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Journal Issue - Volume 8 Issue 7 (1999)

Abstract The GCN4 leucine zipper is a peptide homodimer that has been the subject of a number of experimental and theoretical investigations into the determinants of affinity and specificity. Here, we utilize this model system to investigate electrostatic effects in protein binding using continuum calculations. A particularly novel feature of the computations made here is that they provide an interaction‐by‐interaction breakdown of...

Abstract B*2701 differs from all other HLA‐B27 subtypes of known peptide specificity in that, among its natural peptide ligands, arginine is not the only allowed residue at peptide position 2. Indeed, B*2701 is unique in binding many peptides with Gln2 in vivo. However, the mutation (Asp74Tyr) responsible for altered selectivity is far away from the B pocket of the peptide binding site to which Gln/Arg2 binds. Here, we present a...

Abstract A 12‐residue peptide designed to form an α‐helix and self‐associate into an antiparallel 4‐α‐helical bundle yields a 0.9 Å crystal structure revealing unanticipated features. The structure was determined by direct phasing with the “Shake‐and‐Bake” program, and contains four crystallographically distinct 12‐mer peptide molecules plus solvent for a total of 479 atoms. The crystal is formed from nearly ideal α‐helices hydrogen...

Abstract We report the 0.75 Å crystal structure of a racemic mixture of the 12‐residue designed peptide “Alpha‐1” (Acetyl‐ELLKKLLEELKG), the L‐enantiomer of which is described in the accompanying paper. Equivalent solutions of the centrosymmetric bilayers were determined by two direct phasing programs in space groups P1 and P1. The unit cell contains two L‐alpha‐helices and two D‐alpha‐helices. The columnar‐sheet bilayer motif seen...

Abstract Immunoglobulins of human heavy chain subgroup III have a binding site for Staphylococcal protein A on the heavy chain variable domain (VH), in addition to the well‐known binding site on the Fc portion of the antibody. Thermodynamic characterization of this binding event and localization of the Fv‐binding site on a domain of protein A is described. Isothermal titration calorimetry (ITC) was used to characterize the interaction between...

Abstract The oligopeptide‐binding protein OppA provides a useful model system for studying the physical chemistry underlying noncovalent interactions since it binds a variety of readily synthesized ligands. We have studied the binding of eight closely related tripeptides of the type Lysine‐X‐Lysine, where X is an abnormal amino acid, by isothermal titration calorimetry (ITC) and X‐ray crystallography. The tripeptides fall into three...

Abstract Conformational changes occurring within the NS3 protease domain from the hepatitis C virus Bk strain (NS31–180) under different physico‐chemical conditions either in the absence or in the presence of its cofactor Pep4A were investigated by limited proteolysis experiments. Because the surface accessibility of the protein is affected by conformational changes, when comparative experiments were carried out on NS31–180 either at different...

Abstract Effective inhibitors of matrix metalloproteinases (MMPs), a family of connective tissue‐degrading enzymes, could be useful for the treatment of diseases such as cancer, multiple sclerosis, and arthritis. Many of the known MMP inhibitors are derived from peptide substrates, with high potency in vitro but little selectivity among MMPs and poor bioavail‐ability. We have discovered nonpeptidic MMP inhibitors with improved...

Abstract Insulin‐like growth factor (IGF‐1) contains three disulfide bonds. In the presence of denaturant and thiol catalyst, IGF‐1 shuffles its native disulfide bonds and denatures to form a mixture of scrambled isomers. The composition of scrambled IGF varies under different denaturing conditions. Among the 14 possible scrambled IGF isomers, the yield of the beads‐form isomer is shown to be directly proportional to the strength of...

Abstract The pressure‐induced unfolding of lysozyme was investigated in an aqueous guanidinium chloride solution by means of ultraviolet spectroscopy. Assuming a two‐state transition model, volume changes were calculated from the slope of free energy vs. pressure plots over a temperature range of 10 to 60 °C. Between 25 and 60 °C, almost constant volume changes were observed in the transition region, which was reflected in almost...

Abstract Production of folded and biologically active protein from Escherichia coli derived inclusion bodies can only be accomplished if a scheme exists for in vitro naturation. Motivated by the need for a rapid and statistically meaningful method of determining and evaluating protein folding conditions, we have designed a new fractional factorial protein folding screen. The screen includes 12 factors shown by previous experiments to enhance...

Abstract Apomyoglobin from sperm whale is often used for studies of ligand binding, protein folding, and protein stability. In an effort to describe its conformational properties in solution, homonuclear and heteronuclear (13C and 15N) NMR methods were applied to the protein in its native state. Assignments were confirmed for nuclear Overhauser effects (NOEs) involving side chain and backbone protons in the folded regions of the structure....

Abstract Conformational free energy calculations have been carried out for proline‐containing alanine‐based pentadecapeptides with the sequence Ac‐(Ala)n‐Pro‐(Ala)m‐NHMe, where n + m = 14, to figure out the positional preference of proline in α‐helices. The relative free energy of each peptide was calculated by subtracting the free energy of the extended conformation from that of the α‐helical one, which is used here as a measure of...

Abstract The change in heat capacity ΔCp for the folding of ribonuclease A was determined using differential scanning calorimetry and thermal denaturation curves. The methods gave equivalent results, ΔCp = 1.15 ± 0.08 kcal mol–1 K–1. Estimates of the conformational stability of ribonuclease A based on these results from thermal unfolding are in good agreement with estimates from urea unfolding analyzed using the linear extrapolation method. ...

Abstract Refolding of b*C40A/C82A/P27A is comprised of several kinetically detectable folding phases. The slowest phase in refolding originates from trans → cis isomerization of the Tyr47–Pro48 peptide bond being in cis conformation in the native state. This refolding phase can be accelerated by the peptidyl‐prolyl cis/trans isomerase human cytosolic cyclophilin (Cyp18) with a kcat/KM of 254, 000 M–1 s–1. The fast refolding phase is not influenced by the...

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