative hydrogen-bond network and a salt bridge, play a role in determining the frequency of spontaneous channel gating.      

Evidence for an initiation site for hen lysozyme folding from the reduced form using its dissected peptide fragments

We prepared two dissected fragments of hen lysozyme and examinedwhether or not these two fragments associated to form a native-likestructure. One (Fragment I) is the peptide fragment Asn59–homoserine-105containing Cys64–Cys80 and Cys76–Cys94. The other(Fragment II) is the peptide fragment Lys1–homoserine-58connected by two disulfide bridges, Cys6–Cys127 and Cys30–Cys115,to the peptide fragment Asn106–Leu129. It was found thatthe Fragment I immobilized in the cuvette formed an equimolarcomplex with Fragment II (K_d = 3.3x10^–4 M at pH 8 and25°C) by means of surface plasmon resonance. Moreover, fromanalyses by circular dichroism spectroscopy and ion-exchangechromatography of the mixture of Fragments I and II at pH 8under non-reducing conditions, it was suggested that these fragmentsassociated to give the native-like structure. However, the mutantFragment I in which Cys64–Cys80 and Cys76–Cys94are lacking owing to the mutation of Cys to Ala, or the mutantfragment in which Trp62 is mutated to Gly, did not form thenative-like species with Fragment II, because the mutant FragmentI derived from mutant lysozymes had no local conformation dueto mutations. Considering our previous results where the preferentialoxidation of two inside disulfide bonds, Cys64–Cys80 andCys76–Cys94, occurred in the refolding of the fully reducedFragment I, we suggest that the peptide region correspondingto Fragment I is an initiation site for hen lysozyme folding.     

甜菜碱对溶菌酶去折叠热力学和复性动力学的影响

应用荧光分析技术考察了甜菜碱对溶菌酶在盐酸胍溶液中稳定性的影响;并提出将甜菜碱作为添加剂应用于彻底变性还原溶菌酶的复性;同时应用表观竞争反应动力学模型分析了不同浓度甜菜碱对溶菌酶复性动力学的影响特性。结果表明,甜菜碱对溶菌酶具有稳定作用,可以作为添加剂促进变性还原溶菌酶的复性。甜菜碱可以抑制复性过程溶菌酶分子间的聚集,同时提高溶菌酶的复性速率,从而提高溶菌酶的复性收率。     

The role of cysteine oxidation in the thermal inactivation of T4 lysozyme

Wild-type T4 lysozyme contains unpaired cysteine residues atpositions 54 and 97. To investigate the role these residuesplay in the thermal inactivation of the wild-type, we constructeda double mutant with these cysteines replaced with valine andserine. This molecule, T4 lysozyme (C54V/C97S), is more stablethan the wild-type to inactivation at 70°C at pH 6.5 and8.0. Guanidine hydrochloride reactivation experiments and SDS-PAGEon the inactivated products show that the wild-type is susceptibleto varying degrees of oxidative damage, depending on bufferconditions, while the cysteine-minus mutant inactivates onlyby other pathways. The products of thermal, oxidative inactivationof the wild-type are disulfide-linked oligomers. The dependenceof inactivation rate on temperature suggests that the formationof these aggregates depends on prior thermal unfolding of theT4 lysozyme molecule.