Point mutation of alanine (31) to valine prohibits the folding of reduced lysozyme by sulfhydryl--disulfide interchange

In the preceding paper in this issue, we described the overproduction of one mutant chicken lysozyme in Escherichia coil.Since this lysozyme contained two amino acid substitutions (Ala³¹ValandAsn¹⁰⁶Ser)in addition to an extra methionine residue at theNH₂-terminus the substituted amino acid residues were convertedback to the original ones by means of oligonucleotide-directedsite-specific mutagenesis and in vitro recombination. Thus fourkinds of chicken lysozyme [Met^–1 Val³¹Ser¹⁰⁶-, Met^–1Ser¹⁰⁶-,Met^–1 Val³¹-and Met^–1 (wild type)] wereexpressed in E. coli. From the results of folding experimentsof the reduced lysozymes by sulfhydryl-disulfide interchangeat pH 8.0 and 38°C, follow ed by the specific activity measurementsof the folded en zymes, the following conclusions can be drawn:(i) an extra methionine residue at the NH₂-terminus reducesthe folding rate but does not affect the lysozyme activity ofthe folded enzyme; (ii) the substitution of Asn¹⁰⁶ by Ser decreasesthe activity to 58% of that of intact native lysozyme withoutchanging the folding rate; and (iii) the substitution of Ala³¹Val prohibits the correct folding of lysozyme. Since the wildtype enzyme (Met^–1-lysozyme) was activated in vitro withoutloss of specific activity, the systems described in this study(mutagenesis, overproduction, purification and folding of inactivemutant lysozymes) may be useful in the study of folding pathways,expression of biological activity and stability of lysozyme.     

Stabilization of lysozyme by the introduction of Gly-Pro sequence

Three mutant lysozymes where the Asp¹⁰¹ – Gly¹⁰² sequenceof lysozyme was converted to Asp¹⁰¹–Pro¹⁰², Gly¹⁰¹–Pro102and Pro¹⁰¹–Gly¹⁰² were prepared to investigate the effectof proline residues on the stabilization of proteins. The freeenergy changes of lysozymes for the unfolding in aqueous solutionat pH 5.5 and 35°C were 10.0, 10.1, 11.0 and 7.7 kcal/molfor wild type, Asp¹⁰¹Pro¹⁰², Gly¹⁰¹Pro¹⁰² and Pro¹⁰¹Gly¹⁰² lysozymerespectively. When the energy level in the unfolded state ofwild type lysozyme was fixed at a standard level, the energylevels in the folded state of Asp¹⁰¹Pro¹⁰² and Pro¹⁰¹Gly¹⁰²lysozymes were found to be higher than that of wild type lysozymeon the basis of G_D(H₂O) and entropy losses of their polypeptidechains in the unfolded state. The presence of some strain inthe folded state of these lysozymes was supported by both thecalculation of conformational energy for a trans-L-prolyl residue[Schimmel, P.R. and Flory,P.J. (1968) J. Mol. Biol, 34, 105–120] and the analysis of structures of energy-minimizedmutant lysozymes. Therefore, it is concluded that the formationof the Gly-Pro sequence is effective in avoiding possible strainin the folded state of a protein caused by the introductionof proline residue(s).     

Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K

The catalytic contribution of His48 in the active site of porcinepancreatic phospholipase A2 was examined using site-directedmutagenesis. Replacement of His48 by lysine (H48K) gives riseto a protein having a distorted lipid binding pocket. Activityof this variant drops below the detection limit which is 10⁷-foldlower than that of the wild-type enzyme. On the other hand,the presence of glutamine (H48Q) or asparagine (H48N) at thisposition does not affect the structural integrity of the enzymeas can be derived from the preserved lipid binding propertiesof these variants. However, the substitutions H48Q and H48Nstrongly reduce the turnover number, i.e. by a factor of 10⁵.Residual activity is totally lost after addition of a competitiveinhibitor. We conclude that proper lipid binding on its ownaccelerates ester bond hydrolysis by a factor of 10². With theselected variants, we were also able to dissect the contributionof the hydrogen bond between Asp99 and His48 on conformationalstability, being 5.2 kJ/mol. Another hydrogen bond with His48is formed when the competitive inhibitor (R)-2-dodecanoylamino-hexanol-1-phosphoglycolinteracts with the enzyme. Its contribution to binding of theinhibitor in the presence of an interface was found to be 5.7kJ/mol.     

Conformational properties of the guanine-binding site of ribonuclease T1 inferred from the X-ray structure and protein engineering

Recognition by ribonuclease T₁ of guanine bases via multidentatehydrogen bonding and stacking interactions appears to be mediatedmainly by a short peptide segment formed by one stretch of aheptapeptide, Tyr⁴²-Asn⁴³-Asn⁴⁴-Tyr⁴⁵-Gly⁴⁶-Gly⁴⁷-Phe⁴⁸. Thesegment displays a unique folding of the polypeptide chain—consistingof a reverse turn, Asn⁴⁴-Tyr⁴⁵-Glu⁴⁶-Gly⁴⁷, stabilized by ahydrogen-bond network involving the side chain of Asn⁴⁴, themain-chain atoms of Asn⁴⁴, Gly⁴⁷ and Phe⁴⁸ and one water molecule.The segment is connected to the C terminus of a ß-strandand expands into a loop region between Asn⁴³ and Ser⁵⁴. Lowvalues for the crystallographic thermal parameters of the segmentindicate that the structure has a rigidity comparable to thatof a ß-pleated sheet. Replacement of Asn⁴⁴ with alanineleads to a far lower enzymatic activity and demonstrates thatthe side chain of Asn⁴⁴ plays a key role in polypeptide foldingin addition to a role in maintaining the segment structure.Substitution of Asn⁴³ by alanine to remove a weak hydrogen bondto the guanine base destabilized the transition state of thecomplex by 6.3 kJ/mol at 37°C. In contrast, mutation ofGlu⁴⁶ to alanine to remove a strong hydrogen bond to the guaninebase caused a destabilization of the complex by 14.0 kJ/mol.A double-mutant enzyme with substitutions of Asn⁴³ by a histidineand Asn⁴⁴ by an aspartic acid, to reproduce the natural substitutionsfound in ribonuclease M_s, showed an activity and base specificitysimilar to that of the wild-type ribonuclease M_s. The segmenttherefore appears to be well conserved in several fungal ribonucleases.     

The double catalytic triad, Cys25-Hisl59-Aspl58 and Cys25-Hisl59-Asnl75, in papain catalysis: role of Aspl58 and Asnl75

The 1.65 Å X-ray structure of papain, which exhibits aCys25-Hisl59-Asnl75 triad, does not correspond to the catalyticallyactive ion pair state since Cys25 is oxidized to cystelc acidand Hisl59 is predominantly neutral. Thus, stochastic boundarymolecular dynamics simulations starting from the 1.65 ÅX-ray structure of papain have been performed for Cys25 andHisl59 in the SH-ImH⁺, SH-Im, S^-ImH⁺ and S^--Im states and forAspl58 mutated to Asn, Glu and Gly in the ion pair state. Bycomparing the resulting averaged structures and analyzing thetrajectories of certain interatomic distances, important differencesin the active-site geometry of papain have been found. In particular,the initial Cys25(S^-)-Hisl59(ImH⁺)-Asnl75(C=O) triad found inthe X-ray structure is retained in all the structures exceptthe wild type and Aspl58 - Asn ion pair states where there isa confonmational transition to form the triad, Cys25(S^-)-Hisl59(ImH⁺)-Aspl58(COO^-). Both triads, Cys25(S^-)-Hisl59(ImH⁺)-Aspl58(COO^-)and Cys25(S^-)-Hisl59(ImH⁺)-Asnl75(C=O) are postulated to participatein catalysis and their roles are discussed. Thus, catalysisdoes not take place from a single sterk position but a two-statemechanism     

Improved insulin stability through amino acid substitution

Insulin analogs designed to decrease self-association and increaseabsorption rates from subcutaneous tissue were found to havealtered stability. Replacement of H^B10 with aspartic acid increasedstability while substitutions at B²⁸ and/or B²⁹ were eithercomparable to insulin or had decreased stability. The principalchemical degradation product of accelerated storage conditionswas a disulfidelinked multimer that was formed through a disulfideinterchange reaction which resulted from ß-eliminationof the disulfides. The maintenance of the native state of insulinwas shown to be important in protecting the disulfides fromreduction by dithiothreitol and implicitly from the disulfideinter change reaction that occurs during storage. To understandhow these amino acid changes alter chemical stability, the intramolecularconformational equilibria of each analog was assessed by equilibriumdenaturation. The Gibbs free energy of unfolding was comparedwith the chemical stability during storage for over 20 analogs.A significant positive correla tion (R²=0.8 and P < 0.0005)exists between the conformational stability and chemical stabilityof these analogs, indicating that the chemical stability ofinsulin's disulfides is under the thermodynamic control of theconformational equilibria.     

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 net energetic contribution of interhelical electrostatic attractions to coiled-coil stability

The net energetic contribution of interhelical electrostaticattractions to coiled-coil stability has been quantitated usingde novo designed synthetic coiled-coils. The synthesized modelcoiled-coil (EK), denoted by amino acid res-idues in positionse and g, which contains only interhelical ionic interactionswithout any possible (i, i + 3) and (i, i + 4) intrahelicalionic interaction, consists of two identical 35 residue polypeptidechains with a heptad repeat KgLaG-bAcLdEeKf. Three mutant coiled-coilswere prepared where five Glu residues at e positions in EK weremutated to Gin residues (QK); five Lys residues at g positionswere altered to Gin residues (EQ) or these mutations were effectedat both positions e and g (QQ). The stabilities of the fourcoiled-coils were determined by measuring the ellipticitiesat 220 nm as a function of urea concentration at 20C. By usinga double-mutant cycle analysis it was possible to isolate theenergetic contribution of interhelical ionic attractions tocoiled-coil stability from the other contributions such as helicalpreference and hydro-phobicity. The 0.37 0.01 kcal/mol ofenergetic contribution of one interhelical ion pair to the coiled-coilstability was obtained from three independent comparisons. Thisfinding suggests that a large number of weak interhelical electrostaticinteractions on the surface of a protein can make a substantialcontribution to protein stability. In addition, the energeticcontributions of a single mutation E^– Q, K⁺Q, Q E andE^– Ewere also determined (G = 0.22, 0.26, 0.46 and 0.65kcal/mol for the single mutations, respectively). The greatercontribution of a protonated Glu residue to coiled-coil stabilitycompared with an ionized Glu residue (0.65 kcal/mol) can outweighthe relatively smaller contribution of an interhelical ion pair(0.37 kcal/mol), which clearly explains why most coiled-coilsare more stable at acidic pH compared with neutral pH even wheninterhelical salt bridges contribute to the coiled-coil stabilityat neutral pH.     

Folding and conformational studies on SCR1-3 domains of human complement receptor 1

Short consensus repeats SCR3 and SCR1-3 are soluble recombinantproteins, consisting of the third and first three N-terminaldomains of complement receptor 1, respectively, which retainsome anti-complement activity. The conformational stabilitiesand folding/unfolding of SCR3 and SCR1-3 have been studied usingcircular dichroism and equilibrium and pre-equilibrium fluorescencespectroscopy. Denaturation by guanidinium hydrochloride (GdnHCl)is rapid and completely reversible. Reduction of disulphidebridges in the folded proteins by ß-mercaptoethanolleads to an increase in fluorescence intensity. The fluorescenceintensity of the folded proteins is {small tilde}7.5% of thatof the respective unfolded proteins. The data can be approximatedto a two-state transition between native and denatured formsof the proteins. SCR3 has a conformational stability in waterof 12–13 kJ/mol whereas that of SCR1-3 is 19.5–19.9kJ/mol depending upon the technique utilized. The heat capacitychange associated with the unfolding of SCR1-3 was obtainedby a series of GdnHCl unfolding experiments over a range oftemperatures and was found to be 6.6 kJ/K.mol or 33.8 J/K.mol_residue.The refolding process of SCR3 was found to be simple, describedby a single exponential equation, whereas that of SCR1-3 wasfound to be complex and could be fitted to a double exponentialequation indicating the presence of folding intermediates.     

1.85 A structure of anti-fluorescein 4-4-20 Fab

The crystal complex of fluorescein bound to the high-affinityanti-fluorescein 4-4-20 Fab {K_a = 10¹⁰ M^–1 at 2°C)has been determined at 1.85 Å. Isomorphous crystals oftwo isoelectric forms (p1 = 7.5 and 7.9) of the antifluorescein4-4-20 Fab, an IgG2A [Gibson et al (1988)Proteins: Struct. FunctGenet., 3, 155–160], have been grown. Both complexes crystallizewith one molecule in the asymmetric unit in space group P1,with a = 42.75 Å, b =43.87 Å, c = 58.17 Å, = 95.15° , ß = 86.85° and = 98.01°.The final structure has an R value of 0.188 at 1.85 Åresolution. Interactions between bound fluorescein, the complementarity-determiningregions (CDRs) of the Fab and the active-site mutants of the4-4-20 single-chain Fv will be discussed. Differences were foundbetween the structure reported here and the previously reported2.7 Å 4-4-20 Fab structure [Herron et al. (1989) Proteins:Struct. Fund., 5, 271–280]. Our structure determinationwas based on 26 328 unique reflections — four times theamount of data used in the previous report. Differences in thetwo structures could be explained by differences in interpretingthe electron density maps at the various resolutions. The r.m.s.deviations between the variable and constant domains of thetwo structures were 0.77 and 1.54 Å, respectively. Fourregions of the light chain and four regions of the heavy chainhad r.m.s. backbone deviations of >4 Å. The most significantof these was the conformation of the light chain CDR 1.     

Stereochemical modeling of disulfide bridges. Criteria for introduction into proteins by site-directed mutagenesis

A computer modeling procedure for assessing the stereochemicalsuitability of pairs of residues in proteins as potential sitesfor introduction of cystine disulfide crosslinks has been developed.Residue pairs with C – C distances of 6.5 Å andC^beta;–C^ß distances of 4.5 Å are chosenfor geometrical fixation of S atoms using the program MODIP.The stereochemistry of the modeled disulfides is evaluated usinglimits for the structural parameters of the various torsionangles and S–S bond length in the disulfide bridge. Theability of the procedure to correctly model disulfides has beenchecked with examples of cystine peptides of known crystal structuresand 103 disulfide bridges from 25 available protein crystalstructures determined at 2 Å resolution. An analysis ofresults on three proteins with engineered disulfides, T4 lysozyme,dihydrofolate reductase and subtilisin, is presented. Two positionsfor the introduction of ‘stereochemically optimal’disulfides are identified in subtilisin.     

Altering the association properties of insulin by amino acid replacement

The importance of Pro^B28 and Lys^B29 on the self-associationof insulin was established by systematically truncating theC terminus of the B chain. The relationship between structureand association was further explored by making numerous aminoacid replacements at B²⁸ and B²⁹ Association was studied bycircular dichroism, size-exclusion chromatography and ultracentrifugation.Our results show that the location of a prolyl residue at B²⁸is critical for high-affinity self-association. Removal of Pro^B28in a series of C-terminal truncated insulins, or amino acidreplacement of Pro²⁸, greatly reduced association. The largestdisruption to association was achieved by replacing Lys^B29 withPro and varying the amino acid at B²⁸ Several of the analogswere predominantly monomers in solutions up to 3 mg/ml. Theseamino acid substitutions decreased association by primarilydisrupting the formation of dimers. Such amino acid substitutionsalso substantially reduced the Zn-induced insulin hexamer formation.The formation of monomeric insulins through amino acid replacementswas accompanied by conformational changes that may be the causefor decreased association. It is demonstrated that self-associationof insulin can be drastically altered by substitution of oneor two key amino acids.     

Site-directed mutagenesis of pseudoazurin from Alcatigenes faecalis S-6; Pro80Ala mutant exhibits marked increase in reduction potential

Pseudoazurin (a blue copper protein or cupredoxin) of a denitrifyingbacterium Alcaligenes faecalis S-6 is a direct electron carrierfor a Cu-containing nitrite reductase (NIR) of the same organism.Site-directed mutagenesis of the pseudoazurin was carried outusing an Escherichia coli expression system. Replacement ofTyr74 by Phe to remove an internal hydrogen bond in the ß-barrelcaused a slight decrease in heat stability as well as a requirementfor a higher concentration of Cu²⁺ for production in the E.colihost. Exchange of Ala for Pro80 adjacent to His81, one of thefour ligands binding a type I Cu atom, caused a marked increasein reduction potential by 139 mV without change in the opticalabsorption spectrum. The ability of the pseudoazurin to transferelectrons to NIR was markedly diminished but the apparent K_mof NIR for pseudoazurin was not affected by the mutation. X-raydiffraction data collected on the oxidized and reduced formsof the Pro80Ala mutant show that a water molecule occupies thepocket created by the absent side chain. This observation suggeststhat the increase in reduction potential may be caused due tothe increased solvent accessibility to the Cu atom. The electrondensity difference maps on these structures (at 2.0 Å)show that this water moves during the change in oxidation state,and that there are small, but localized, conformational changes>6.5 Å from the copper site, as well as movement ofboth the Cu²⁺ and the cysteinate sulfur.     

3-D structure of the D153G mutant of Escherichia coli alkaline phosphatase: an enzyme with weaker magnesium binding and increased catalytic activity

The substitution of aspartate at position 153 in Escherichiacoli alkaline phosphatase by glycine results in a mutant enzymewith 5-fold higher catalytic activity (k_cat but no change inKm at pH 8.0 in 50 mM Tris-HCl. The increased k_cat is achievedby a faster release of the phosphate product as a result ofthe lower phosphate affinity. The mutation also affects Mg²⁺binding, resulting in an enzyme with lower metal affinity. The3-D X-ray structure of the D153G mutant has been refined at2.5 Å to a crystallographic Rfactor of 16.2%. An analysisof this structure has revealed that the decreased phosphateaffinity is caused by an apparent increase in flexibility ofthe guanidinium side chain of Argl66 involved in phosphate binding.The mutation of Aspl53 to Gly also affects the position of thewater ligands of Mg²⁺, and the loop Glnl52–Thrl55 is shiftedby 0.3 Å away from the active site. The weaker Mg²⁺ bindingof the mutant compared with the wild type is caused by an alteredcoordination sphere in the proximity of the Mg²⁺ ion, and alsoby the loss of an electrostatic interaction (Mg²⁺.COO^-Aspl53)in the mutant Its ligands W454 and W455 and hydroxyl of Thrl55,involved in the octahedral coordination of the Mg²⁺ ion, arefurther apart in the mutant compared with the wild-type     

The Trp-cage: optimizing the stability of a globular miniprotein

The Trp-cage, as the smallest miniprotein, remains the subject of numerous computational and experimental studies of protein folding dynamics and pathways. The original Trp-cage (NLYIQWLKDGGPSSGRPPPS, Tm = 42 degrees C) can be significantly stabilized by mutations; melting points as high as 64 degrees C are reported. In helical portions of the structure, each allowed replacement of Leu, Ile, Lys or Ser residues by Ala results in a 1.5 (+/-0.35) kJ/mol fold stabilization. No changes in structure or fluxionality of the core results upon stabilization. Contrary to the initial hypothesis, specific Pro/Trp interactions are not essential for core formation. The entropic advantage of Pro versus Ala (DeltaDeltaS(U) = 11 +/- 2 J/mol K) was measured at the solvent-exposed P17 site. Pro-Ala mutations at two of the three prolines (P12 and P18) that encage the indole ring result in less fold destabilization (2.3-3.4 kJ/mol). However, a P19A mutation reduces fold stability by 16 kJ/mol reflecting a favorable Y3/P19 interaction as well as Trp burial. The Y3/P19 hydrophobic staple interaction defines the folding motif as an 18-residue unit. Other stabilizing features that have been identified include a solvent-exposed Arg/Asp salt bridge (3.4-6 kJ/mol) and a buried H-bonded Ser side chain ( approximately 10 kJ/mol).     

A mini-protein designed by removing a module from barnase: molecular modeling and NMR measurements of the conformation

A globular domain can be decomposed into compact modules consistingof contiguous 10–30 amino acid residues. The correlationbetween modules and exons observed in different proteins suggeststhat each module was encoded by an ancestral exon and that moduleswere combined into globular domains by exon fusion. Barnaseis a single domain RNase consisting of 110 amino acid residuesand was decomposed into six modules. We designed a mini-proteinby removing the second module, M2, from barnase in order togain an insight into the structural and functional roles ofthe module. In the molecular modeling of the mini-protein, weevaluated thermodynamic stability and aqueous solubility togetherwith mechanical stability of the model. We chemically synthesizeda mini-barnase with ¹⁵N-labeling at 10 residues, whose correspondingresidues in barnase are all found in the region around the hydrophobiccore. Circular dichroism and NMR measurements revealed thatmini-barnase takes a non-random specific conformation that hasa similar hydrophobic core structure to that of barnase. Thisresult, that a module could be deleted without altering thestructure of core region of barnase, supports the view thatmodules act as the building blocks of protein design.     

The design of a biochip: a self-assembling molecular-scale memory device

A design for a biochip memory device based on known materialsand existing principles is presented. The fabrication of thismemory system relies on the self-assembly of the nucleic acidjunction system, which acts as the scaffolding for a molecularwire consisting of polyacetylene-like units. A molecular switchto control current is described which is based on the formationof a charge - transfer complex. A molecular-scale bit is presentedwhich is based on oxidation - reduction potentials of metalatoms or clusters. The readable ‘bit’ which canbe made of these components has a volume of 3x10⁷ ų andshould operate at electronic speeds over short distances.     

Interactions of (Ala*Ala*Lys*Pro)n and (Lys*Lys*Ser*Pro)n with DNA. Proposed coiled-coil structure of AlgR3 and AlgP from Pseudomonas aeruginosa

The proteins, AlgR3 and AlgP, are involved in the regulationof alginate synthesis in Pseudomonas. They contain multiplerepeats of Ala^*Ala^*Lys^*Pro as do several other proteins thatresemble histones. The interactions of synthesis oligopeptidescomposed of repeated Ala^*Ala^*Lys^*Pro or Lys^*Lys^*Ser^*Pro unitswith DNA were studied by fluorescence of the Fmoc (9-fluorenylmethyloxycarbonyl)group attached to the N-termini of the peptides. DNA quenchingof the Fmoc fluorescence of the peptides was used to estimatethe apparent association constants for the interaction of Fmoc(AAKP)_nOH(n = 2, 4, 8, 18, 32) and of Fmoc(KKSP)_nOH (n = 2, 4, 8, 16,20, 32) with DNA. The Fmoc(AAKP)_nOH peptides bind to DNA onlyat low ionic strength; the Fmoc(KKSP)_n OH peptides interactwith DNA at both low (0.05 M KCl) and high (0.2 M KCl) saltAt low ionic strength an increase in the number of the repeatunits causes an increase in the apparent association constantup to {small tilde}2 x 10⁶ M^–1 for both types of peptidesat N 24. The insertion of an AAKTA unit into the middle ofthe Fmoc(AAKP)₈OH peptide increases its affinity to DNA. Wepropose a model of (AAKP)_n and of its interaction with DNA.The repeat unit consists of a single turn of -helix followedby a bend necessitated by Pro. The resultant coiled-coil formsa right-handed superhelix with 10 AAKPs per repeat distanceof {small tilde}33 Å. With only slight modification ofthe canonical parameters of this model the AAKP super helixfits into the major groove of B-form DNA with one AAKP tetramerper base pair repeat of 3.4 Å. The -amine nitrogen ofLys can form a polar hydrogen bond with a phosphate oxygen atomof the DNA backbone. A better fit is obtained when the modelis modified to accommodate [(AAKP)₅AAKTA]_n as actually observedin AlgR3. We suggest that this coiled-coil represents a generalmotif for other protein–DNA interactions.     

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

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

Development of an optimized refolding process for recombinant Ala-Glu-IGF-1

Denatured and reduced N-terminal extended insulin-like growthfactor-1 (AE-IGF-1) was purified from Escherichia coli extractsand subjected to in vitro folding. The renaturation processwas shown to be a function of the redox potential of the solution.Folding by different methods had no significant effect on therenaturation. A maximal yield of 60% (w/w)was obtained. Thefolded AE-IGF-1 was enzymatically converted to IGF-1. The majorby-product (20% w/w) was identified as scrambled IGF-1. Enzymaticdigestion at alkaline and acidic pH suggested two possible disulphidebond arrangements: (i) Cys⁶–Cys⁴⁷, Cys¹⁸–Cys⁶¹,Cys⁴⁸–Cys⁵²; or (if) Cys⁶–Cys⁵², Cys^l8–Cys⁶¹,Cys⁴⁷ and Cys⁴⁸ being in their reduced forms. Energy minimizationand molecular modelling suggested that the scrambled IGF-1,having reduced cysteines at positions 47 and 48, was the energeticallymost stable conformation of the two.