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.     

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.     

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 de novo protein with grafted biological function: transferring of interferon blast-transforming activity to albebetin

The de novo protein albebetin has been designed recently toform a predetermined tertiary fold that has not yet been observedin natural proteins. An eight amino acid fragment (131–138)of human interferon ₂ carrying the blasttransforming activityof the protein was attached to the N-terminus of albebetin nextto its initiatory methionine residue. The gene of chimeric proteinwas expressed in a wheat germ cell-free translation system andsynthesized protein was tested for its compactness and stability.Its ability for receptor binding was also studied. We have shownthat albebetin with attached octapeptide is practically as compactas natural proteins of corresponding molecular weight and possesseshigh stability toward the urea-induced unfolding. It binds murinethymocyte receptor at a high affinity and activates the thymocyteblast transformation efficiently at a concentration of 10^-11M.     

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     

Cooperative deformation of a de novo designed protein

A de novo protein design has been made to understand the uniquepacking of natural proteins that have a ß/-barrelfold. A carefully designed 207 amino acid sequence was synthesizedusing an Escherichia coli expression system and the structuraland thermodynamic characteristics of the purified protein werestudied. At neutral pH the protein is soluble and monomeric,with large amounts of secondary structure and a hydrophobiccore, although the broad resonance peaks of its NMR spectrumsuggest that the designed protein does not have a unique structurewith tightly packed side chains. In an H–D exchange experiment,no amido protons of the designed protein exchanged slowly withdeuterons. At acidic pH, thermal unfolding was observedwitha remarkable change in the excess heat capacity measured directlyby a differential scanning microcalorimeter. The enthalpy andentropy differences at 110°C, extrapolated from analyzedthermodynamic parameters, are 1/3 of the common values for naturalproteins. These measurements indicate that the folding is significantlycooperative as expected, but that the protein is still looselypacked.     

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.     

Comparative modeling of the three CP modules of the {beta}-chain of C4BP and evaluation of potential sites of interaction with protein S

A computer model of the ß-chain of C4b-binding protein(C4BP) was constructed, using the backbone fold of the NMR structuresof the sixteenth CP module of factor H (H16) and of a pair ofmodules consisting of the fifteenth and sixteenth CPs of factorH (H15-16). The characteristic hydrophobic core responsiblefor dictating the three-dimensional structure of the CP familyis conserved in the amino acid sequence of C4BP ßl, ß2and ß3. The distribution of the electrostatic potentialshows that the model is mainly covered by a negative contour.Interestingly, a positive area is observed in the C-terminalregion of the first CP module, enclosing peptide 31-45, knownto be a binding site for protein S. This observation suggeststhat electrostatic interactions can be of importance for theinteraction of C4BP to protein S. A solvent-accessible hydrophobicpatch, located nearby and involving the peptide 31-45, was alsofound in the model, further confirming that this area is involvedin the interaction with protein S. The contribution of ß-chainresidues 31-45 to the affinity for protein S was studied furtherby means of synthetic mutant peptides. The results suggest thatboth electrostatic and hydrophobic interactions are importantfor the binding to protein S.     

Modular mutagenesis of a TEM-barrel enzyme: the crystal structure of a chimeric E.coli TIM having the eighth {beta}{alpha}-unit replaced by the equivalent unit of chicken TIM

Abstract The crystal structure of a hybrid Escherichia coli triosephosphateisomerase (TIM) has been determined at 2.8 Å resolution.The hybrid TIM (ETIM8CHI) was constructed by replacing the eighthß-unit of E.coli TIM with the equivalent unit of chickenTIM. This replacement involves 10 sequence changes. One of thechanges concerns the mutation of a buried alanine (Ala232 instrand 8) into a phenylalanine. The ETIM8CHI structure showsthat the A232F sequence change can be incorporated by a side-chainrotation of Phe224 (in helix 7). No cavities or strained dihedralsare observed in ETIM8CHI in the region near position 232, whichis in agreement with the observation that ETIM8CHI and E.coliTIM have similar stabilities. The largest CA (C-alpha atom)movements, 3 Å, are seen for the C-terminal end of helix8 (associated with the outward rotation of Phe224) and for theresidues in the loop after helix 1 (associated with sequencechanges in helix 8). From the structure it is not clear whythe k_cat of ETIM8CHI is 10 times lower than in wild type E.coliTIM     

Functionally essential, invariant glutamate near the C-terminus of strand {beta}5 in various ({alpha}/{beta})8-barrel enzymes as a possible indicator of their evolutionary relatedness

Twelve different (/ß)₈-barrel enzymes belonging tothree structurally distinct families were found to contain,near the C-terminus of their strand ß5, a conservedinvariant glutamic acid residue that plays an important functionalrole in each of these enzymes. The search was based on the ideathat a conserved sequence region of an (/ß)₈-barrelenzyme should be more or less conserved also in the equivalentpart of the structure of the other enzymes with this foldingmotif owing to their mutual evolutionary relatedness. For thispurpose, the sequence region around the well conserved fifthß-strand of a-amylase containing catalytic glutamate(Glu230, Aspergillus oryzae -amylase numbering), was used asthe sequence-structural template. The isolated sequence stretchesof the 12 (/ß)₈-barrels are discussed from both thesequence-structural and the evolutionary point of view, theinvariant glutamate residue being proposed to be a joining featureof the studied group of enzymes remaining from their ancestral(/ß)₈-barrel     

The solution structure and conformational dynamics of tumor necrosis factor-{alpha} and a (Cys69 - Asp; Cys101 - Arg) analog as examined by IR spectroscopy and hydrogen exchange

An analog of human tumor necrosis factor- (TNF-) was createdwith Cys69 and CyslOl replaced with Asp and Arg respectively.We have undertaken a comparative study of the solution conformationand dynamics of the native and analog molecules using a combinationof Fourier transform IR spectroscopy and hydrogen-deuterium(H-D) exchange kinetics. IR spectroscopic results indicate thatthe analog molecule adopts a gross structure similar to thatof the native molecule but significant differences in the conformationof the ß-sheets are observed. Increased bandwidthsobserved for several of the amide I components also suggesta less rigid structure for the analog molecule. Further, bymonitoring the frequency shifts of the individual amide I componentbands as a function of hydrogen exchange, we have enhanced ourability to assign these components to individual protein secondarystructures, particularly the high frequency ß-strandmode. Hydrogen exchange kinetic studies indicate that the Asp–Arganalog adopts a looser, more flexible solution structure relativeto the natural sequence molecule.     

Search for the most stable folds of protein chains: II. Computation of stable architectures of {beta}-proteins using a self-consistent molecular field theory

In a preceding paper we presented a novel approach to computationof 3-D folds of protein chains from their amino acid sequences.This approach is a physically correct generalization of the‘threading’ methods. It is based on a self-consistentmolecular field theory and on a physical theory of protein foldingpatterns, which make it possible to examine all the varietyof ‘potentially stable’ folding patterns and allthe variety of the chain conformations within each of them andto determine the thermodynamically stable structure. In thispaper, we apply this approach to single out stable folding patternsand conformations for the chains of ß-sandwich proteinsand show that the similarity of the calculated and observedstructures is usually rather close.     

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.     

Molecular modeling of the amyloid-{beta}-peptide using the homology to a fragment of triosephosphate isomerase that forms amyloid in vitro

The main component of the amyloid senile plaques found in Alzheimer'sbrain is the amyloid-ß-peptide (Aß), a proteolyticproduct of a membrane precursor protein. Previous structuralstudies have found different conformations for the Aßpeptide depending on the solvent and pH used. In general, theyhave suggested an -helix conformation at the N-terminal domainand a ß-sheet conformation for the C-terminal domain.The structure of the complete Aß peptide (residues 1–40)solved by NMR has revealed that only helical structure is presentin Aß. However, this result cannot explain the large ß-sheetAß aggregates known to form amyloid under physiologicalconditions. Therefore, we investigated the structure of Aßby molecular modeling based on extensive homology using theSmith and Waterman algorithm implemented in the MPsrch program(Blitz server). The results showed a mean value of 23% identitywith selected sequences. Since these values do not allow a clearhomology to be established with a reference structure in orderto perform molecular modeling studies, we searched for detailedhomology. A 28% identity with an /ß segment of a triosephosphateisomerase (TIM) from Culex tarralis with an unsolved three-dimensionalstructure was obtained. Then, multiple sequence alignment wasperformed considering Aß, TIM from C.tarralis and anotherfive TIM sequences with known three-dimensional structures.We found a TIM segment with secondary structure elements inagreement with previous experimental data for Aß. Moreover,when a synthetic peptide from this TIM segment was studied invitro, it was able to aggregate and to form amyloid fibrils,as established by Congo red binding and electron microscopy.The Aß model obtained was optimized by molecular dynamicsconsidering ionizable side chains in order to simulate Aßin a neutral pH environment. We report here the structural implicationsof this study.     

Nuclear magnetic resonance studies and molecular dynamics simulations of the solution conformation of a 'designed', {alpha}-helical peptide

The complete three-dimensional structure in methanol of an amphipathica-helical peptide, that has been designed by taking into accountthe three-dimensional structures of small haemolytic peptides,secondary structure prediction algorithms and the well documentedliterature on -helix stabilizing factors, has been elucidatedby two-dimensional NMR spectroscopy. Initially various two-dimensionalspectra (COSY, TOCSY, and NOESY) allowed the complete sequencespecific assignment of all signals in the ¹H spectrum. Consequentlytrial structures were generated which were then subjected tomolecular dynamics simulations using 121 NOE-derived distancesand 25 vicinal coupling constant values as structural restraintsto give a final set of calculated structures. These structuresare in complete agreement with the results of a circular dichroismstudy and reveal that the peptide adopted a highly ordered a-helicalconformation. Details of the structure which throw light onfuture peptide/protein design are discussed.     

Stabilization of the autoproteolysis of TNF-alpha converting enzyme (TACE) results in a novel crystal form suitable for structure-based drug design studies

The crystallization of TNF-alpha converting enzyme (TACE) has been useful in understanding the structure-activity relationships of new chemical entities. However, the propensity of TACE to undergo autoproteolysis has made enzyme handling difficult and impeded the identification of inhibitor soakable crystal forms. The autoproteolysis of TACE was found to be specific (Y352-V353) and occurred within a flexible loop that is in close proximity to the P-side of the active site. The rate of autoproteolysis was found to be proportional to the concentration of TACE, suggesting a bimolecular reaction mechanism. A limited specificity study of the S(1)' subsite was conducted using surrogate peptides and suggested substitutions that would stabilize the proteolysis of the loop at positions Y352-V353. Two mutant proteases (V353G and V353S) were generated and proved to be highly resistant to autoproteolysis. The kinetics of the more resistant mutant (V353G) and wild-type TACE were compared and demonstrated virtually identical IC(50) values for a panel of competitive inhibitors. However, the k(cat)/K(m) of the mutant for a larger substrate (P6 - P(6)') was approximately 5-fold lower than that for the wild-type enzyme. Comparison of the complexed wild-type and mutant structures indicated a subtle shift in a peripheral P-side loop (comprising the mutation site) that may be involved in substrate binding/turnover and might explain the mild kinetic difference. The characterization of this stabilized form of TACE has yielded an enzyme with similar native kinetic properties and identified a novel crystal form that is suitable for inhibitor soaking and structure determination.     

Roles of catalytic residues in {alpha}-amylases as evidenced by the structures of the product-complexed mutants of a maltotetraose-forming amylase

The crystal structures of the four product-complexed singlemutants of the catalytic residues of Pseudomonas stutzeri maltotetraose-forming-amylase, E219G, D193N, D193G and D294N, have been determined.Possible roles of the catalytic residues Glu219, Asp193 andAsp294 have been discussed by comparing the structures amongthe previously determined complexed mutant E219Q and the presentmutant enzymes. The results suggested that Asp193 predominantlyworks as the base catalyst (nucleophile), whose side chain atomlies in close proximity to the C1-atom of Glc4, being involvedin the intermediate formation in the hydrolysis reaction. WhileAsp294 works for tightly binding the substrate to give a twistedand a deformed conformation of the glucose ring at position–1 (Glc4). The hydrogen bond between the side chain atomof Glu219 and the O1-atom of Glc4, that implies the possibilityof interaction via hydrogen, consistently present throughoutthese analyses, supports the generally accepted role of thisresidue as the acid catalyst (proton donor).     

Generation of a Ni(II) binding site by introduction of a histidine cluster in the structure of human glutathione transferase Al-1

Two mutant forms of human glutathione transferase (GST) Al–1with affinity for metal ions were constructed by introductionof His residues by site–directed mutagenesis. A mutant,2–His, contained the mutations Lys84Gln, Asp85His andGlu88His, and another, 5–His, contained the mutationsTyr79His, Asn80His, Lys84His, Asp85His and Glu88HLs. The mutantproteins were obtained in good yields (40–150 mg per 3I culture) by heterologous expression in Escherichia coli. Themutant enzymes possessed novel binding affinities for Ni(II)and Zn(II) ions, as demonstrated by immobilized metal ion affinitychromatography. The mutant with two novel His residues (2–Hismutant) did not bind as tightly to immobilized Nifll) as didthe mutant with five novel His residues (5–His mutant).When tested for affinity to immobilized Zn(II), only the 5–Hismutant remained bound to the column. The affinity of the 5–Hismutant for Ni(II) ions in solution was determined by bindingexperiments in an aqueous polymeric two–phase system.Analysis of the binding curve showed two binding sites per enzymesubunit and a dissociation constant of 6.7 1 . 6 M. The kineticconstants k_cat, K_m and k_cat/k_m for the reaction with glutathioneand l–chloro–2,4–dinitrobenzene were determinedby steady–state kinetic analysis and the parameter valuesfor the mutant forms were found to be indistinguishable fromthose obtained for the wild–type GST Al–1. The differencesin surface charge in the mutant proteins as compared with thewild–type enzyme did not alter the pH dependence of k_cat.The results provide an alternative method for purification offully active recombinant GST Al–1 by the introductionof novel metal binding sites. The data also showed that twoHis residues are sufficient for Ni(II) binding.