摘要:

AbstractThe introduction of conformational constraints into a flexible peptide hormone can be exploited to develop models for the conformation required for receptor binding and activity. In this review, we illustrate this approach to analog design using our work on antagonists of gonadotropin‐releasing hormone (GnRH). Design of a conformationally constrained, competitive antagonist of GnRH, cyclo[Δ3,4Pro‐D4ClPhe‐DTrp‐Ser‐Tyr‐DTrp‐NMeLeu‐Arg‐Pro‐βAla], led to the prediction of its bioactive conformation. Template forcing experiments show that this conformation is accessible to other active GnRH analogs. Two‐dimensional NMR studies verified the predicted conformation in solution. The predicted binding conformation has recently been used to design two new analogs incorporating side chain–side chain linkages suggested by the conformational model:These analogs were synthesized and the one predicted to be most similar to the parent conformation had equivalent potency while the second, designed to refine the conformational hypothesis, was found to exhibit enhanced potency, thus confirming the original binding conformation hypothesis. These compounds and their derivatives now provide a new class of GnRH antagonists possessing both high biological potency and limited conformational flexibility, thus making them ideal for both biophysical and s

ISSN:0887-3585    

DOI:10.1002/prot.340080403

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractNAD‐linked aldehyde dehydrogenases (A1DH) (EC 1.2.1.3) catalyze the irreversible oxidation of a wide variety of aldehydes to their respective carboxylic acids. Crystals of a class 3 A1DH (from anEscherichia coliexpression system) suitable for X‐ray analysis have been obtained. These crystals, which can be grown to a size of 0.8 × 0.3 × 0.2 mm, diffract to 2.5 Å resolution. Analysis of the diffraction pattern indicates that the crystals belong to the monoclinic space groupP21, with cell parametersa= 65.11 Å,b= 170.67 Å,c= 47.15 Å, and β = 110.5°. Assuming one dimer per asymmetric unit, the valueVmis calculated to be 2.45 and the solvent content of the crystal is estimated to be 50%. A self‐rotation function study produced significant rotation peaks (58% of the origin) on theK= 180 section at ψ = 90° and ϕ = 71° and 341°, indicating that the pseudo‐dimer axis is (or is very nearly) perpen

ISSN:0887-3585    

DOI:10.1002/prot.340080404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractHuman growth hormone (hGH), a 191 residue protein containing two disulfide bonds, was fused to the carboxyl‐terminal domain of the gene III protein, a minor coat protein exposed at one end of the filamentous phage M13. The gene fusion was cloned into a plasmid containing origins of replication forEscherichia coliand filamentous phage and was packaged into phagemid particles upon infection by an M13KO7 helper phage. Transcription of the hGH‐gene III fusion was controlled so that usually no more than one copy of the fusion protein was displayed along with the four copies of the wild‐type gene III protein. The hGH‐gene III fusion protein was properly folded, as judged by reactivity with six hGH monoclonal antibodies whose epitopes are sensitive to the folded conformation of hGH. Moreover, the hGH–gene III phagemid particles were enriched over 5000‐fold from non‐hGH phage, and 8‐fold from a mutant hGH phagemid following a single hGH‐specific elution step from hGH receptor‐coated beads. The hGH phagemid should be useful for isolating new receptor binding mutants of hGH. More generally, this expression system may allow other large proteins with discontinuous binding epitopes to be displayed, and binding selections applied to their mutated gene III fusions o

ISSN:0887-3585    

DOI:10.1002/prot.340080405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractThe structure of thymidylate synthase (TS) fromEscherichia coliwas solved from cubic crystals witha= 133 Å grown under reducing conditions at pH 7.0, and refined toR= 22% at 2.1 Å resolution. The structure is compared with that fromLactobacillus caseisolved toR= 21% at 2.3 Å resolution. The structures are compared using a difference distance matrix, which identifies a common core of residues that retains the same relationship to one another in both species. After subtraction of the effects of a 50 amino acid insert present inLactobacillus casei, differences in position of atoms correlate with temperature factors and with distance from the nearest substituted residue. The dependence of structural difference on thermal factor is parameterized and reflects both errors in coordinates that correlate with thermal factor, and the increased width of the energy well in which atoms of high thermal factor lie. The dependence of structural difference on distance from the nearest substitution also depends on thermal factors and shows an exponential dependence with half maximal effect at 3.0 Å from the substitution. This represents the plastic accommodation of the protein which is parameterized in terms of thermal B factor and distance from a mutational cha

ISSN:0887-3585    

DOI:10.1002/prot.340080406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractBy exhaustive structural comparisons, we have found that about one‐third of the α‐helix–turn–β–strand polypeptides in α–β barrel domains share a common structural motif. The chief characteristics of this motif are that first, the geometry of the turn between the α‐helix and the β‐strand is somewhat constrained, and second, the β‐strand contains a hydrophobic patch that fits into a hydrophobic pocket on the α‐helix. The geometry of the turn does not seem to be a major determinant of the α–β unit, because the turns vary in length from four to six residues. However, the motif does not occur when there are few constraints on the geometry of the turn–for instance, when the turns between the α‐helix and the β‐strands are very long. It also occurs much less frequently in flat‐sheet α–β proteins, where the topology is much less regular and the amount of twist on the sheet varies considerably more than in the barrel proteins. The motif may be one of the basic building

ISSN:0887-3585    

DOI:10.1002/prot.340080407

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractFrom protein sequence comparison data found in the literature, a library was organized using peptide fragment sequences which are common to related proteins. Each of the fragments was then examined for its occurrence in all the protein superfamilies defined by the NBRF‐PIR data base. We have selected those fragment peptides that appear exclusively in one or a few superfamilies, and thus made a library of fragment peptides that characterize specific superfamilies. Such characteristic peptides are, in general, five to seven residues long and contain unusually high proportions of glycine and cysteine. This collection is a useful resource for the classification and functional prediction of protein molecule

ISSN:0887-3585    

DOI:10.1002/prot.340080408

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractThe structure of a small rubredoxin from the bacteriumDesulfovibrio desulfuricanshas been determined and refined at 1.5 Å resolution. The hairpin loop containing seven residues in other rubredoxins is missing in this 45 residue molecule, and once that fact was determined by amino acid sequencing studies, refinement progressed smoothly to anRvalue of 0.093 for all reflections from 5 to 1.5 Å resolution. Nearly all of the water molecules in the well‐ordered triclinic unit cell have been added to the crystallographic model. As in the other refined rubredoxin models, the Fe‐S4complex is slightly distorted from ideal tetrahedral coordin

ISSN:0887-3585    

DOI:10.1002/prot.340080409

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractThe structure of the methyl‐α‐D‐mannopyranoside–LOL I complex has been solved by the molecular replacement method using the refined saccharide‐free LOL I coordinates as starting model. The methyl‐α‐D‐mannopyranoside–LOL I complex was refined by simulated annealing using the program X‐PLOR. The finalR‐factor value is 0.182 [Fo>1σ(Fo)]. The isostructural methyl‐α‐D‐glucopyranoside‐LOL I complex was refined by X‐Ray coupled energy minimization using the methyl‐α‐D‐mannopyranoside‐LOL I structure as a starting model to anRfactor of 0.179 (all data). In both crystal forms, each dimer binds two molecules of sugar in pockets found near the calcium ions. The two saccharide moieties, which are in the C1 chair conformation, establish the same hydrogen bond pattern with the lectin. However, the van der Walls contacts are different between the O2, C2, C6, and O6 atoms of the two molecules and the backbone atoms of residues 208–211. Mannose, due to its axial C2 conformation, encloses the backbone atoms of the protein in a clamplike way. Van der Waals energy calculations suggest that this better complementarity of the mannoside molecule with the lectin cou

ISSN:0887-3585    

DOI:10.1002/prot.340080410

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractStructure determination of small proteins using NMR data is most commonly pursued by combining NOE derived distance constraints with inherent constraints based on chemical bonding. Ideally, one would make use of a variety of experimental observation, not just distance constraints. Here, coupling constant constraints have been added to molecular mechanics and molecular dynamics protocols for structure determination in the form of a psuedoenergy function that is minimized in a search for an optimum molecular conformation. Application is made to refinement of a structure for a 77 amino acid protein involved in fatty acid synthesis,Escherichia coliacyl carrier protein (ACP). 543JHNαcoupling constants, 12 coupling constants for stereospecifically assigned side chain protons, and 450 NOE distance contraints were used to calculate the 3‐D structure of ACP. A three‐step protocol for a molecular dynamics calculation is described, in analogy to the protocol previously used in molecular mechanics calculations. The structures calculated with the molecular mechanics approach and the molecular dynamics apporach using a rigid model for the protein show similar molecular energies and similar agreement with experimental distance and coupling constant constraints. The molecular dynamics approach shows some advantage in overcoming local minimum problems, but only when a two‐state averaging model for the protein was used, did molecular energies drop signifi

ISSN:0887-3585    

DOI:10.1002/prot.340080411

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractA generalized model is presented of agonist binding to ligand‐gated ion channels (LGICs). Broad similarity in the structure of agonists suggests that the binding sites of LGICs may have evolved from a protobinding site. Aligned sequence data identified as a candidate for such a site a highly conserved 15 residue stretch of primary structure in the N‐terminal extracellular region of all known LGIC subunits. We modeled this subregion, termed the cys‐loop, as a rigid, amphiphilic β‐hairpin and propose that it may form a major determinant of a conserved structural binding cleft.In the model of the binding complex (1) an invariant aspartate residue at position 11 of the cys‐loop is the anionic site interacting with the positively charged amine group of agonists, (2) a local dipole within the π‐electron system of agonists is favorably oriented in the electrostatic field of the invariant aspartate, (3) the ε ring‐proton of a conserved aromatic residue at the turn of thecys‐loop interacts orthogonally with the agonist α‐electron density at its electronegative center, and (4) selective recognition is partly a result of the type of amino acid residue at position 6 of the cys‐loop. Additionally, the formation of a hydrogen bond between the electronegative atom of the π‐electron system of agonist and a complementary group in the receptor may be important in the high‐

ISSN:0887-3585    

DOI:10.1002/prot.340080412

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY