An aldol approach to a building block corresponding to the C21–C26-part of FK506

Starting from D-tartrate 2 , the chiral aldehyde 10 was prepared in 8 steps. Key steps include the reductive opening of the p-methoxybenzyl acetal 4 and the elongation of the aldehyde 7 via Wittig and hydroboration reaction providing the alcohol 9 . Subsequent Evans aldol reaction provided compound 12 which corresponds to the C21–C26 part of the immunosuppressive FK506.     

Structural and kinetic studies on ligand binding in wild-type and active-site mutants of penicillin acylase

Penicillin acylase catalyses the condensation of Calpha-substituted phenylacetic acids with beta-lactam nucleophiles, producing semi-synthetic beta-lactam antibiotics. For efficient synthesis a low affinity for phenylacetic acid and a high affinity for Calpha-substituted phenylacetic acid derivatives is desirable. We made three active site mutants, alphaF146Y, betaF24A and alphaF146Y/betaF24A, which all had a 2- to 10-fold higher affinity for Calpha-substituted compounds than wild-type enzyme. In addition, betaF24A had a 20-fold reduced affinity for phenylacetic acid. The molecular basis of the improved properties was investigated by X-ray crystallography. These studies showed that the higher affinity of alphaF146Y for (R)-alpha-methylphenylacetic acid can be explained by van der Waals interactions between alphaY146:OH and the Calpha-substituent. The betaF24A mutation causes an opening of the phenylacetic acid binding site. Only (R)-alpha-methylphenylacetic acid, but not phenylacetic acid, induces a conformation with the ligand tightly bound, explaining the weak binding of phenylacetic acid. A comparison of the betaF24A structure with other open conformations of penicillin acylase showed that betaF24 has a fixed position, whereas alphaF146 acts as a flexible lid on the binding site and reorients its position to achieve optimal substrate binding.     

Contribution of a proline residue and a salt bridge to the stability of a type I reverse turn in chymotrypsin inhibitor-2

The contributions of the components of a type I reverse turnto the stability of chymotrypsin inhibitor-2 (Lys43-Pro44-Gly45)have been determined by protein engineering methods. A double-mutantcycle was used to determine the interaction between Lys43 andGlu45 by replacing them with alanine. We also mutated Pro44,which gives the geometry of the turn, to alanine and analysedthe stability of the resulting mutants compared with wild-typechymotrypsin inhibitor-2, using equilibrium denaturation inducedby guanldinium chloride. There are decreases in stability (inkcal/mol) of 0.64 = 0.06 for Lys43 - Ala, 0.57 ± 0.15for Glu45 - Ala, 0.95 ± 0.06 for Lys43 - Ala/Glu45 -Ala and 1.93 ± 0.09 for Pro44 - Ala. The free energyof interaction between Lys43 and Glu45 is calculated to be only0.25 ± 0.09 kcal/mol. From the changes in denaturationmidpoint, T_m measured by circular dkhroism, we estimate theenergy of interaction between Lys43 and Glu45 to be 0.36 ±0.07 kcal/mol whereas the contribution of Pro44 is -2.0 kcal/mol.The contribution of the salt bridge to the stability of theprotein is very small and the residue Pro44 plays the key rolein stabilizing the turn     

Protein modification from mutational analysis of an autologous peptide fragment

In the -complementation of ß-galactosidase an N-terminalpeptide fragment (-peptide) of the wild-type enzyme interactswith a defective ß-galactosidase enzyme to restorecapacity for subunit assembly and activity. We have used previouslya random mutagenesis and screening approach to identify a pentapeptideresidue tract in the -peptide that was highly tolerant of residuesubstitution, with some mutations conferring improved function.This tract is of clear importance for -peptide function butis apparently dispensible in the intact parental enzyme. Toinvestigate this further, we selected tract mutations and placedthem into intact ß-galactosidase, at the correspondingN-terminal position as in the -peptide. We then tested whethersuch specific tract sequences conferred properties to the wholeenzyme which could be predicted from the behaviour of the defectiveenzyme complemented with the corresponding mutant -peptide.This was shown for mutations which positively or negativelyaffected enzyme stability. Additionally, a subset of mutationswhich affected complementation efficiency in vivo were predictedto affect the formation of higher-order structures in the intactprotein, and this was observed experimentally. Mutations whichdecreased peptide complementation dramatically decreased thelevel of formation of multimers in the intact protein and amutation which increased peptide complementation produced markedenhancement of multimer formation in a protein with a preexistingimpairment in higher-order structure formation. Such subtleeffects are difficult to detect directly in the whole proteinby randomization/selection approaches, but in the complementingpeptide the role of the residues within the pentapeptide tractis effectively amplified. Identification of residue tracts exhibitingfunctional tolerance to amino acid substitution in an activepeptide fragment can thus be combined with transferral of potentiallyuseful mutant peptide sequences back into the intact protein.Manipulation of a complementation system in this manner affordsa sensitive approach towards targeted improvement of proteins.     

The active site of carboxypeptidase Taq possesses the active-site motif His-Glu-X-X-His of zinc-dependent endopeptidases and aminopeptidases

Carboxypeptidase (CPase) Taq possesses the His–Glu–X–X–Hissequence, which is the consensus sequence in the active siteof zinc-dependent endopeptidases and amino-peptidases, at positions276–280. Amino acid replacement of the conserved His andGlu drastically diminished the activity of CPase Taq, and thezinc content of the enzyme was also greatly reduced when eitherof the two His residues was replaced with Arg or Tyr. The resultsindicate that this sequence actually functions as the activesite in CPase Taq, showing that CPase Taq is a novel type ofzinc-dependent CPase that possesses the His–Glu–X–X–Hisactive-site motif.     

Identification of important functional environs in protein tertiary structures from the analysis of residue variation in 3-D: application to cytochromes c and carboxypeptidases A and B

A simple methodology is described to apply to aligned proteinsequence sets for which at least one representative 3-D C structureis known. The evolutionary variation observed at each residueposition in the sequence alignment is qualified by taking intoaccount the residue variation that has occurred at other positionslocated within 7 A (according to the probable chain fold). Thisexpresses the evolutionary behaviour of any residue positionin the more appropriate context of its immediate surroundingsand distinguishes between invariant residues on the basis ofthe variation of their environment. The highest mechanisticsignificance is attached to conserved residues in conservedsurroundings, but the quantitative nature of the analysis meansthat all residue vicinities can be ranked and merged accordingto the degree of conservation that they exhibit and the residuepositions that comprise them. Therefore, with the aid of thechain fold, contour maps can be constructed that show gradedfoci of evolutionary conservation in the underlying superstructureof the protein type, and the irregular shapes and extents oflarge conserved areas. To test the methodology, it was appliedto cytochromes c and the carboxypeptidases A and B.     

A mutational analysis of receptor binding sites of interleukin-1{beta}:differences in binding of human interleukin-1{beta} muteins to human and mouse receptors

The 3-D crystal structure of interleukin-1ß(IL-1ß)has been used to define its receptor binding surface by mutationalanalysis. The surface of IL-1ß was probed by site-directedmutagenesis. A total of 27 different IL-1ß muteinswere constructed, purified and analyzed. Receptor binding measurementson mouse and human cell lines were performed to identify receptoraffinities. IL-1ß muteins with modified receptor affinitywere evaluated for structural integrity by CD spectroscopy orX-ray crystallography. Changes in six surface loops, as wellas in the C- and N-termini, yielded muteins with lower bindingaffinities. Two muteins with intact binding affinities showed10- to 100-fold reduced biological activity. The surface regioninvolved in receptor binding constitutes a discontinuous areaof 1000 Å2 formed by discontinuous polypeptide chain stretches.Based on these results, a subdivision into two distinct localareas is proposed. Differences in receptor binding affinitiesfor human and mouse receptors have been observed for some muteins,but not for wild-type IL-1ß. This is the first timea difference in binding affinity of IL-1ß muteinsto human and mouse receptors has been demonstrated     

Engineering of a thioglycoligase: randomized mutagenesis of the acid-base residue leads to the identification of improved catalysts

Thioglycoligases are recently introduced variants of retaining glycosidases in which the acid-base catalyst has been mutated, rendering them capable of thioglycoside synthesis. The original acid-base mutant of Agrobacterium sp. beta-glucosidase (E170A) was previously shown to be an effective thioglycoligase carrying out glycosyltransfer from 2,4-dinitrophenyl glycosides to several different thio sugar acceptors. Here we report the generation of a screen for improved thioglycoligases, randomized mutagenesis of the acid-base catalyst E170 and identification of variants superior to E170A. Furthermore we have established a coupled assay allowing kinetic analysis of isolated variants and found that Abg E170Q is 5-fold faster than Abg E170A when 2,4-dinitrophenyl glucoside is used as donor and 100-fold faster when glucosyl azide is used. To demonstrate its utility, different acceptor and donor sugar combinations were employed to produce thio-linked di- or trisaccharides in high yields, showing the considerable versatility of the system for the synthesis of carbohydrate mimetics.     

Autocatalytic processing of pro-papaya proteinase IV is prevented by crowding of the active-site cleft

The DNA coding for pro-papaya proteinase IV (PPIV) has beencloned and expressed in Escherichia coli. Heterologous expressionof the protein, followed by refolding in vitro, yields an enzymaticallyactive pro-enzyme which fails to autodigest to form the matureprotein. Mutagenesis of the active site of papain to simulatethat of PPIV yields a proenzyme which also fails to autoactivate.Complementarymutagenesis of the pro-region/mature boundary ofPPIV, to introduce its own substrate recognition sequence, has,however, produced a pro-enzyme that will autocatalytically cleave.This is the first report of enzymatic activity in a recombinantpro-cysteine proteinase, and the first time that such a proteinhas been shown to fail to autocatalytically cleave because ofits stringent substrate specificity.     

Alteration of the amino acid substrate specificity of clostridial glutamate dehydrogenase by site-directed mutagenesis of an active-site lysine residue

Two residues, K89 and S380, thought to interact with the -carboxylgroup of the substrate L-glutamate, have been altered by site-directedmutagenesis of clostridial glutamate dehydrogenase (GDH). Thesingle mutants K89L and S380V and the combined double mutantK89L/S380V were constructed. All three mutants were satisfactorilyoverproduced in soluble form. However, only the K89L mutantwas retained by the dye column normally used in purifying thewild-type enzyme. All three mutant enzymes were purified tohomogeneity and tested for substrate specificity with 24 aminoacids. The single mutant S380V showed no detectable activity.The alternative single mutant K89L showed an activity towardsL-glutamate that was decreased nearly 2000-fold compared withwild-type enzyme, whereas the activities towards the monocarboxylicsubstrates -aminobutyrate and norvaline were increased 2- to3-fold. A similar level of activity was obtained with methionine(0.005 U/mg) and norleucine (0.012 U/mg), neither of which giveany activity with the wild-type enzyme under the same conditions.The double mutant showed decreased activity with all substratescompared with the wild-type GDH. In view of its novel activities,the K89L mutant was investigated in greater detail. A strictlylinear relationship between reaction velocity and substrateconcentration was observed up to 80 mM L-methionine and 200mM L-norleucine, implying very high K_m values. Values of k_cat/K_m,for L-methionine and L-norleucine were 6.7x10^–2 and 0.15s^–1M^–1, respectively. Measurements with dithiobisnitrobenzoicacid showed that the mutant enzymes all reacted with a stoichiometryof one -SH group per subunit and all showed protection by coenzyme,indicating essentially unimpaired coenzyme binding. With glutamateor 2-oxoglutarate as substrate the K_m values for the vestigialactivity in the mutant enzyme preparations were strikingly closeto the wild-type K_m values. Both for wild-type GDH and K89L,L-glutamate gave competitive product inhibition of 2-oxoglutaratereduction but did not inhibit the reduction of 2-oxocaproatecatalysed by K89L enzyme. This suggests that the low levelsof glutamate/2-oxoglutarate activity shown by the mutant enzymeare due to trace contamination. Since stringent precautionswere taken, it appears possible that this reflects the levelof reading error during overexpression of the mutant proteins.CD measurements indicate that the S380V mutant has an alteredconformation, whereas the K89L enzyme gave an identical CD spectrumto that of wild-type GDH; the spectrum of the double mutantwas similar, although somewhat altered in intensity. The resultsconfirm the key role of K89 in dicarboxylate recognition byGDH.     

Expression of immunoglobulin and scavenger receptor superfamily domains as chimeric proteins with domains 3 and 4 of CD4 for ligand analysis

One approach to the analysis of leucocyte cell surface proteinsis to express their domains with part of another protein asa carrier. We report the use of two immunoglobulin superfamily(IgSF) domains from rat CD4 (CD4d3+4) in producing domains fromvarious superfamilies as chimeric proteins in Chinese hamsterovary cell lines. Four types of construct were successfullyexpressed containing: (i) the two IgSF domains of CD48; (ii)the IgSF domain of mb-1 which is part of the B cell antigenrecognition complex; (iii) a T cell receptor V domain; and (iv)the N-terminal domain of CD5 which belongs to the scavengerreceptor superfamily. This CD5 chimeric protein was antigenkfor a panel of CD5 mAbs showing that mAbs with functional effectsreacted with the N-terminal domain of CD5. The CD48 chimericprotein has been used both as multivalent complexes producedby crosslinking with mAbs recognizing CD4 and in a monomericform to analyse the kinetics of the interaction between CD48and CD2 [van der Merwe et al. (1993) EMBO J., 12, 4945–4954].     

Redesigning a sweet protein: increased stability and renaturability

Monellin is one of two natural proteins from African berrieswith potent sweet taste. Monellin is the smaller of the two,and consists of two peptides. The protein loses sweetness whenheated above 50°C under acidic pH. Based on the crystalstructure of monellin we have fused the two chains into a singlechain using several different linkers copied and ‘transplanted’from the same molecule. One of the newly designed proteins isas potently sweet as the natural one, is more stable upon temperatureor pH changes, and renatures easily even after heating to 100°Cat low pH.     

Site-directed and deletion mutational analysis of the receptor binding domain of the interleukin-6 receptor targeted fusion toxin DAB389-IL-6

We have used site-directed and in-frame deletion mutationalanalysis in order to explore the structural features of theIL–6 portion of the diphtheria toxin-related interleukin–6(IL–6) fusion toxin DAB₃₈₉-IL–6 that are essentialfor receptorbinding and subsequent inhibition of protein synthesisin target cells. Deletion of the first 14 amino acids of theIL–6 component of the fusion toxin did not alter eitherreceptor binding affinity or cytotoxk potency. In contrast,both receptor binding and cytotoxic activity were abolishedwhen the C–terminal 30 amino acids of the fusion toxinwere deleted. In addition, we explored the relative role ofthe disulfide bridges within the IL–6 portion of DAB₃₈₉-IL–6in the stabilization of structure required for receptor-binding.The analysis of mutants in which the substitution of eitherCys⁴⁴⁰, Cys⁴⁴⁶, Cys⁴⁶⁹ or Cys⁴⁷⁹ to Ser respectively, demonstratesthat only the disulfide bridge between Cys⁴⁶⁹ and Cys⁴⁷⁹ isrequired to maintain a functional receptor binding domain. Inaddition, the internal in-frame deletion of residues 435–451,which includes Cys⁴⁴⁰ and Cys⁴⁴⁶, was found to reduce, but notabolish receptor binding affinity. These results further demonstratethat the disulfide bridge between Cys⁴⁴⁰ and Cys⁴⁴⁶ is not essentialfor receptor-binding. However, the reduced cytotoxic potencyof DAB₃₈₉-IL6(435–451) suggests that the conformationand/or receptor binding sites associated with this region ofthe fusion toxin is/are important for maintaining the wild typereceptor binding affinity and cytotoxic potency.     

Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability

Folding stabilities of camelized human antibody VH domains werestudied through the determination of their melting points inthermodenaturation experiments. The melting point of a VH domainoriginating from a synthetic library of human VHs, which hadbeen optimized for the use as small recognition units throughthe mimicking of camelid antibody heavy chains occurring naturallywithout light chain, was 56.6C compared with 71.2C of theoriginal human VH. Its stability was improved (melting point61.6C) through three mutations to mimic camelid VHs even further:Va137 was replaced by phenylalanine and two cysteines were introducedat positions 33 and 100b. The resulting VH folded properly andformed a second intradomain disulphide between the extra cysteines.The new mutations were then built constitutively into a phage-displayVH library, from which antigen-specific VHs were selected. Twowere analysed for stability with melting points of 72.6 and75.3C. Thus secondary camelization enabled the isolation ofVHs with improved folding stabilities exceeding even that ofthe original human VH. This indicates an effect on folding stabilityfor some mutations specific in the light chain lacking camelidheavy chains.     

Free energy calculations of the mutation of Ile96 -> Ala in barnase: contributions to the difference in stability

Free energy calculations were carried out to determine the relativeunfolding free energy of the Ile96 wild type and Ala96 mutantbarnases. The total calculated free energies suggest that substitutionof Ile96 with Ala destabilizes barnase by 3.9 kcal/mol, whichis in good agreement with the independently determined experimentalvalues of 4.0 and 3.3 kcal/mol and a previous simulation. However,a decomposition of the free energy finds the dominant contributionsto this free energy arising from the noncovalent Interactionsbetween the perturbed group and distant residues of barnasein the sequence and water molecules and only a very small contributionfrom covalent interactions. This is in contrast to the previoussimulation, using the dual topology methodology, which produceda decomposition with an {small tilde}60% free energy contributionfrom changes in covalent interactions. The use of the singletopology employed in the present calculations and the dual topologyemployed in the previous study are analyzed in order to understandthe contrast between the present results and the results ofthe previous study.     

Application of scaled particle theory to model the hydrophobic effect: implications for molecular association and protein stability

The energetics of alkane dissolution and partition between waterand organic solvent are described in terms of the energy ofcavity formation and solute-solvent interaction using scaledparticle theory. Thermodynamic arguments are proposed that allowcomparison of experimental measurements of the surface areawith values calculated from an all-atom representation of thesolute. While the surface tension relating to the accessiblesurface is shape dependent, it is found that for the molecularsurface it is not. This model rationalizes the change in surfacetension between the microscopic (20–30 cal/mol/A2) andmacroscopic (70–75 cal/mol/A2) regimes without the needto invoke Flory-Huggins theory or to apply other corrections.The difference in the values arises (i) to a small extent asa result of the curvature dependence of surface tension and(ii) to a large extent due to the difference in the molecularsurface derived from the experiment and that calculated froman extended all-atom model. The model suggests that the primarydriving force for alkane association in water is due to thetendency of water to reduce the solute cavity surface. It isargued that to model the energetics of alkane association, thesurface tension should be related to the molecular surface (ratherthan the accessible surface) with a surface tension near themacroscopic limit for water. This model is compared with resultsfrom theoretical simulations of the hydrophobic effect for twowell-studied systems. The implications for antibody– antigeninteractions and the effect of hydrophobic amino acid deletionon protein stability are discussed. The approach can be usedto model the solute cavity formation energy in solution as afirst step in the continuum modelling of biomolecular interactions     

Automating the identification and analysis of protein {beta}-barrels

ßBarrels are widespread and well-studied featuresof a great many protein structures. In this paper an unsuper-visedmethod for the detection of P-barrels is developed based ontechniques from graph theory. The hydrogen bonded connectivityof ß-sheets is derived using standard pattern recognitiontechniques and expressed as a graph. Barrels correspond to topologicalrings in these connectivity graphs and can thus be identifiedusing ring perception algorithms. Following from this, the characteristictopological structure of a barrel can be expressed using a novelform of reduced nomenclature that counts sequence separationsbetween successive members of the ring set These techniquesare tested by applying them to the detection of barrels in anon-redundant subset of the Brookhaven database. Results indicatethat topological rings do seem to correspond uniquely to ß-barrelsand that the technique, as implemented, finds the majority ofbarrels present in the dataset.     

Progesterone binding to uteroglobin: two alternative orientations of the ligand

Progesterone binding to a homodimer of uteroglobin takes placein a hydrophobic cavity formed by the two subunits. Previousmutational analyses have shown that the tyrosine (21 and 21')and threonine (60 and 60') residues of the uteroglobin dimerare directly involved in progesterone binding. To analyze thecontribution of each of the two tyrosines and threonines inthe dimer, we have constructed a covalently linked uteroglobindimer (UG^cl) by fusing two uteroglobin cDNAs via a syntheticlinker sequence. Escherichia coli expressed UG^cl bound progesteronewith the same affinity as the native dimeric protein. Replacementof both tyrosines by phenylalanines abolished progesterone binding.Replacement of either the C-terminal tyrosine 21 or the N-terminaltyrosine 21' separately, reduced the affinity for progesterone3- to 4-fold, suggesting that both tyrosines participate inprogesterone binding. In contrast, substitutions of the threonineresidues of the C- or Nterminal moities had no effect, whereasthe replacement of both threonines reduced the affinity forprogesterone 2- to 3-fold. These data, together with computermodels, suggest that progesterone docks in the internal bindingpocket of uteroglobin in two different orientations.     

Exchange of domains of glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus and the mesophilic bacterium Clostridium difficile: effects on catalysis, thermoactivity and stability

The glutamate dehydrogenase gene from the hyperthermophilicarchaeon Pyrococcus furiosus has been functionally expressedin Escherichia coli under the control of the X, PL promoter.The P.furiosus glutamate dehydrogenase amounted to 20% of thetotal E.coli cell protein, and the vast majority consisted ofhexamers. Following activation by heat treatment, an enzymecould be purified from E.coli that was indistinguishable fromthe glutamate dehydrogenase purified from P.furiosus. Hybridgenes, that consisted of the coding regions for the homologousglutamate dehydrogenases from P.furiosus and the mesophilicbacterium Clostridium difficile, were constructed and successfullyexpressed in E.coli. One of the resulting hybrid proteins, containingthe glutamate binding domain of the C.difficile enzyme and thecofactor binding domain of the P.furiosus enzyme, did not showa detectable activity. In contrast, the complementary hybridcontaining the P.furiosus glutamate and the C.difficile cofactorbinding domain was a catalytically active hexamer that showeda reduced substrate affinity but maintained efficient cofactorbinding with the specificity found in the Clostridium symbiosumenzyme. Compared with the C.difficile glutamate dehydrogenase,the archaeal-bacterial hybrid is slightly more thermoactive,less thermostable but much more stable towards guanidinium chloride-inducedinactivation and denaturation     

Inverse protein folding by the residue pair preference profile method: estimating the correctness of alignments of structurally compatible sequences

The residue pair preference profile (R3P) method is an inversefolding method that combines environmental profiles and pairpreference profiles. The method uses statistical preferencesfor residue pairs which score the likelihood of finding a profiledresidue to be paired with a residue within its local environmentAll pairs are characterized by their dihedral angles, secondarystructure and number of neighboring residues as a function ofresidue type. Each residue pair preference is expressed forall 20 amino acids of the profiled residue and is weighted bythe compatibility of the environment residue with its own localenvironment The R3P method produces an initial profile-sequencealignment which is then refined by converting the initial profileinto a profile of a target sequence threaded into the structureof the initial profile. We have tested this method by evaluatingalignments of sequences with known 3-D structures using structuralsuperposition alignments as reference. R3P-sequence alignmentsare 50% correct on average for sequences whose 3-D structurepairs superimpose with an r.m.s. deviation of 1.97 Å.The average improvement in correctness during this iterativerefinement is 14%. The R3P-sequence alignments are comparedwith sequence-sequence and 3-D profile-sequence alignments.When all three methods are combined, on average 50% of the alignmentsare correct for pairs of 3-D structures that superimpose within2.12 Å. A 3-D model of HisA is predicted with the combinedmethod.