Structure of {alpha}-{alpha}-hairpins with short connections

This paper presents the results of detailed stereochemical analysisof structures and sequences of --hairpins with short connections.It is shown that --hairpins of each given type have very similarpatterns of hydrophobic, hydrophilic and glycine residues intheir amino acid sequences. These results can be used in theprediction of --hairpin conformation as well as in protein designand engineering.     

Chemical engineering of a three-fingered toxin with anti-{alpha}7 neuronal acetylcholine receptor activity

Though it possesses four disulfide bonds the three-fingeredfold is amenable to chemical synthesis, using a Fmoc-based method.Thus, we synthesized a three-fingered curaremimetic toxin fromsnake with high yield and showed that the synthetic and nativetoxins have the same structural and biological properties. Bothwere characterized by the same 2D NMR spectra, identical highbinding affinity (K_d = 22 ± 5 pM) for the muscular acetylcholinereceptor (AChR) and identical low affinity (K_d = 2.0 ±0.4 µM) for     

Site-directed mutagenesis in hemoglobin: test of functional homology of the F9 amino acid residues of hemoglobin {alpha} and {beta} chains

The cysteine residue at F9(93) of the human hemoglobin (Hb A)ß chain, conserved in mammalian and avian hemoglobins,is located near the functionally important 1–ß2interface and C-terminal region of the ß chain and isreactive to sulfhydryl reagents. The functional roles of thisresidue are still unclear, although regulation of local bloodflow through allosteric S-nitrosylation of this residue is proposed.To clarify the role of this residue and its functional homologyto F9(88) of the chain, we measured oxygen equilibrium curves,UV-region derivative spectra, Soret-band absorption spectra,the number of titratable -SH groups with p-mercuribenzoate andthe rate of reaction of these groups with 4,4'-dipyridine disulfidefor three recombinant mutant Hbs with single amino acid substitutions:AlaCys at 88 (rHb A88C), CysAla at 93ß (rHb C93ßA)and CysThr at 93ß (rHb C93ßT). These Hbs showedincreased oxygen affinities and impaired allosteric effects.The spectral data indicated that the R to T transition upondeoxygenation was partially restricted in these Hbs. The numberof titratable -SH groups of liganded form was 3.2–3.5for rHb A88C compared with 2.2 for Hb A, whereas those for rHbC93ßA and rHb C93ßT were negligibly small. The reductionof rate of reaction with 4,4'-dipyridine disulfide upon deoxygenationin rHb A88C was smaller than that in Hb A. Our experimentaldata have shown that the residues at 88 and 93ß have definiteroles but they have no functional homology. Structure–functionrelationships in our mutant Hbs are discussed.     

Hyperthermostable mutants of Bacillus licheniformis {alpha}-amylase: multiple amino acid replacements and molecular modelling

We have identified previously two critical positions for thethermostability of the highly thermostable -amylase from Bacilluslicheniformis. We have now introduced all 19 possible aminoacid residues to these two positions, His 133 and Ala209. Themost favourable substitutions were to Ile and Val, respectively,which both increased the half-life of the enzyme at 80°Cby a factor of 3. At both positions a stabilizing effect ofhydrophobic residues was observed, although only in the caseof position 133 could a clear correlation be drawn between thehydrophobicity of the inserted amino acid and the gain in proteinstability. The construction of double mutants showed a cumulativeeffect of the most favourable and/or deleterious substitutions.Computer modelling was used to generate a 3-D structure of thewild-type protein and to model substitutions at position 209,which lies in the conserved (/ß)₈ barrel domain of-amylase; Ala209 would be located at the beginning of the thirdhelix of the barrel, in the bottom of a small cavity facingthe fourth helix. The model suggests that replacement by, forexample, a valine could fill this cavity and therefore increaseintra- and interhelical compactness and hydrophobic interactions.     

Characterization of the interactions of a bifunctional inhibitor with {alpha}-thrombin by molecular modelling and peptide synthesis

A potent thrombin inhibitor, [D-Phe⁴⁵, Arg⁴⁷] hirudin 45-65,that contains an active site-directed sequence D-Phe-Pro-Arg-Pro,an exosite specific fragment hirudin 55-65 (H55-65) and a linkerportion hirudin 49-54, was designed based on the hirudin sequence[DiMaio et al. (1990) J. Biol. Chem., 265, 21698-21798]. A three-dimensionalmodel of the complex between the B-chain of human thrombin andthe inhibitor [D-Phe45, Arg47⁴⁵, Arg⁴⁷] hirudin 45-65 was constructedusing molecular modelling starting from the X-ray Ca coordinatesof the thrombin-hirudin complex and the NMR-derived structureof the thrombin-bound hirudin 55 - 65. The contribution of theH49 -54 fragment to the thrombin - inhibitor interaction wasdeduced by examining a series of analogs containing single glycinesubstitution and analogs with reduced number of residues withinthe linker. The results were consistent with the molecular modellingobservations i.e. the H49-54 fragment serves the role of a spacerin the binding interaction and could be replaced by four glycineresidues. The studies on the interaction of the exosite-directedportion of the inhibitor with thrombin using a series of syntheticH55 -65 analogs demonstrated that residues AspH55 to ProH60play a major role in binding to human thrombin where the sidechains of PheH56, IleH59 and GluH57 showed critical contributions.Molecular modelling suggested that these side chains may contributeto inter- and intramolecular hydrophobic and electrostatic interactions,respectively.     

Efficient generation of a reshaped human mAb specific for the {alpha} toxin of Clostridium perfringens

We have used the technique of antibody reshaping to producea humanized antibody specific for the a toxin of Clostridiumperfringens. The starting antibody was from a mouse hybridomafrom which variable (V) region nucleo-tide sequences were determined.The complementarity-determining regions (CDRs) from these Vregions were then inserted into human heavy and light chainV region genes with human constant region gene fragments subsequentlyadded. The insertion of CDRs alone into human frameworks didnot produce a functional reshaped antibody and modificationsto the V region framework were required. With minor frameworkmodifications, the affinity of the original murine mAb was restoredand even exceeded. Where affinity was increased, an alteredbinding profile to overlapping peptides was observed. Computermodelling of the reshaped heavy chain V regions suggested thatamino acids adjacent to CDRs can either contribute to, or distort,CDR loop conformation and must be adjusted to achieve high bindingaffinity.     

The elusive role of the N-terminal extension of {beta}A3- and {beta}Al-crystallin

ß-Crystallins are structural lens proteins with aconserved two-domain structure and variable N- and C-terminalextensions. These extensions are assumed to be involved in quaternaryinteractions within the ß-crystallin oligomers orwith other lens proteins. Therefore, the production of ßA3-and ßAl-crystallin from the single ßA3/A1mRNA by dual translation initiation is of interest. These crystallinsare identical, except that ßAl has a much shorterN-terminal extension than ßA3. This rare mechanismhas been conserved for over 250 million years during the evolutionof the ßA3/A1 gene, suggesting that the generationof different N-terminal extensions confers a selective advantage.We therefore compared the stability and association behaviourof recombinant ßA3- and ßAl-crystallin.Both proteins are equally stable in urea- and pH-induced denaturationexperiments. Gel filtration and analytical ultracentrifugationestablished that ßA3 and ßA1 both form homodimers.In the water-soluble proteins of bovine lens, ßA3and ßA1 are present in the same molecular weight fractions,indicating that they oligomerize equally with other ß-crystallins.¹H-NMR spectroscopy showed that residues Met1 to Asn22 of theN-terminal extension of ßA3 have great flexibilityand are solvent exposed, excluding them from protein interactionsin the homodimer. These results indicate that the differentN-terminal extensions of ßA3 and ßA1 donot affect their homo- or heteromeric interactions.     

Family of G protein {alpha} chains: amphipathic analysis and predicted structure of functional domains

The G proteins transduce hormonal and other signals into regulationof enzymes such as adenylyl cyclase and retinal cGMP phosphodiesterase.Each G protein contains an subunit that binds and hydrolyzesguanine nucleotides and interacts with ß subunitsand specific receptor and effector proteins. Amphipathic andsecondary structure analysis of the primary sequences of fivedifferent chains (bovine _s, _t1 and _t2, mouse _i, and rat _o)predicted the secondary structure of a composite chain (_avg).The chains contain four short regions of sequence homologousto regions in the GDP binding domain of bacterial elongationfactor Tu (EF-Tu). Similarities between the predicted secondarystructures of these regions in _avg and the known secondary structureof EF-Tu allowed us to construct a three-dimensional model ofthe GDP binding domain of _avg. Identification of the GDP bindingdomain of _avg defined three additional domains in the compositepolypeptide. The first includes the amino terminal 41 residuesof _avg, with a predicted am phipathic helical structure; thisdomain may control binding of the chains to the ßcomplex. The second domain, containing predicted ßstrands and helices, several of which are strongly amphipathic,probably contains sequences responsible for interaction of chains with effector enzymes. The predicted structure of thethird domain, containing the carhoxy terminal 100 amino acids,is predominantly ß sheet with an amphipathic helixat the carboxy terminus. We propose that this domain is reponsiblefor receptor binding. Our model should help direct further experimentsinto the structure and function of the G protein chain.     

Stabilization and activation of {alpha}-chymotrypsin in water-organic solvent systems by complex formation with oligoamines

Formation of enzyme–oligoamine complexes was suggestedas an approach to obtain biocatalysts with enhanced resistancetowards inactivation in water–organic media. Complex formationresults in broadening (by 20–40% v/v ethanol) of the rangeof cosolvent concentrations where the enzyme retains its catalyticactivity (stabilization effect). At moderate cosolvent concentrations(20–40% v/v) complex formation activates the enzyme (by3–6 times). The magnitude of activation and stabilizationeffects increases with the number of possible electrostaticcontacts between the protein surface and the molecules of oligoamines(OA). Circular dichroism spectra in the far-UV region show thatcomplex formation stabilizes protein conformation and preventsaggregation in water–organic solvent mixtures. Two populationsof the complexes with different thermodynamic stabilities werefound in -chymotrypsin (CT)–OA systems depending on theCT/OA ratio. The average dissociation constants and stoichiometriesof both low- and high-affinity populations of the complexeswere estimated. It appears that it is the low-affinity siteson the CT surface that are responsible for the activation effect.     

Model complexes of tumor necrosis factor-{alpha} with receptors Rl and R2

The biological activities of tumor necrosis factor- (TNF-) aremediated by two different receptors, TNFR1 and TNFR2. To analyzethe receptor binding site(s) of TNF-, molecular models havebeen built of the complexes of TNF- with the extracellular regionsof receptors Rl and R2, based on the known crystal structuresof TNF- and lymphotoxin bound to Rl. The model structure ofR2 from residues 18-160 was built by analogy to the crystalstructure of Rl in complex with lymphotoxin. The amino acidsequences of Rl and R2 show 27.5% identity over this regionand were aligned with five insertions and three deletions. Thereare 18 conserved cysteines that form disulfides. R2 has lostone pair of cysteines compared with Rl, but two new cysteineswere modeled as forming a new disulfide bond. Both symmetricand asymmetric trimers of TNF- were used to model the complexeswith TNFR1 and R2. An analysis of differences in the model complexesshowed good agreement with data on the differential bindingof TNF mutants to its two receptors.     

Preparation and crystal structure of the recombinant {alpha}1/{alpha}2 catalytic heterodimer of bovine brain platelet-activating factor acetylhydrolase Ib

The intracellular form of mammalian platelet activating factoracetylhydrolase found in brain (PAF-AH Ib) is thought to playa critical role in control in neuronal migration during cortexdevelopment. This oligomeric complex consists of a homodimerof the 45 kDa (ß) LIS1 protein, the product of thecausative gene for type I lissencephaly, and, depending on thedevelopmental stage and species, one of three possible pairsof two homologous ~26 kDa     

Mutational analysis of the N-capping box of the {alpha}-helix of chymotrypsin inhibitor 2

The N-terminus of the helix of the chymotrypsin inhibitor 2from barley (CI2) has an N-capping box (Ser at the first positionin the helix and Glu at position 4) as well as a frequentlyfound Glu at position 3. The energetic importance of this motifhas been studied by determining the free energy of unfoldingof the wild-type and protein mutants derived from those residuesusing guanidinium chloride-induced denaturation and differentialscanning microcalorimetry. Mutating N-cap residue Ser31 to eitherAla or Gly destabilizes CI2 by 0.8-1 kcal mol^–1. Truncationof the box in the mutants SA31EA33EA34 or SG31EA33EA34 destabilizesthe protein by 1.5–2 kcal mol^–1. The N-capping boxis an important motif in stabilizing proteins and delineatingthe beginning of -helices in the pathway of protein folding.     

The Influence of an Adsorbate Layer on Adatom Diffusion and Island Nucleation: Fe on \hbox{Si(111)-}\sqrt{3} \times \sqrt{3} \hbox{-Au}

Using scanning tunneling microscopy, the influence of a thin Au layer on the diffusion of Fe adatoms and the subsequent island nucleation on a Si(111) surface is investigated. The adsorbate induces thestructure that increases the surface mobility of subsequently deposited Fe atoms, resulting in the formation well-defined nanoclusters. Surprisingly, the domain walls—inherent to the reconstruction—do not influence the surface diffusion, which demonstrates that the passivation is of much more importance for the self-assembly than the surface corrugation. Using the decoupling of the diffusion and nucleationonthe surface and the reactionwiththe surface and conventional nucleation theory, the activation energy for surface diffusionE_d = 0.61 eV and the critical cluster sizei = 3 are determined, which reveal the microscopic details of the diffusion and nucleation processes.     

2-溴-3-{4-[2-(5-乙基-2-吡啶基)-乙氧基]苯基}丙烯酸甲酯的合成

以5－乙基－2吡啶基乙醇为起始原料,与对氟硝基苯缩合,Pd/C催化加氢,得4－[2－（5－乙基－2－吡啶基）－乙氧基]苯胺,再经重氮化和桑德迈尔反应,制得2－溴－3－{4－[2－（5－乙基－2－吡啶基）－乙氧基]苯基}丙烯酸甲酯,总收率为65．6％,最终产物经IR和NMR结构确证。     

First zinc complex of an amino acid pyrazole conjugate: synthesis and crystal structure of {Zn[3-(Ac-Phe)-5-methyl-pyrazole]2}2(ClO4)4

The zinc(II) complex, of the dimeric {Zn[3-(Ac-Phe)-5-methyl-pyrazole]₂}₂(ClO₄)₄ (5) was obtained by the reaction of the amino acid–pyrazole conjugate, 3-(Ac-Phe)-5-methyl-pyrazole (4) and Zn(ClO₄)₂. An acetone solvate of this complex was analyzed by single crystal X-ray crystallography, which establishes the dimeric nature of the complex with a large Zn–Zn separation of 5.551(6) Å and exbihiting coordination to the distorted trigonal bipyramidal zinc centers through the amino acid CO and the pyrazole N.     

Structural Pitstops and Turnoffs on the Way to the Birefringent 2-D Layer Structure $$\{\hbox{(tmeda)M[Hg(CN)}_{2}]_{2}\}[\hbox{HgCl}_{4}]$$ (M=Cu,Ni)

The reaction of N,N,N′,N′-tetramethylethylenediamine (tmeda) and NiCl₂ with the soft, Lewis acidic Hg(CN)₂ and HgCl₂ in ethanol formed the 2-D layer structure {(tmeda)Ni[Hg(CN)₂]₂}[HgCl₄] (1), isostructural to the Cu(II) analogue. Complex 1 crystallizes in the tetragonal, non-centric m space group and contains a 2-D cationic layer of {(tmeda)Ni[Hg(CN)₂]₂}²⁺ units in which the six-coordinate Ni(II) centres are bridged by four Hg(CN)₂ groups and capped by a tmeda ligand. This array is interspersed with a layer of [HgCl₄]²⁻ anions, which form bridging Hg–Cl bonds with the Hg(CN)₂ units. The formation of 1 is very sensitive to reaction conditions; the addition of water to the mixture yields the related “structural pitstop” 2-D array {(tmeda)Ni(H₂O)[Hg(CN)₂]}{[Hg(CN)Cl]₂Cl₂}·H₂O (2), in which the halide migration among Hg(II) centres is incomplete. The larger zero-field splitting D-values of 6.91(1) cm⁻¹ for 1 vs. 2.85(4) cm⁻¹ for 2 indicate that some weak antiferromagnetic interactions are likely present in 1. The reaction of tmeda/Cu(ClO₄)₂·6H₂O with Hg(CN)₂ yields [Cu(tmeda)(μ-OH)(ClO₄)]₂[Hg(CN)₂(H₂O)₂][Hg(CN)₂] (3) which is composed of [Cu(tmeda)(μ-OH)(ClO₄)]₂ dimers in which the anions cis-bridge the copper(II) centres in the axial positions as well as bind to two adjacent Hg(CN)₂ moieties; the perchlorate anion is acting as a rare η⁴–μ₄–ClO₄ ligand. N-cyano interactions also exist between the Hg(II) centres; overall, a 2-D corrugated sheet structure which stacks via Cl–O–Hg bridges to yield a 3-D array is formed. The χ_M T value for 3 decreases with decreasing temperature; a maximum in χ_M vs. T at 20 K is also observed. This is consistent with antiferromagnetic interactions within the copper(II) dimer, which were fit with the Bleaney-Bowers model to yield J=−23.1(1) cm⁻¹, g=2.113(5) and a paramagnetic impurity P=0.017(1).Special Issue to honour Professor Richard J. Puddephatt     

Mathematical modelling of protein diffusion in microcapsules: A comparison with experimental results

The objective of this study was to develop a general diffusion model for describing mass transport phenomena and membrane diffusivities in alginate—polylysine (PLL) microcapsules. Good agreement between calculated and experimental protein concentration profiles was obtained based on a microcapsule model, consisting of a capsule membrane containing a partially impermeable alginate gel core with a decreasing gel pore size towards the centre of the capsule. The apparent size of the impermeable gel core and the capsule membrane permeability were directly dependent on the size of the diffusing protein and the alginate-PLL reaction time. The presence of this impermeable core may hinder the commercial and clinical use of these microcapsules in cell culture engineering and cell transplantation by affecting cell viability.     

Second-generation octarellins: two new de novo ({beta}/{alpha})8 polypeptides designed for investigating the influence of {beta}-residue packing on the {alpha}/{beta}-barrel structure stability

The sequence of octarellin I, the first de novo (ß/)₈polypeptide, was revised according to several criteria, amongothers the symmetry of the sequence, ß-residue volumeand hydrophobicity, and charge distribution. These considerationsand the overall conclusions drawn from the first design ledto two new sequences, corresponding to octarellins II and III.Octarellin II retains perfect 8-fold symmetry. Octarellin IIIhas the same sequence as octarellin II, except for the ß-strandswhich exhibit a 4-fold symmetry. The two proteins were producedin Escherichia coli. Infrared and CD spectral analyses of octarellinsII and III reveal a high secondary structure content. Non-denaturinggel electrophoresis, molecular sieve chromatography and analyticalultracentrifugation suggest that both of these second-generationartificial polypeptides exist as a mixture of a monomer anda dimer form. Octarellins II and III are at least 10 times moresoluble than octarellin I. Ureainduced unfolding followed byfluorescence emission suggests that the tryptophan residues,designed to be buried in the (ß/)₈, are indeed packedin the hydrophobic core of both proteins. However, octarellinIII displays a higher stability towards urea denaturation, indicatingthat introducing 4-fold symmetry into the ß-barrelmight be important for stability of the overall folding.