Insertion of an elastase-binding loop into interleukin-1{beta}

The protease-binding sequence EAIPMSIPPE from 1-antitrypsinhas been inserted into the cytokine interleykin-1ß,replacing residues 50–53. The resulting mutant proteinwas cleaved specifically at a singly site by elastase and chymotrypsin,but not by trypson. The cleavage by elastase was shown to bebetween Met and Ser of the inserted loop. In contrast, wild-typeinterleukin is not sus-ceptible to cleavage by any of theseenzymes. The mutant protein acts as an inhibitor of elastase,with a K1 of 30 µM. The wild type displays no such inhibitoryactitvity. The overall structure of the mutant, as demonstratedbyu CD, appears to be indistinguishabel from that fo the wildtype. These results indicate that the protease-binding regionfo 1-antitrypson can be recognized and is active even withinthe context of an entirely differentproteinstructure. Giventhat interleukinm-1ß binds to, and is intenalizedby, many types of cells, this hybrid protein also demonstratesthe feasibility of using interleukin-1ß as a deliverysystem for useful therapeutic agents.     

Cloning of rabbit interleukin-1{beta}: differential evolution of IL-1{alpha} and IL-1{beta} proteins

We have cloned the rabbit IL-1ß cDNA, which encodesa 268 amino acid precursor similar in length to other sequencedIL-1 precursors. Comparison of all published IL-1 and IL-1ßsequences respectively indicates that the IL-1 gene family isevolving faster than the IL-1ß family, and that thetwo genes diverged –270 million years ago. Surprisingly,there are differences in the regions preferentially conservedwithin the two families. The IL-1 family is most conserved atthe amino terminus whereas the IL-1ß family is mostconserved in the carboxy-terminal half. This is despite thefact that the carboxy-terminal half encodes the active portionof both molecules and would be expected to adopt a similar ß-sheetstructure in IL-1 as in the published X-ray structure of matureIL-1ß. These findings suggest that differences inthe function and properties of the IL-1 and IL-1ßprecursor molecules may have been conserved. These differencesmay therefore provide an explanation for the existence of twoIL-1 molecules.     

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     

A point mutation that decreases the thermal stability of human interferon {gamma}

We have identified a mutation of human gamma-interferon (IFN)causing a temperature-sensitive phenotype. We used a randomizedoligonucleotide to mutagenize a synthetic human IFN gene, thenscreened the resulting mutants produced in Escherichia colifor proteins with altered biological activity. One mutant proteinselected for detailed characterization exhibited < 0.3% ofthe specific biological activity of native IFN in an antiviralactivity assay performed at 37°C. However, the protein boundthe human IFN receptor with native efficiency at 4°C. Sequencingthe plasmid DNA encoding this protein snowed that the mutationchanged the lysine residue at amino acid 43 to glutamic acid(IFN/K43E). Site-specific mutagenesis at amino acid 43 showedthat this protein's phenotype resulted from positioning a negativecharge at position 43. Structural characterization of IFN/K43Eusing CD demonstrated that the protein had native conformationat 25°C, but assumed an altered conformation at 37°C.IFN/K43E in this altered conformation bound poorly to the IFNreceptor at 37°C, providing a rationale for the mutant'sdecreased antiviral activity.     

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.     

Estimating the twist of {beta}-strands embedded within a regular parallel {beta}-barrel structure

The parallel ß-barrel is a recurrent structural motiffound in a large variety of different enzymes belonging to thefamily of /ß-proteins. It has been shown previouslythat the hyperboloid can be considered as a scaffold describingthe parallel ß-barrel structure. To assess restraintson ß-strand twist imposed by a given scaffold geometry,the notion of scaffold twist, T_s, is introduced. From T_s, theß-strand twist (Tw_ß) expected for a givenscaffold geometry can be derived and it is verified that thiscomputed twist can be used to identify ß-barrels characterizedby good hydrogen bonding. It is noted that Tw_ß isonly slightly affected for ß-barrels differing inthe number (N) of ß-strands, suggesting that restraintson main-chain conformation of ß-strands are not likelyto account for the N = 8 invariability observed in natural parallelß-barrels thereby strengthening previous work rationalizingthis constancy.     

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.     

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.     

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.     

Analysis and prediction of inter-strand packing distances between {beta}-sheets of globular proteins

Any two ß-strands belonging to two different ß-sheetsin a protein structure are considered to pack interactivelyif each ß-strand has at least one residue that undergoesa loss of one tenth or more of its solvent contact surface areaupon packing. A data set of protein 3-D structures (determinedat 2.5 Å resolution or better), corresponding to 428 proteinchains, contains 1986 non-identical pairs of ß-strandsinvolved in interactive packing. The inter-axial distance betweenthese is significantly correlated to the weighted sum of thevolumes of the interacting residues at the packing interface.This correlation can be used to predict the changes in the inter-sheetdistances in equivalent ß-sheets in homologous proteinsand, therefore, is of value in comparative modelling of proteins.     

Interspecific luciferase {beta} subunit hybrids between Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi

Bacterial luciferase (EC 1.14.143) is a heterodimer composedof and ß-chains encoded by luxA and luxB, respectively.Although some interspecific combinations of these subunits leadto active enzyme, others do not. The ß subunits ofVibrio fischeri and Photobacterium leiognathi form active enzymewith the subunits of V.fischeri, P.leiognathi and Vibrio harveyi,while the ß subunit from V.harveyi only complementsthe subunit of V.harveyi. Inactivity is caused by a lack ofdimerization of the ß subunit of V.harveyi with the subunits of V.fischeri and P.leiognathi. These observationsserved as the basis for a search to discover which segment ofthe ß polypeptide confers the ability to dimerizewith the subunits of V.fischeri and P.leiognathi. Intragenicß subunit hybrids were made between V.harveyi, V.fischeriand P.leiognathi. Unique restriction sites were introduced intothe respective luxB genes to divide them into four roughly equalsegments. In all, 78 hybrids were constructed by in vitro techniques.The N-terminal segment of the peptide contains the signals thatdifferentiate between the ß subunits of V.fischeriand P.leiognathi and the ß subunit of V.harveyi, andallow the former to dimerize with their a subunits. The secondsegment has no major effect on enzyme activity but does exhibitsome context effects. Important interactions were found betweenthe third and fourth segments of the polypeptide with respectto enzymatic activity.     

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.     

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.     

Permuteins of interleukin 1{beta}--a simplified approach for the construction of permutated proteins having new termini

A technique for the rapid and simple generation of permutatedversions of the interleukin-1ß (IL-1ß) geneis described. In this method, the human IL-1ß cDNAis twice amplified by the polymerase chain reaction (PCR) andthe resulting DNA fragments are ligated in tandem. Between thetwo genes, the DNA sequence encodes a short four amino acidloop to link the native N- and C-terminal ends of the IL-1ßprotein. By using PCR amplification from this starting template,a new version of the IL-1ß cDNA was obtained thatencodes a permutated form of the IL-1ß protein wherethe new N- and C-terminal amino acids correspond to residues65 and 64 of the native IL-1ß sequence, respectively.The name ‘permutein’ is proposed to describe proteinsgenerated by this technology. The molecular profile (IL-1 receptorbinding, biologic activity and solution properties) of the IL-1permutein produced by this technology, permutein 65/64, is shownto be identical to that of native IL-1ß The approachshould be useful to define further the structural features ofthis protein that are important for its function.     

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.     

Structure--activity relationships in human interleukin-1{alpha}: identification of key residues for expression of biological activities

To identify the sites important for the different biologicalactivities of human interleukin-l (hIL-1), 56 single-amino acid-substitutedmutants of hIL-l were produced in Escherichia coli using site-directedmutagenesis, and were examined for their biological activitiessuch as mouse lymphocyte activating factor activity (LAF activity),cytostatic activity against human melanoma cells A-375 (A375activity) and prostaglandin E2 (PGE2) inducing activity in humanosteosarcoma cells MG-63 (PEI activity). Two amino acid residues,Asp26 and Asp151, were found to be important for these activities.The replacement of Asp26 by Val caused a decrease in LAF andA375 activities by one or two orders of magnitude and a slightdecrease in A375 activity. The Tyr or Phe substitution for Asp151caused decreases in LAF and A375 activities by one or two ordersof magnitude and complete loss of PEI activity. The change fromAsp151 to Lys or Arg resulted in marked decrease in LAF activityand complete loss of A375 and PEI activities. Since Asp26 andAsp151 are close to each other in the three-dimensional structure,the region involving these amino acids seems to be importantfor the biological activities of hIL-1.     

Study of cysteine residues in the {alpha} subunit of Escherichia coli tryptophan synthase. 1. Role in conformational stability

To eludicate the role in conformational stability of Cys residuesburied in the interior of a protein, the thermodynamic propertiesof denaturation of mutant subunit of Escherichia coli tryptophansynthase, in which Ser, Ala, Val or Gly was substituted foreach of the three Cys residues, were analyzed using calorimetry.The mutants were less stable than the wild type, indicatingthat Cys residues contribute greatly to the stability of the subunit. In most cases, a large decrease in denaturation enthalpywas observed, compensated for by the denaturation entropy toa major extent. The extent of changes in the denaturation Gibbsenergy and denaturation enthalpy varied greatly depending onboth substituting residues and positions. Of all the mutantproteins, the Cys154Ser mutant showed the greatest decreasein denaturation enthalpy; its denaturation enthalpy was halfthat of the wild type, and was considerably repaired by addinga ligand of the subunit. Because the enthalpy of ligand bindingto Cys154Ser in the native state did not change. it seems thatthe decrease in the denaturation enthalpy of Cys154Ser and itsrecovery by ligand binding are caused by conformational changesin the denatured state due to the mutation.     

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.     

Correlation of surface properties with conformational stabilities of wild-type and six mutant tryptophan synthase {alpha}-subunits substituted at the same position

The surface properties of wild-type and six mutant -subunitsof tryptophan synthase substituted at the same position, 49,which is buried in the interior, were measured by surface tension,foaming and emulsifying properties to correlate the surfaceproperties with the stabilities. The conformational stabilitiesof the seven -subunits differed dramatically depending on thecharacteristics of the substituting residues [Yutani et al.(1987) Proc. Natl. Acad. Sci., 84, 4441–4444]. The mutantproteins substituted by isoleucine and phenylalanine in placeof glutamic acid at position 49 were more stable than the otherproteins and showed higher surface tension and lower foamingand emulsifying properties than the wild-type and other mutantproteins. Good correlations were observed between these surfaceproperties and values of the Gibbs free energy of unfoldingin water, of the proteins. This indicates that the surface propertiesof the -subunits of tryptophan synthase depend closely on theconformational stabilities.     

Native-like {beta}-hairpin structure in an isolated fragment from ferredoxin: NMR and CD studies of solvent effects on the N-terminal 20 residues

The conformational properties of protein fragments have beenwidely studied as models of the earliest initiation events inprotein folding. While native-like -helices and ß-turnshave been identified, less is known about the factors that underlyß-sheet formation, in particular ß-hairpins,where considerably greater long-range order is required. TheN-terminal 20 residue sequence of native ferredoxin I (fromthe blue-green alga Aphanothece sacrum ) forms a ß-hairpinin the native structure and has been studied in isolation byNMR and CD spectroscopy. Local native-like interactions aloneare unable to stabilize significantly a folded conformationof the 20-residue fragment in purely aqueous solution. However,we show that the addition of low levels of organic co-solventspromotes formation of native-like ß-hairpin structure.The results suggest an intrinsic propensity of the peptide toform a native-like ß-hairpin structure, and that theorganic co-solvent acts in lieu of the stabilizing influenceof tertiary interactions (probably hydrophobic contacts) whichoccur in the folding of the complete ferredoxin sequence. Thestructure of the isolated hairpin, including the native-likeregister of interstrand hydrogen bonding interactions, appearsto be determined entirely by the amino acid sequence. The solventconditions employed have enabled this intrinsic property tobe established.