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Journal Issue - Volume 6 Issue 8 (August 1997)

Abstract The structure of cruzain, an essential protease from the parasite Trypanosoma cruzi, was determined by X‐ray crystallography bound to two different covalent inhibitors. The cruzain S2 specificity pocket is able to productively bind both arginine and phenylalanine residues. The structures of cruzain bound to benzoyl‐Tyr‐Ala‐fluoromethyl ketone and benzoyl‐Tyr‐Ala‐fluoromethyl ketone at 2.2 and 2.1 Å, respectively, show a pH‐dependent...

Abstract β‐Crystallins are oligomeric eye lens proteins that are related to monomeric γ‐crystallins by domain swapping: like γ‐crystallins, they are comprised of two similar domains but they differ in having long sequence extensions. βB2, a major component of β‐crystallin oligomers, self‐associates to a homodimer in solution. In two crystal structures of native βB2, the protein is a 222‐symmetric tetramer of eight domains. It has...

Abstract We have determined by X‐ray crystallography the structures of several variants of staphylococcal nuclease with long flexible straight chain and equivalent length cyclic unnatural amino acid side chains embedded in the protein core. The terminal atoms in the straight side chains are not well defined by the observed electron density even though they remain buried within the protein interior. We have previously observed this...

Abstract Cooperative unfolding penalties are calculated by statistically evaluating an ensemble of denatured states derived from native structures. The ensemble of denatured states is determined by dividing the native protein into short contiguous segments and defining all possible combinations of native, i.e., interacting, and non‐native, i.e., non‐interacting, segments. We use a novel knowledge‐based scoring function, derived from...

Abstract Murine macrophage inflammatory protein‐2 (MIP‐2), a member of the α‐ chemokine family, is one of several proteins secreted by cells in response to lipopolysaccharide. Many of the α‐chemokines, such as interleukin‐8, gro‐α/MGSA, and neutrophil activating peptide‐2 (NAP‐2), are associated with neutrophil activation and chemotaxis. We describe the expression, purification, and characterization of murine MIP‐2 from Pichia...

Abstract The env gene of SIV and HIV‐1 encodes a single glycoprotein gp160, which is processed to give a noncovalent complex of the soluble glycoprotein gp120 and the transmembrane glycoprotein gp41. The extracellular region (ectodomain), minus the N‐terminal fusion peptide, of gp41 from HIV‐1 (residues 27‐154) and SIV (residues 27‐149) have been expressed in Escherichia coli. These insoluble proteins were solubilized and subjected to a simple...

Abstract We present a Bayesian statistical analysis of the conformations of side chains in proteins from the Protein Data Bank. This is an extension of the backbone‐dependent rotamer library, and includes rotamer populations and average x angles for a full range of ø,ψ values. The Bayesian analysis used here provides a rigorous statistical method for taking account of varying amounts of data. Bayesian statistics requires the assumption of a...

Abstract Selective deamidation of proteins and peptides is a reaction of great interest, both because it has a physiological role and because it can cause alteration in the biological activity, local folding, and overall stability of the protein. In order to evaluate the thermodynamic effects of this reaction in proteins, we investigated the temperature‐induced denaturation of ribonuclease A derivatives in which asparagine 67 was...

Abstract Changes in the temperature, pH, ionic strength, or denaturant concentration of aqueous solutions of the monomeric non‐α‐helical peptide acety1YEAAAKEAPAKEAAAKAamide generate changes in its dichroic spectrum characteristic for a conformational transition. This transition has the characteristic features of a residue PII/unstructured conformational equilibrium in which PII denotes an extended left‐handed helical conformation...

Abstract Using a protein design algorithm that considers side‐chain packing quantitatively, the effect of explicit backbone motion on the selection of amino acids in protein design was assessed in the core of the streptococcal protein G β1 domain (Gβ1). Concerted backbone motion was introduced by varying Gβ1′s supersecondary structure parameter values. The stability and structural flexibility of seven of the redesigned proteins were...

Abstract A systematic study of the membrane‐associated regions in the plasma membrane Ca2+ pump of erythrocytes has been performed by hydrophobic photolabeling. Purified Ca2+ pump was labeled with 3‐(trifluorometnyl)‐3‐(m‐[ 125I]iodophenyl)‐diazirine ([125I]TID), a generic photoactivatable hydrophobic probe. These results were compared with the enzyme labeled with a strictly membrane‐bound probe, [3H]bis‐phosphatidylethanolamine (trifluoromethyl)...

Abstract The hyperthermophilic bacterium Thermotoga maritima is capable of gaining metabolic energy utilizing xylan. XynA, one of the corresponding hydrolases required for its degradation, is a 120‐kDa endo‐l,4‐D‐xylanase exhibiting high intrinsic stability and a temperature optimum —90 °C. Sequence alignments with other xylanases suggest the enzyme to consist of five domains. The C‐terminal part of XynA was previously shown to be ...

Abstract Virtually all studies of the protein‐folding reaction add either heat, acid, or a chemical denaturant to an aqueous protein solution in order to perturb the protein structure. When chemical denaturants are used, very high concentrations are usually necessary to observe any change in protein structure. In a solution with such high denaturant concentrations, both the structure of the protein and the structure of the solvent...

Abstract The C‐terminal oligomerization domain of chicken cartilage matrix protein is a trimeric coiled coil comprised of three identical 43‐residue chains. NMR spectra of the protein show equivalent magnetic environments for each monomer, indicating a parallel coiled coil structure with complete threefold symmetry. Sequence‐specific assignments for 1H‐, 15N‐, and 13C‐NMR resonances have been obtained from 2D 1H NOESY and TOCSY spectra, and...

Abstract Chaperone proteins demonstrate the paradoxical ability to bind ligands rapidly and with high affinity but with no apparent sequence specificity. To learn more about this singular property, we have mapped the binding frame of the chaperone SecB from E. coli on the oligopeptide‐binding protein. Similar studies performed on the maltose‐binding and galactose‐binding proteins revealed centrally positioned binding frames of ∼160...

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