Weak points of antiparallel {beta}-sheets. How are they filled up in globular proteins?

A lack of knowledge about the construction of tight packingis now the main obstacle for a successful design of artificialproteins. In this paper we examine a way of close packing antiparallelß-sandwihes. We show that there are some ‘weakpoints’ at the surfaces of ß-sheets, which cannotbe filled by the surrounding aliphatic side chains that arethe most abundant. Theoretically, these ‘weak points‘can be filled either by aromatic side chains of the same sheetor by the residues of the other parts of the protein molecule.The analysis of protein structures shows that both possibilitiesare used by nature and that there are many cases when these‘weak points’ are not filled by any atom. They remainfree and form a majority of the defects of close packing inprotein globules.     

On the use of machine learning to identify topological rules in the packing of {beta}-strands

The machine learning program GOLEM was applied to discover topologicalrules in the packing ofß-sheets in /ß-domainproteins. Rules (constraints) were determined for four featuresof ß-sheet packing: (i) whether a ß-strandis at an edge; (ii) whether two consecutive ß-strandspack parallel or anti-parallel; (iii) whether twoß-strandspack adjacently; and (iv) the winding direction of two consecutiveß-strands. Rules were found with high predictive accuracyand coverage. The errors were generally associated with complicationsin domain folds, especially in one doubly wound domains. Investigationof the rules revealed interesting patterns, some of which wereknown previously, others that are novel. Novel features include(i) the relationship between pairs of sequential strands isin general one of decreasing size; (ii) more sequential pairsof strands wind in the direction out than in; and (iii) it takesa larger alteration in hydrophobicity to change a strand fromwinding in the direction out than in. These patterns in thedata may be the result of folding pathways in the domains. Therules found are of predictive value and could be used in thecombinatorial prediction of protein structure, or as a generaltest of model structures, e.g. those produced by threading.We conclude that machine learning has a useful role in the analysisof protein structures.     

Secondary structure characterization of {beta}-lactamase inclusion bodies

The secondary structure of proteins in E.coli inclusion bodieswas investigated via Raman spectroscopy. Inclusion bodies werepurified from cells expressing different forms of RTEM ß-lactamaseand grown at either 37 or 42° C. All of the solid phaseinclusion body samples examined gave amide I band spectra thatwere perturbed from that of the native, purified protein inboth solution and powder forms; secondary structure estimatesindicated significant decreases in a-helix and increases inß-sheet contents in the inclusion body samples. The structureestimates for inclusion bodies isolated from 37°C cultureswere similar, regardless of aggregate localization in the E.colicytoplasmic or periplasmic spaces or ß-lactamase precursorcontent. Inclusion bodies obtained from 42°C cells exhibiteda further reduction of ß-helix and augmentation of ß-sheetcontents relative to those from 37°C cultures. These resultsare consistent with the paradigm for inclusion body formationvia the self-association of intra-cellular folding intermediateshaving extensive secondary structure content. Further, the overallsecondary structure content of inclusion bodies is not significantlyaffected by subcellular compartmentalization, but may be alteredat increased temperatures     

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.     

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.     

An SS1-SS2 {beta}-barrel structure for the voltage-activated potassium channel

To examine the feasibility of a ß structure for thepore-lining region of the voltage-gated potassium channel, wehave characterized a family of 12 antiparallel ß-barrels.Each is comprised of four identical pairs of ß-strandsorganized with approximate 4-fold symmetry about a channel axis.The Cand N-termini of the ß-strand pairs are assumedto be at the extracellular end of the channel, and each pairis connected by a hairpin turn at the intracellular end of thechannel. The models differ in the residues located in the hairpinturn and in the orientation of the two strands of each pairin the barrel, i.e. whether the C-terminus of a pair is clockwise(CW) or counterclockwise (CCW) from the N-terminus when thechannel is viewed from outside the cell. Following known structureprecedents and potential energy predictions, the barrel is assumedto be right-twisting in all cases. All models have crowded layersof inward-projecting aromatic sidechains near the center ofthe channel which could regulate channel selectivity. The modelswith an odd number of amino acids in the hairpin turn have theadvantage of predicting that F433 points into the barrel, butthe disadvantage that V438 does not. Of these models, two ofthe models are most consistent with the external tetraethylanunonhim(TEA) block data, and of those, one (T439 CCW 3:5) is most consistentwith the internal TEA block data.     

Predicted {beta}-structure stability parameters under experimental test

Recently, ß-structure stability parameters have beenmeasured for all 20 natural amino acids, separately for theedge and separately for the internal ß-sheet positions.Theoretical estimations of all these stability parameters havebeen obtained earlier on the basis of the molecular theory.Comparison shows a good concordance between the recent experimentalresults and the earlier theoretical predictions.     

Molecular dynamics simulations of the whey protein {beta}-lactoglobulin

Molecular dynamics simulations have been used to model the motionsand conformational behavior of the whey protein bovine ß-lactoglobulin.Simulations were performed for the protein by itself and complexedto a single retinol ligand located in a putative interior bindingpocket. In the absence of the retinol ligand, the backbone loopsaround the opening of this ulterior pocket shifted inward topartially close off this cavity, similar to the shifts observedin previously reported molecular dynamics simulations of theuncomplexed form of the homologous retinol binding protein.The protein complexed with retinol does not exhibit the sameconformational shifts. Conformational changes of this type couldserve as a recognition signal allowing in vivo discriminationbetween the free and retinol complexed forms of the 3-lactoglobulinmolecule. The unusual bending of the single a-helix observedin the simulations of retinol binding protein were not observedin the present calculations     

Alcohol-induced changes of {beta}-lactoglobulin - retinol-binding stoichiometry

It has been demonstrated using CD that ethanol induces importantsecondary structure changes of ß-lactoglobulin. CDspectra indicate that ß-lactoglobulin secondary structure,which is mainly composed of ß-strands, becomes mostly-helical under the influence of the solvent polarity changes.The midpoint of ß-strand/-helix transition in ß-lactoglobulinis observed at dielectric constant {small tilde}60 (35% ethanol;v/v). According to CD measurements, the ethanol-dependent secondarystructure changes are reversible. The alkylation of lysines-NH₂ in ß-lactoglobulin weakens the central ß-barrelstructure, since the ß-strand/-helix transition midpointof alkylated ß-lactoglobulin is shifted to lower ethanolconcentration (25% ethanol; v/v). ß-Lactoglobulinstructural changes are triggering the dissociation of the ß-lactoglobulin- retinol complex as judged from complete quenching of its fluorescencein ethanol concentration >30% (v/v). However, in 20% ethanol(v/v), ß-lactoglobulin still retains most of its nativesecondary structure as shown by CD and, in this condition, oneß-lactoglobulin molecule binds an additional secondretinol molecule. This suggests that the highly populated speciesobserved around 20% ethanol (v/v) might represent an intermediatestate able to bind two molecules of retinol.     

Thermostable variants of bovine {beta}-lactoglobulin

The thermal stability of bovine ß-lactoglobulin (BLG)has been enhanced by the introduction of an additional disulfidebond. Wild-type BLG has two disulfide bonds, C106–C119and C66–C160, with a free cysteine at position 121. Wehave designed, with the aid of molecular modeling calculations,two mutants of a recombinant BLG (rBLG), L104C and A132C. Moleculardynamics simulations were performed at 300K to study the effectof these alterations on the conformation of the protein. Thesemutants were then created by site-directed mutagenesis and purifiedfrom Escherichia coli carrying a tac expression vector usinga two-step renaturation method. Formation of disulfide linkagesin the correct arrangement, as designed, was confirmed by peptidemapping. In contrast to wild-type rBLG, which polymerizes attemperatures >65°C, neither of the mutant proteins polymerized.The conformational stability of the L104C and A132C mutant proteinsagainst thermal denaturation has been substantially increased(8- 10°C) as compared with wild-type rBLG. Furthermore,the A132C rBLG exhibits an enhanced stability against denaturationby guanidine hydrocnloride as compared with the wild-type orL104C rBLG     

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.     

Mutant forms of {beta}-galactosidase with an altered requirement for magnesium ions

By random approaches we have previously isolated many variantsof Escherichia coli ß-galactosidase within a shortcontiguous tract near the N-terminus (residues 8–12 ofwildtype enzyme), some of which have increased stability towardsheat and denaturants. The activity of these mutants was originallyanalysed and quantitated in situ in activity gels without theaddition of magnesium ions to the buffer system. We now showthat the improved stability is only observable under such conditionsof limiting magnesium ion concentrations or in the presenceof appropriate concentrations of a metal chelator. In the presenceof EDTA, purified preparations of one of these mutant enzymeswere much more resistant to denaturants than wild-type, butthis differential was completely nullified in the presence of1 mM Mg²⁺. However, the stability of this mutant enzyme in EDTAwas lower than that shown by it, or the wild-type enzyme, inthe presence of magnesium ions. In addition, certain alterationswithin another N-terminal tract (residues 27–31 of wild-type)resulted in enzymes with greater dependence on Mg²⁺ than naturalß-galactosidase. We conclude that a small number ofresidue changes in a large protein can profoundly modulate therequirement for metal ion stabilization, allowing partial abrogationof this need in certain cases. Thus, some enzymes which requiredivalent metal ions for structural purposes only may be engineeredtowards metal independence.     

Synthesis, purification and initial structural characterization of octarellin, a de novo polypeptide modelled on the {alpha}/{beta}-barrel Proteins

We have attempted to construct an artificial polypeptide thatfolds like the eight-stranded parallel ß-barrel structures.Our approach consists of repeating eight times a unit peptidedesigned to adopt a ‘ß-strand/-helix’pattern. A first ‘test’ sequence for this structuralunit was deduced from a series of parameters defined after ananalysis of three natural /ß-barrel proteins and includingprincipally the lengths of the secondary structure elements,the /ß packing and the fitting on average Garnierprofiles. The gene encoding this structural unit was synthesized,cloned and expressed in Escherichia coli either as a monomeror as direct repeats of 2–12 units. Preliminary structuralcharacterization of the 7-, 8- and 9-fold unit polypeptidesby circular dichroism measurements indicates the presence ofthe predicted amount of -helix in the three proteins. Furtheranalysis by urea-gradient gel electrophoresis demonstrates that,in the conditions tested, only the 8-fold unit polypeptide formsa compact structure through a cooperative and rapid two-statefolding transition involving long-range molecular interactions.     

Spectroscopic investigation of structure in octarellin (a de novo protein designed to adopt the {alpha}/{beta}-barred packing)

We present here a spectroscopic structural characterizationof octarellin, a recently reported de novo protein modelledon /ß-barrel proteins [K. Go raj, A.Renard and J.A.Martial(1990) Protein Engng, 3, 259–266]. Infrared and Ramanspectra analyses of octarellin‘s secondary structure revealthe expected percentage of -helices (30%) and a higher ß-sheetcontent (40%) than predicted from the design. When the Ramanspectra obtained with octarellin and native triosephosphateisomerase (a natural /ß-barrel) are compared, similarpercentages of secondary structures are found. Thermal denaturationof octarellin monitored by CD confirms that its secondary structuresare quite stable, whereas its native-like tertiary fold is not.Tyrosine residues, predicted to be partially hidden from solvent,are actually exposed as revealed by Raman and UV absorptionspectra. We conclude that the attempted /ß-barrelconformation in octarellin may be loosely packed. The criteriaused to design octarellin are discussed and improvements suggested.     

Display of {beta}-lactamase on the Escherichia coli surface: outer membrane phenotypes conferred by Lpp'-OmpA'-{beta}-lactamase fusions

Bacterial cell-surface exposure of foreign peptides and solubleproteins has been achieved recently by employing a fusion proteinmethodology. An Lpp'–OmpA(46–159)–Bla fusionprotein has been shown previously to display the normally periplasmicenzyme ß-lactamase (Bla) on the cell surface of theGram-negative bacterium Escherichia coli. Here, we have investigatedthe role of the OmpA domain of the tripartite fusion proteinin the surface display of the passenger domain (Bla) and havecharacterized the effects of the fusion proteins on the integrityand permeability of the outer membrane. We show that in additionto OmpA(46–159), a second OmpA segment, consisting ofamino acids 46–66, can also mediate the display of Blaon the cell surface. Other OmpA domains of various lengths (aminoacids 46–84, 46–109, 46–128, 46–141and 46–145) either anchored the Bla domain on the periplasmicface of the outer membrane or caused a major disruption of theouter membrane, allowing the penetration of antibodies intothe cell. Detergent and antibiotic sensitivity and periplasmicleakage assays showed that changes in the permeability of theouter membrane are an unavoidable consequence of displayinga large periplasmic protein on the surface of E.coli. This isthe first systematic report on the effects that cell surfaceengineering may have on the integrity and permeability propertiesof bacterial outer membranes.     

An engineered Staphylococcus aureus PCI {beta}-lactamase that hydrolyses third-generation cephalosporins

The ß-lactamase from Staphylococcus aureus PCI hasbeen cloned into an Escherichia coli vector for site-directedmutagenesis and high-level protein expression. A mutant enzymehas been produced in which Ala238 is replaced by a serine, andIle239 is deleted (A238S:I239del). The engineered enzyme hydrolysesthird-generation cephalosporins substantially more rapidly thanthe parental enzyme does, while hydrolysis of benzylpenicillinis slower with the mutant than with the wild-type and nativeenzymes. The mutant P-lactamase has been crystallized and thestructure determined and refined at 2.8 A resolution. The dispositionof the ß-strand which forms the side of the activesite is altered in comparison with the native S.aureus ß-lactamasestructure, widening the active site cleft and providing spaceto accommodate the bulky side-chains of the third-generationcephalosporins.     

Introduction of a stabilizing 10 residue {beta}-hairpin in Bacillus subtilis neutral protease

A 10 residue ß-hairpin, which is characteristic ofthermostable Bacillus neutral proteases, was engineered intothe thermolabile neutral protease of Bacillus subtilis. Therecipient enzyme remained fully active after introduction ofthe loop. However, the mutant protein exhibited autocatalyticnicking and a 0.4°C decrease in thermostability. Two additionalpoint mutations designed to improve the interactions betweenthe enzyme surface and the introduced ß-hairpin resultedin reduced nicking and increased thermostability. After theintroduction of both additional mutations in the loopcontainingmutant, nicking was largely prevented and an increase in thermostabilityof 1.1°C was achieved.     

Strong inhibition of fibrinogen binding to platelet receptor {alpha}IIb{beta}3 by RGD sequences installed into a presentation scaffold

In order to probe the structural constraints on binding of RGDsequences to the platelet receptor _IIbß₃ we have usedrecombinant DNA techniques to install the RGD sequence into‘presentation scaffolds’, small proteins of known3-D structure chosen to present guest sequences in constrainedorientations. Using Escherichia coli expression systems we madesequence variants in which loop residues of the immunoglobulinV_L domain REI and of human interleukin-1ß were replaced(without changing polypeptide length) by the RGD sequence atpositions predicted, based on small molecule studies, to orientthe RGD moiety into an active conformation. These variants donot compete for fibrinogen binding to _IIbß₃ up toalmost 1 mM concentration. Unfolded or proteolytically fragmentedforms of these same proteins do compete, however, showing thatthe RGD sequences in the mutants must be prohibited from bindingby constraints imposed by scaffold structure. To suppress theeffects of such structural constraints we constructed two sequencevariants in which RGD-containing sequences 42–57 or 44–55from the snake venom platelet antagonist kistrin were inserted(this increasing the length of the loop) into the third complementaritydetermining loop of REI. Both of these variants compete stronglyfor fibrinogen binding with IC₅₀s in the nM range. These results,plus data on kistrin-related peptides also presented here, suggestthat the molecular scaffold REI is capable of providing to aninstalled sequence a structural context and conformation beneficialto binding. The results also suggest that in order to bind wellto _IIbß₃, RGD sequences in protein ligands must eitherproject significantly from the surface of the scaffold and/orretain a degree of conformational flexibility within the scaffold.Molecular scaffolds like REI should prove useful in the elucidationof structure-function relationships and the discovery of newactive sequences, and may also serve as the basis for noveltherapeutic agents.     

The role of the sequence extensions in {beta}-crystallin assembly

The modular construction of the eye lens ß-crystallinsmakes them good candidates for protein engineering to ascertainthe rules of assembly of oligomers. X-ray studies have shownthat although the polypeptide chains of ßB2-crystallinand -crystallins fold to form similar N- and C-terminal domains,the conformation of the connecting peptides are such that the-crystallins are monomers and the ß-crystallin isa dimer. Unlike -crystallins, the numerous -crystallins haveextensions of variable sequence from the globular domains. Wehave tested the effect of removing the N- and C-terminal extensionsfrom rat ßB2-crystallin using a bacterial expressionsystem. Abundant proteins were produced in Escherichia coliusing the pET or pQE vectors. Full-length and truncated proteinswere purified and checked for refolding using circular dichroism.Sizing of the truncated proteins using gel filtration chroma-tographyshowed that the absence of either the N- or C-terminal extensiondoes not affect dimerization of ßB2-crystallin.     

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