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.     

Modeling the intact form of the {alpha}1-proteinase inhibitor

The structure of the intact form of the serpin ₁-proteinaseinhibitor has been modeled based on the assumption that thecentral strand s4A of the six-stranded ß-sheet A ofthe cleaved inhibitor is not incorporated into the sheet ofintact ₁-proteinase inhibitor. This strand was removed fromits position in the center of the sheet by suitable rotationsabout the backbone dihedrals of Lys343 using molecular graphics.The resulting structure was then annealed using molecular dynamics(MD) while applying progressive distance restraints to the reactivepeptide bond (Met358-Ser359) for 50 ps. During this time, thedisrupted ß-sheet reformed to create a five-strandedß-sheet with strands 3 and 5 in a parallel arrangement.This change and accompanying structural rearrangements are largelyconfirmed by the X-ray structure of plakalbumin, whose structurereflects the overall structure of intact serpins. The successfulmodeling experiment demonstrates the utility of MD for makinggross structural predictions based on related structures. Thebinding loop of the intact form is modeled to allow dockingwith serine proteinases, in particular thrombin, which mosthighly constrains the possible conformations of the bindingloop.     

The differences in conformation between {alpha}-human atrial natriuretic polypeptide, {alpha}-hANP, and its derivative, Met(O)-{alpha}-hANP, in solution

The differences in conformation between -human atrial natriureticpolypeptide (-hANP) and its inactive analog, Met(O)--hANP, havebeen analyzed by nuclear magnetic resonance spectroscopy. Allproton resonances for both peptides were assigned by means ofthe sequential assignment procedure. The three-dimensional structureof -hANP in solution had previously been determined by distancegeometry calculation using distance constraints derived fromnuclear Overhauser effects (NOEs). Here, the three-dimensionalstructure of Met(O)--hANP was determined. The conformationaldifferences between these two molecules were as follows: threesegments of -hANP, Serl–Cys7, Arg11–Ala17 and Gln18–Tyr28,have some ordered structures. In Met(O)--hANP the Gln18-Tyr28region has a similar conformation, while the remaining two regionsdo not have the ordered structure found in -hANP. It is suggestedthat the conserved conformation of the Gln18–Tyr28 regionis required for binding to the ANP receptor and that the slightbiological activity of Met(O)-a-hANP is due to loss of the orderedstructures evoked in the Serl–Cys7 and Arg11–Ala17regions of -hANP.     

C-terminal truncations of a thermostable Bacillus stearothermophilus {alpha}-amylase

A series of truncated proteins from a thermostable Bacillusstearothermophilus -amylase was prepared to study the importanceof the extension in the C-terminus compared with other liquefyingBacillus -amylases. The mutations introducing new translationtermination sites shortened the 515 amino acid residue-longwild type enzyme by 17, 32, 47, 73 or 93 residues. The longerthe truncation, the lower the specific activity of the enzyme.Only the two longest mutant proteins were active: the specificactivity of the 498 residue variant was 97% and protein 483was 36% that of the parental enzyme. The K_m values of starchhydrolysis changed from 1.09 for wild type enzyme to 0.35 and0.21 for mutants 498 and 483, respectively, indicating alteredsubstrate binding. The mutant enzymes had almost identical pHand temperature optima with the wild type amylase, but enhancedthermal stability and altered end product profile. The consequencesof the truncation to the structure and function of the enzymeswere explored with molecular modeling. The liquefying amylasesseem to require {small tilde}480 residues to be active, whereasthe C-terminal end of B.stearothermophilus amylase is requiredfor increased activity.     

Expression of goat {alpha}-lactalbumin in Escherichia coli and its refolding to biologically active protein

A cDNA encoding the mature region of goat -lactalbumin and the3'-non-coding region was fused to cDNA of the N-terminal halfof porcine adenylate kinase which had been placed under thecontrol of the tac promoter in an expression vector in Escherichiacoli. In addition, a methionine codon was inserted between thetwo cDNAs. When the plasmid carried the full-length 3'-non-codingregion, little accumulation of the fused protein was observed.However, the deletion of two-thirds of the 3'-non-coding regionproduced significant expression of the fused protein in E.colistrain JM105. Since goat -lactalbumin contains no methionineresidue, the mature goat -lactalbumin was isolated by CNBr digestionof the fused insoluble protein and refolded using thioredoxin.The homogeneous and biologically active goat -lactalbumin waspurified by Ca²⁺ ion-dependent hydrophobic chromatography.     

A simple method for predicting transmembrane {alpha} helices with better accuracy

The prediction of a protein's structure from its amino acidsequence has been a long-standing goal of molecular biology.In this work, a new set of conformational parameters for membranespanning helices was developed using the information from thetopology of 70 membrane proteins. Based on these conformationalparameters, a simple algorithm has been formulated to predictthe transmembrane helices in membrane proteins. A FORTRAN programhas been developed which takes the amino acid sequence as inputand gives the predicted transmembrane -helices as output. Thepresent method correctly identifies 295 transmembrane helicalsegments in 70 membrane proteins with only two overpredictions.Furthermore, this method predicts all 45 transmembrane helicesin the photosynthetic reaction center, bacteriorhodopsin andcytochrome c oxidase to an 86% level of accuracy and so is betterthan all other methods published to date.     

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.     

Recombinant-derived interleukin-1{alpha} stabilized against specific deamidation

Recombinant-derived human interleukln-1 (IL-1), purified fromEscherichia coli, was resolved by isoelectric focusing on polyacrylamidegels into two species of isoelectric points (pI) 5.45 and 5.20,which constituted 75% and 25% of the total IL-1 protein respectively.The pI 5.45 and pI 5.20 species were separated by chromatofocusingand subjected to N-terminal sequence analysis. The pI 5.45 speciescontained the expected Asn residue at position 36 of the matureprotein sequence whereas the pI 5.20 species contained an Aspresidue at the same position. A mutant protein in which Asn-36was substituted for a Ser residue was isolated from E.coli andshown to be homogeneous on isoelectric focusing analysis witha pI = 5.45. ¹H-n.m.r. and circular dichroism analyses of wild-typeand the mutant IL-1 indicated a similar conformation which wasalso indicated by the identical receptor binding affinitiesof IL-1 with Asn, Asp or Ser in position 36. The mutant proteinwas stabilized against specific base catalysed and temperature-induceddeamidation, and may be more suitable than the wild-type positionfor physical and structural studies.     

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.     

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.     

Molecular model-building of amylin and {alpha}-calcitonin gene-related polypeptide hormones using a combination of knowledge sources

Amylin is the major component of the amyloid found in the pancreasesof noninsulin-dependent diabetics (type 2 diabetes). It is a37 amino acid polypeptide and has been shown to have 46% sequenceidentity with the neuropeptide -calcitonin gene-related peptide(-CGRP). Both amylin and -CGRP are known to be potent inhibitorsof glycogen synthesis in stripped rat soleus muscle. Secondarystructure prediction and tertiary structure model-building showthe two polypeptides to have an -helix/ß-strand motifsimilar to that observed in the insulin B-chain. The resultshave been supported by CD spectroscopy, although there is nosequence similarity between insulin and amylin/-CGRP. Aggregationstates have been predicted based on the dimeric and hexamericarrangements seen in porcine insulin. Rat and hamster amylinhave a changed sequence motif in the ß-strand whichresults in lack of amyloid formation and type 2 diabetes. This,we propose, is caused by disruption of hydrogen bonding whichprevents the formation of the dimer.     

Independent self-assembly of cadmium-binding {alpha}-fragment of metallothionein in Escherichia coli without participation of {beta}-fragment

We examined the independent self-assembly of the - and ß-fragmentsof human metallothionein (MT) into cadmiumbinding conformationin an Escherichia coli expression system, in addition to wild-typeMT expression. The expressed -fragment formed independentlythe structure of a metal-binding cluster without the aid ofthe ß-fragment. The -fragment and wild-type MT expressedin E.coli were purified and analyzed for their biochemical andspectroscopic properties. The apparent cadmium binding of the-fragment was approximately 12-fold greater than that for thewild-type MT, whereas in other respects the studied biochemicalproperties were similar. In contrast, we were unable to obtainany independently expressed ß-fragment as the cadmium-bindingform in this study. Possible explanations for this phenomenonare discussed.     

Improvement of turn structure prediction by molecular dynamics: a case study of {alpha}1-purothionin

Because of the problems in predicting a correct conformationfor loop regions in homology-based prediction, disagreementsare often found between the predicted models and the refinedX-ray structures of the same protein in loop regions. Such asituation has been encountered for ₁-purothionin (₁-PT). Hence,attempts have been made to improve the predicted model of ₁PTby limited molecular dynamics using both AMBER and XPLOR. Withmolecular dynamics, the previously predicted incorrect turnregion reverts to the correct conformation as seen in the X-rayrefined structure. In contrast to the model which is not subjectedto molecular dynamics, the improved model refines with the X-raydata of ₁PT in fewer cycles, without any manual rebuilding andwith comparable or better refinement statistics. Also, the improvedmodel serves as a better starting model in the determinationof the structure with the molecular replacement methods.     

Computer simulations of signal transduction mechanism in {alpha}1B-adrenergic and m3-muscarinic receptors

Molecular dynamices simulations of the hamster _1Badrenergicand the rat m3-muscarinic seven-helix bundle receptor modelshave been carried out. The free, agonist-bound and antagonist-boundforms have been considered. Moreover, three mutant forms ofthe m3-muscarinic recep-tor (N507A, N507D and N507S) have alsobeen simulated; among these, the N507S mutant shows a constitutiveactivity. A comparative structural/dynamics analysis has beenperformed to elucidate (i) the perturbations induced by thefunctionally different ligands upon binding to their targetreceptor, (ii) the features of the three single-point mutantswith respect to the receptor wild type and (iii) the propertiesshared by the agonist-boundforms of the _1B-adrenergic receptorand the m3-muscarinic receptor and by the constitutively activemutant N507S. The consistency obtained between the structuralrearrangement of the transmembrane seven-helix bundle modelsconsidered, and the experimental pharmacological efficaciesof the ligands and of the mutants, constitute an important validationof the 3-D models obtained and allow the inference of the mechanismof ligand- or mutation-induced receptor activation at the molecularlevel.     

Structure-function validation of high lysine analogs of {alpha}-hordothionin designed by protein modeling

Cereal grains and legume seeds, which are key protien sourcesfor the vegetarian diet, are generally deficient in essentialamino acids. Maize, in particular, is deficient in lysine. Theinherent lack of lysine-rich protiens in maize has necessitatedthe search for heterologous protiens enriched in this aminoacid, the isolation of the corresponding gene and its ultimateintroduction into maize through plant transformation techniques.However, a rate-limiting step to this strategy has been theavailability of plant-derived lysine-rich protiens. An appealingsolution to the problem is to artificially increase the lysinecontent of a given protein by mutating appropriate residuesto lysine. Here, we expound this strategy, starting with theprotein hordothionin that is derived from barley seeds andconsists of five lysine residues in a total of 45 amino acids(11 % lysine). To facilitate rational substitutions, the 3-Dstructure of the protein has been determined by homology modelingwith crambin. based on this model, we have identified surfaceresidues amenable to substitution with lysine. Furthermore,the acceptability of the mutations has been validated throughthe syntheses and characterization of the derivatives. To thisend, our approach has permitted the creation of a modified -hordothionin protein that has a lysine content of 27 % andretains the antifungal activity of the wild-type protein.     

Chemical synthesis, expression and product assessment of a gene coding for biologically active human tumour necrosis factor {alpha}

A gene encoding human tumour necrosis factor (TNF-) has beenchemically synthesized, cloned and expressed to yield a biologicallyactive protein in Escherichia coli. The 480-bp gene was assembledby enzymic ligation of 32 oligonucleotides, cloned directlyinto M13mp18 for sequence verification and expressed in thebroad host range high-level expression vector pMMB66EHST. Expressedrecombinant TNF- was shown to have the correct molecular weight,processed N-terminal sequence, antibody cross-reactivity andtumour cell killing activity. The expression product of thesynthetic gene has been purified to homogeneity by a two-stepion-exchange procedure and the purified material shown to beactive.     

The relative positions of alanine residues in the hydrophobic core control the formation of two-stranded or four-stranded {alpha}-helical coiled-coils

The objective of this study was to investigate the positionaleffect of hydrophobic interactions in the -helical interfacein controlling the formation of two-stranded and four-strandedcoiled-coils. Two disulfide-bridged antiparallel coiled-coilswere designed which differ only in the position of a singleAla residue in the middle heptad: in peptide 2H the Ala residuesare in register (in the same rung), while in peptide 4H theyare not. Data from size-exclusion chromatography and sedimentationequilibrium experiments showed that under benign conditionspeptides 2H and 4H were two-stranded and four-stranded coiled-coilsrespectively. These results, in conjunction with molecular modelingstudies, suggest that when four Ala residues are in the sameplane of a potential four-stranded coiled-coil, the small sidechains of Ala would create a large cavity in the hydrophobicinterface of the potential four-stranded structure which isdestabilizing and favors the two-stranded, disulfide-bridgedcoiled-coil. In contrast, an alternating Leu-Ala hydrophobicpacking in the two planes distributes the potential cavity overa larger region, which may be partially filled by minor adjustmentsof the neighboring Leu side chains. As a result, there is stillsufficient hydrophobic contact to maintain the four-strandedstructure.     

Stability, activity and flexibility in {alpha}-lactalbumin

-Lactalbumins and the type-c lysozymes are homologues with similarfolds that differ in function and stability. To determine ifthe lower stability of -lactalbumin results from specific substitutionsrequired for its adaptation to a new function, the effects oflysozyme-based and other substitutions on thermal stabilitywere determined. Unblocking the upper cleft in -lactalbuminby replacing Tyr103 with Ala, perturbs stability and structurebut Pro, which also generates an open cleft, is compatible withnormal structure and activity. These effects appear to reflectalternative enthalpic and entropic forms of structural stabilizationby Tyr and Pro. Of 23 mutations, only three, which involve substitutionsfor residues in flexible substructures adjacent to the functionalsite, increase stability. Two are lysozyme-based substitutionsfor Leu110, a component of a region with alternative helix andloop conformations, and one is Asn for Lys114, a residue whosemicroenvironment changes when -lactalbumin interacts with itstarget enzyme. While all substitutions for Leu110 perturb activity,a Lys114 to Asn mutation increases T_m by more than 10°Cand reduces activity, but two other destabilizing substitutionsdo not affect activity. It is proposed that increased stabilityand reduced activity in Lys114Asn result from reduced flexibilityin the functional site of -lactalbumin.     

Inhibition of {beta}S-chain dependent polymerization by synergistic complementation of contact site perturbations of {alpha}-chain: application of semisynthetic chimeric {alpha}-chains

Mouse _1–30-horse _31–141 chimeric -chain, a semisyntheticsuper-inhibitory -chain, inhibits ß^S-chain dependent polymerizationbetter than both parent -chains. Although contact site sequencedifferences are absent in the _1–30 region of the chimericchain, the four sequence differences of the region _17-22 couldinduce perturbations of the side chains at ₁₆, ₂₀ and ₂₃, thethree contact sites of the region. A synergistic complementationof such contact site perturbation with that of horse _31–141probably results in the super-inhibitory activity of the chimeric-chain. The inhibitory contact site sequence differences, bythemselves, could also exhibit similar synergistic complementation.Accordingly, the polymerization inhibitory activity of Hb Le-Lamentin(LM) mutation [His20()Gln], a contact site sequence difference,engineered into human–horse chimeric -chain has been investigatedto map such a synergistic complementation. Gln20() has littleeffect on the O₂ affinity of HbS, but in human–horse chimeric-chain it reduces the O₂ affinity slightly. In the chimeric-chain, Gln20() increased sensitivity of the ßßcleft for the DPG influence, reflecting a cross-talk betweenthe ₁ß₁ interface and ßß cleft in this semisyntheticchimeric HbS. In the human -chain frame, the polymerizationinhibitory activity of Gln20() is higher compared with horse_1–30, but lower than mouse _1–30. Gln20() synergisticallycomplements the inhibitory propensity of horse _31–141.However, the inhibitory activity of LM–horse chimeric-chain is still lower than that of mouse–horse chimeric-chain. Therefore, perturbation of multiple contact sites inthe _1–30 region of the mouse–horse chimeric -chainand its linkage with the inhibitory propensity of horse _31–141has been now invoked to explain the super-inhibitory activityof the chimeric -chain. The `linkage-map' of contact sites canserve as a blueprint for designing synergistic complementationof multiple contact sites into -chains as a strategy for generatingsuper-inhibitory antisickling hemoglobins for gene therapy ofsickle cell disease.     

Synthesis and characterization of a recombinant fragment of human {alpha}-fetoprotein with antigenic selectivity versus albumin

A DNA sequence coding for human -fetoprotein amino acid sequence38–119 was synthesized and cloned in a bacterial expressionvector. The -fetoprotein sequence was selected as the leasthomologous to albumin, since the two proteins have an overallamino acid identity of %. A chimeric protein was obtained whichwas purified by preparative electrophoresis and characterizedin its primary structure by fast atom bombardment mass spectrometry.About 70% of the -fetoprotein sequence was physically mappedand found to correspond to the amino acids encoded in the syntheticgene. The use of this recombinant protein allowed the selectionof monoclonal antibodies recognizing both the recombinant fragmentand native -fetoprotein. These antibodies should allow the developmentof an immunoassay for -fetoprotein with absolute selectivityversus albumin. This might result in more sensitive clinicaldeterminations, avoiding the possibility of cross-reactions.