The distinct heme coordination environments and heme-binding stabilities of His39Ser and His39Cys mutants of cytochrome b5

A gene mutant library containing 16 designed mutated genes atHis39 of cytochrome b₅ has been constructed by using gene randommutagenesis. Two variants of cytochrome b₅, His39Ser and His39Cysmutant proteins, have been obtained. Protein characterizationsand reactions were performed showing that these two mutantshave distinct heme coordination environments: ferric His39Sermutant is a high-spin species whose heme is coordinated by proximalHis63 and likely a water molecule in the distal pocket, whileferrous His39Ser mutant has a low-spin heme coordinated by His63and Ser39; on the other hand, the ferric His39Cys mutant isa low-spin species with His63 and Cys39 acting as two axialligands of the heme, the ferrous His39Cys mutant is at high-spinstate with the only heme ligand of His63. These two mutantswere also found to have quite lower heme-binding stabilities.The order of stabilities of ferric proteins is: wild-type cytochromeb₅ >> His39Cys > His39Ser. Received April 11, 2003; revised October 13, 2003; accepted October 23, 2003     

Photo-control of peptide helix content by an azobenzene cross-linker: steric interactions with underlying residues are not critical

Photo-control of protein conformation could prove useful forprobing function in diverse biological systems. Recently, wereported photo-switching of helix content in a short peptidecontaining an azobenzene cross-linker between cysteine residuesat positions i and i + 7 in the sequence. In the original sequence,underlying residues at positionsi + 3 and i + 4 were made bulkyas preliminary modelling suggested that this would enhance photo-controlof helix content. To test this hypothesis, peptides with Val,Aib; Ile, Aib; and Ala, Ala at positions i + 3 and i + 4 weresynthesized, cross-linked and characterized. Before cross-linking,the peptides show distinct conformational behaviours: two withdiffering helix/coil mixtures whereas the other has a circulardichroism (CD) spectrum characteristic of ß-sheetand a tendency to aggregate. However, upon cross-linking thepeptides have very similar CD spectra: predominantly randomcoil in the dark but predominantly helical upon irradiation.These results refute the original hypothesis. Steric interactionsbetween the linker and underlying residues do not appear tobe critical for photo-switching behaviour. When the cross-linkingbridge is lengthened by replacing the i, i + 7 cysteine residueswith homocysteine, a lower degree of photo-control of helicityis observed. Furthermore, a non-cross-linking version of theazobenzene reagent is shown not to produce any photo-controlof helicity. We conclude that the intramolecular cross-linkis essential for photo-switching and that it should be applicableto a wide range of peptides and proteins.     

Generation of a broad esterolytic subtilisin using combined molecular evolution and periplasmic expression

Concomitant activity improvement of an evolved enzyme towardtwo very different ester substrates was achieved when a uniquecombination of functional periplasmic enzyme expression in Escherichiacoli, random mutagenesis, DNA shuffling and cell-based kineticscreenings was applied. Specifically, we focused on the conversionof subtilisin E into an enzyme with broader esterase activityas opposed to its native amidase activity. Cell-based microtiterassays were performed on N-acetyl-D,L-phenylalanine p-nitrophenylester (Phe-NPE) and sucrose 1'-adipate (S1'A), as well as onthe tetrapeptide amide substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide.After a single modified cycle of directed molecular evolution,we isolated a number of clones exhibiting increased activitytoward Phe-NPE. In the following rounds of screenings, mutantswith improved activity on Phe-NPE were also tested on S1'A.Three mutants were identified with increased esterolytic activityon Phe-NPE and S1'A, while having similar amidase activity tothat of the parental enzymes. Because the two ester substratesare structurally distinct, we have evolved a more general esterolyticsubtilisin and this may have important applications in synthesis.     

An internal cellulose-binding domain meidates adsorption of an engineered bifunctional xylanase/cellulase

A chimeric xylanase/endoglucanase (XynCenA) with an internalcellulose-binding domain was constructed by fusing the Bacillussubtilis xyn gene fragment to the 5'-end of the Cellulomonasfimi cenA. A polyhistidine-encoding sequence was also fusedto the 5'-end of the xyn gene. The gene fusion was overexpressedin Escherichia coli and the fusion poly-peptide purified fromthe cell extracts using the polyhistldine tail. The hybrid proteinbehaved like the parental endo-glucanase or xylanase when assayedon a number of soluble and insoluble cellulosic substrates orxylans. The presence of two distinct active sites and the internalcellulose-binding domain did not significantly affect the hydrolysisof any of these substrates. However, the fusion protein exhibiteda strong affinity for both mkrocrystaUine cellulose (Avicel)and regenerated chitin. Like the parental endoglucanase, boundXynCenA could not be duted from these porysaccharides with eitherlow or high salt buffer or distilled water. More stringent conditions,such as 1% SDS or 8 M guanidinium hydro-chloride, fully desorbedthe protein. The fusion protein did not adsorb significantlyto insoluble xylan     

Identification of sequence motifs from a set of porteins with related function

The automatic identification of motifs associated with a givenfunction is an important challenge for molecular sequence analysis.A method is presented for the extraction of such patterns fromlarge sets of unaligned sequences with related but general function,for example, a set of heat shock proteins. In such a set ofproteins there can often be several subfamilies each characterizedby one or more distinct motifs. The aim is to develop computationaltools to identify these motifs. The algorithm presented locateshigh frequency words of length k with a given number of positions,r, fixed. Statistics for a binomial distribution are used toassess the significance of the words. The high-frequency wordsare clustered and highly populated clusters retained. The compositionof the clusters is displayed graphically. A set of motifs associatedwith the sequence family can automatically be extracted. Themethod is benchmarked on a set of 106 heat shock sequences anda set of 257 toxin sequences. It is shown to recover previouslyidentified motifs.     

Molecular modeling of coiled-coil {alpha}-tropomyosin: analysis of staggered and in register helix--helix interactions

In register and staggered models of tropomyosin coiled-coilwere built from X-ray C coordinates and refined via moleculardynamics. The two models show similar structural features withthe X-ray structure of GCN4 leucine zipper. Empirical energeticmethods used to compare the in register and staggered modelsindicate that both are equally probable. The two models havesimilar profiles of solvation free energy of folding for residuesat positions a and d of the repeating heptad, indicating thatresidues at these positions are as well buried in an in registerstructure as in a staggered one. Neither the in register northe 14 residues staggered structure can be ruled out based onhydrophobic or e–g' (g–e') electrostatic interactionswhich are not able to distinguish between the two models andare therefore not selective. However, the eg–b'c' electrostaticinteractions, although smaller in magnitude, are in favor ofthe in register model. Furthermore, analysis of hydrophobicand electrostatic interactions along the tropomyosin sequenceshows that bulky residues in positions a and d prevent the formationof inter-chain salt bridges.     

Design of a monomeric human glutathione transferase GSTP1, a structurally stable but catalytically inactive protein

By the introduction of 10 site-specific mutations in the dimerinterface of human glutathione transferase P1-1 (GSTP1-1), astable monomeric protein variant, GSTP1, was obtained. The monomerhad lost the catalytic activity but retained the affinity fora number of electrophilic compounds normally serving as substratesfor GSTP1-1. Fluorescence and circular dichroism spectra ofthe monomer and wild-type proteins were similar, indicatingthat there are no large structural differences between the subunitsof the respective proteins. The GSTs have potential as targetsfor in vitro evolution and redesign with the aim of developingproteins with novel properties. To this end, a monomeric GSTvariant may have distinct advantages.     

Analysis of membrane stereochemistry with homology modeling of sn-glycerol-1-phosphate dehydrogenase

Different enantiomeric isomers, sn-glycerol-1-phosphate andsn-glycerol-3-phosphate, are used as the glycerophosphate backbonesof phospholipids in the cellular membranes of Archaea and theremaining two kingdoms, respectively. In Archaea, sn-glycerol-1-phosphatedehydrogenase is involved in the generation of sn-glycerol-1-phosphate,while sn-glycerol-3-phosphate dehydrogenase synthesizes theenantiomer in Eukarya and Bacteria. The coordinates of sn-glycerol-3-phosphatedehydrogenase are available, although neither the tertiary structurenor the reaction mechanism of sn-glycerol-1-phosphate dehydrogenaseis known. Database searching revealed that the archaeal enzymeshows sequence similarity to glycerol dehydrogenase, dehydroquinatesynthase and alcohol dehydrogenase IV. The glycerol dehydrogenase,with coordinates that are available today, is closely relatedto the archaeal enzyme. Using the structure of glycerol dehydrogenaseas the template, we built a model structure of the Methanothermobacterthermautotrophicus sn-glycerol-1-phosphate dehydrogenase, whichcould explain the chirality of the product. Based on the modelstructure, we determined the following: (1) the enzyme requiresa Zn²⁺ ion for its activity; (2) the enzyme selectively usesthe pro-R hydrogen of the NAD(P)H; (3) the putative active siteand the reaction mechanism were predicted; and (4) the archaealenzyme does not share its evolutionary origin with sn-glycerol-3-phosphatedehydrogenase.     

25℃下Na+,K+,Mg2+//Cl-,NO-3-H2O五元体系液固相平衡

The solubilities of the quinary system Na + ,K + ,Mg 2+ //Cl ,NO 3 -H2O and its two quaternary subsystems, Na + ,K + ,Mg 2+ //NO 3 -H2O and K + ,Mg 2+ //Cl ,NO 3 -H2O,were studied by isothermal method at 25°C and their phase diagrams were plotted.In the equilibrium phase diagram of quaternary system Na + ,K + ,Mg 2+ //NO 3 -H2O, there are one invariant point,three univariant curves and three regions of crystallization with one salt:NaNO3, KNO3 and Mg(NO3)2·6H2O.In the equilibrium phase diagram of quaternary system K + ,Mg 2+ //Cl ,NO 3 -H2O,there are three invariant points,seven univariant curves and five regions of crystallization with one salt:KNO3,KCl, Mg(NO3)2·6H2O,MgCl2·6H2O and KCl·MgCl2·6H2O.In the equilibrium phase diagram of the quinary system Na + , K + ,Mg 2+ //Cl ,NO 3 -H2O,there are four invariant points,and seven regions of crystallization with one salt:NaCl, KCl,NaNO3,KCl·MgCl2·6H2O,KNO3,MgCl2·6H2O and Mg(NO3)2·6H2O.     

Design and characterization of a soluble fragment of the extracellular ligand-binding domain of the peptide hormone receptor guanylyl cyclase-C

The intestinal guanylyl cyclase-C (GC-C) was originally identifiedas an Escherichia coli heat-stable enterotoxin (STa) receptor.STa stimulates GC-C to much higher activity than the endogenousligands guanylin and uroguanylin, causing severe diarrhea. Toinvestigate the interactions of the endogenous and bacterialligands with GC-C, we designed and characterized a soluble andproperly folded fragment of the extracellular ligand-bindingdomain of GC-C. The membrane-bound guanylyl cyclases exhibita single transmembrane spanning helix and a globularly foldedextracellular ligand-binding domain that comprises about 410of 1050 residues. Based on the crystal structure of the dimerized-bindingdomain of the guanylyl cyclase-coupled atrial natriuretic peptidereceptor and a secondary structure-guided sequence alignment,we generated a model of the extracellular domain of GC-C comprisedof two subdomains. Mapping of mutational and cross-link dataonto this structural model restricts the ligand-binding regionto the membrane proximal subdomain. We thus designed miniGC-C,a 197 amino acid fragment that mimics the ligand-binding membraneproximal subdomain. Cloning, expression and spectroscopic studiesreveal miniGC-C to be a soluble and properly folded proteinwith a distinct secondary and tertiary structure. MiniGC-C bindsSTa with nanomolar affinity.     

Minor structural consequences of alternative CUG codon usage (Ser for Leu) in Candida albicans exoglucanase

In some species of Candida the CUG codon is encoded as serineand not leucine. In the case of the exo-ß-1,3-glucanasefrom the pathogenic fungus C.albicans there are two such translationalevents, one in the prepro-leader sequence and the other at residue64. Overexpression of active mature enzyme in a yeast host indicatedthat these two positions are tolerant to substitution. By comparingthe crystal structure of the recombinant protein with that ofthe native (presented here), it is seen how either serine orleucine can be accommodated at position 64. Examination of therelatively few solved protein structures from C.albicans indicatesthat other CUG encoded serines are also found at non-essentialsurface sites. However such codon usage is rare in C.albicans,in contrast to C.rugosa, with direct implications for respectiverecombinant protein production.     

Cassette mutagenesis of Aspergillus awamori glucoamylase near its general acid residue to probe its catalytic and pH properties

Nine single amino add mutations in the active site of Aspergillusawamori glucoamylase were made by cassette mutagenesis to alterthe pH dependence of the enzyme and to determine possible functionsof the mutated residues. The Glul79-Asp mutation expressed inyeast led to a very large decrease in k_cat but to no changein K_m, verifying this residue's catalytic function. Aspl76-Gluand Glul80-Asp mutations affected K_m a more than k_cat, implyingthat Aspl76 and Glul80 are involved in substrate binding orstructural integrity. The Leul77-Asp mutation decreased k_catonly moderately, probably by changing the position of the generalacid catalytic group, and did not affect K_m. The Trpl78-Aspmutation greatly decreased k_cat while increasing K_m, showingthe importance of Trpl78 in the active site. Vall81-Asp andAsnl82-Asp mutations changed kinetk values little, suggestingthat Vall81 and Asnl82 are of minor catalytic and structuralimportance. Finally, insertions of Asp or Gly between residues176 and 177 resulted in almost complete loss of activity, probablycaused by destruction of the active site structure. No largechanges in pH dependence occurred in those mutations where kineticvalues could be determined, in spite of the increase in mostcases of the total negative charge. Increases in activationenergy of maltoheptaose hydrolysis in most of the mutant glucoamylasessuggested cleavage of individual hydrogen bonds in enzyme-substratecomplexes.     

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.     

Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface

Polypeptide library screening technologies are critically dependentupon the characteristics of the expression system employed.A comparative analysis of the lpp–lac, tet and araBAD promoterswas performed to determine the importance of tight regulationand expression level in library screening applications. Thesurface display of single-chain antibody (scFv) in Escherichiacoli as an Lpp–OmpA' fusion was monitored using a fluorescentlytagged antigen in conjunction with flow cytometry. In contrastto the lpp–lac promoter, both tet and araBAD promoterscould be tightly repressed. Tight regulation was found to beessential for preventing rapid depletion of library clones expressingfunctional scFv and thus for maintaining the initial librarydiversity. Induction with subsaturating inducer concentrationsyielded mixed populations of uninduced and fully induced cellsfor both the tet and araBAD expression systems. In contrast,homogeneous expression levels were obtained throughout the populationusing saturating inducer concentrations and could be adjustedby varying the induction time and plasmid copy number. Underoptimal induction conditions for the araBAD system, proteinexpression did not compromise either cell viability or librarydiversity. This expression system was used to screen a libraryof random scFv mutants specific for digoxigenin for clones exhibitingimproved hapten dissociation kinetics. Thus, an expression systemhas been developed which allows library diversity to be preservedand is generally applicable to the screening of E.coli surfacedisplayed libraries.     

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.     

Staphylococcal lactose phosphoenolpyruvate-dependent phosphotransferase system: site-specific mutagenesis on the lacE gene gives evidence that a cysteine residue is responsible for phosphorylation

The lactose-specific phosphocarrier protein enzyme II of thebacterial phosphoenol-pymvate-dependent phosphotransferase systemof Staphylococcus aureus was modified by sitespecific mutagenesison the corresponding lacE gene in order to replace the histidineresidues 245, 274 and 510 and the cysteine residue 476 of theamino acid sequence with a serine residue. The wild-type andmutant genes were expressed in Escherichia coli and the geneproducts were characterized in different in vitro test systems.In vitro phosphorylation studies on mutant derivatives of thelactose-specific enzyme II led to the conclusion that cysteinersidue 476 is the active-site for phosphorylation of this enzymeII by a phospho-enzyme III of the same sugar specificity. Acysteine residue phosphor) lated intermediate was first postulatedfor the mannitol-specific enzyme II of E.coli and studies performedindependently concerning the lactose-specific enzyme II of Lactobacilluscasei are in agreement with the above results.     

Heat labile ribonuclease HI from a psychrotrophic bacterium: gene cloning, characterization and site-directed mutagenesis

The rnhA gene encoding RNase HI from a psychrotrophic bacterium,Shewanella sp. SIB1, was cloned, sequenced and overexpressedin an rnh mutant strain of Escherichia coli. SIB1 RNase HI iscomposed of 157 amino acid residues and shows 63% amino acidsequence identity to E.coli RNase HI. Upon induction, the recombinantprotein accumulated in the cells in an insoluble form. Thisprotein was solubilized and purified in the presence of 7 Murea and refolded by removing urea. Determination of the enzymaticactivity using M13 DNA–RNA hybrid as a substrate revealedthat the enzymatic properties of SIB1 RNase HI, such as divalentcation requirement, pH optimum and cleavage mode of a substrate,are similar to those of E.coli RNase HI. However, SIB1 RNaseHI was much less stable than E.coli RNase HI and the temperature(T_1/2) at which the enzyme loses half of its activity upon incubationfor 10 min was ~25°C for SIB1 RNase HI and ~60°C forE.coli RNase HI. The optimum temperature for the SIB1 RNaseHI activity was also shifted downward by 20°C compared withthat of E.coli RNase HI. Nevertheless, SIB1 RNase HI was lessactive than E.coli RNase HI even at low temperatures. The specificactivity determined at 10°C was 0.29 units/mg for SIB1 RNaseHI and 1.3 units/mg for E.coli RNase HI. Site-directed mutagenesisstudies suggest that the amino acid substitution in the middleof the     

Engineering the active center of the 6-phospho-{beta}-galactosidase from Lactococcus lactis

Several amino acids in the active center of the 6-phospho-ß-galactosidasefrom Lactococcus lactis were replaced by the corresponding residuesin homologous enzymes of glycosidase family 1 with differentspecificities. Three mutants, W429A, K435V/Y437F and S428D/K435V/Y437F, were constructed. W429A was found to have an improvedspecificity for glucosides compared with the wild-type, consistentwith the theory that the amino acid at this position is relevantfor the distinction between galactosides and glucosides. Thek_cat/K_m for o-nitrophenyl-ß-D-glucose-6-phosphate is 8-foldhigher than for o-nitrophenyl-ß-D-galactose-6-phosphatewhich is the preferred substrate of the wild-type enzyme. Thissuggests that new hydrogen bonds are formed in the mutant betweenthe active site residues, presumably Gln19 or Trp421 and theC-4 hydroxyl group. The two other mutants with the exchangesin the phosphate-binding loop were tested for their abilityto bind phosphorylated substrates. The triple mutant is inactive.The double mutant has a dramatically decreased ability to bindo-nitrophenyl-ß-D-galactose-6-phosphate whereas the interactionwith o-nitrophenyl-ß-D-galactose is barely altered. Thisresult shows that the 6-phospho-ß-galactosidase and therelated cyanogenic ß-glucosidase from Trifolium repenshave different recognition mechanisms for substrates althoughthe structures of the active sites are highly conserved.     

Expression and characterization of Lactobacillus 30a histidine decarboxylase in Escherichia coli

The genes coding for histidine decarboxylase from a wild-typestrain and an autoactivation mutant strain of Lactobacillus30a have been cloned and expressed in Escherichia coli. Themutant protein, G58D, has a single Asp for Gly substitutionat position 58. The cloned genes were placed under control ofthe ß-galactosidase promoter and the products arenatural length, not fusion proteins. The enzyme kinetics ofthe proteins isolated from E. coli are comparable to those isolatedfrom Lactobacillus 30a. At pH 4.8 the K_m of wild-type enzymeis 0.4 mM and the k_cat = 2800 min^–1; the correspondingvalues for G58D are 0.5 mM and 2750 min^–1. The wild-typeand G58D have autoactivation half-times of 21 and 9 h respectivelyunder pseudophysiological conditions of 150 mM K⁺ and pH 7.0.At pH 7.6 and 0.8 M K⁺ the half times are 4.9 and 2.9 h. Therelatively slow rate of autoactivation for purified proteinand the differences in cellular and non-cellular activationrates, coupled with the fact that wild-type protein is readilyactivated in wild-type Lactobacillus 30a but poorly activatedin E. coli, suggest that wild-type Lactobacillus 30a containsa factor, possibly an enzyme, that enhances the activation rate.