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Journal Issue - Volume 9 Issue 8 (2000)

Abstract CrmA is an unusual viral serpin that inhibits both cysteine and serine proteinases involved in the regulation of host inflammatory and apoptosis processes. It differs from other members of the serpin superfamily by having a reactive center loop that is one residue shorter, and by its apparent inability to form SDS‐stable covalent complexes with cysteine proteinases. To obtain insight into the inhibitory mechanism of crmA,...

Abstract Decorsin is an antagonist of integrin αIIbβ3 and a potent platelet aggregation inhibitor. A synthetic gene encoding decorsin, originally isolated from the leech Macrobdella decora, was designed, constructed, and expressed in Escherichia coli. The synthetic gene was fused to the stII signal sequence and expressed under the transcriptional control of the E. coli alkaline phosphatase promoter. The protein was purified by size‐exclusion filtration of the...

Abstract Blue copper proteins are type‐I copper‐containing redox proteins whose role is to shuttle electrons from an electron donor to an electron acceptor in bacteria and plants. A large amount of experimental data is available on blue copper proteins; however, their functional characterization is hindered by the complexity of redox processes in biological systems. We describe here the application of a semi quantitative method...

Abstract Human serum albumin (HSA) interacts with a vast array of chemically diverse ligands at specific binding sites. To pinpoint the essential structural elements for the formation of the warfarin binding site on human serum albumin, a defined set of five recombinant proteins comprising combinations of domains and/or subdomains of the N‐terminal part were prepared and characterized by biochemical standard procedures, tryptophanyl...

Abstract The folding kinetics of human common‐type acylphosphatase (cAcP) from its urea‐ and TFE‐denatured states have been determined by stopped‐flow fluorescence techniques. The refolding reaction from the highly unfolded state formed in urea is characterized by double exponential behavior that includes a slow phase associated with isomerism of the Gly53–Pro54 peptide bond. However, this slow phase is absent when refolding is...

Abstract The rubredoxin from the cryptomonad Guillardia theta is one of the first examples of a rubredoxin encoded in a eukaryotic organism. The structure of a soluble zinc‐substituted 70‐residue G. theta rubredoxin lacking the membrane anchor and the thylakoid targeting sequence was determined by multidimensional heteronuclear NMR, representing the first three‐dimensional (3D) structure of a eukaryotic rubredoxin. For the structure calculation...

Abstract Several recent publications illustrated advantages of using sequence profiles in recognizing distant homologies between proteins. At the same time, the practical usefulness of distant homology recognition depends not only on the sensitivity of the algorithm, but also on the quality of the alignment between a prediction target and the template from the database of known proteins. Here, we study this question for several...

Abstract The native form of some proteins such as strained plasma serpins (serine protease inhibitors) and the spring‐loaded viral membrane fusion proteins are in a metastable state. The metastable native form is thought to be a folding intermediate in which conversion into the most stable state is blocked by a very high kinetic barrier. In an effort to understand how the spontaneous conversion of the metastable native form into the...

Abstract The intermolecular contact regions between monomers of the homodimeric DNA binding protein ParR and the interaction between the glycoproteins CD28 and CD80 were investigated using a strategy that combined chemical cross‐linking with differential MALDI‐MS analyses. ParR dimers were modified in vitro with the thiol‐cleavable cross‐linker 3,3′‐dithio‐bis(succinimidylproprionate) (DTSSP), proteolytically digested with trypsin and analyzed...

Abstract Chemical and thermal denaturation of calmodulin has been monitored spectroscopically to determine the stability for the intact protein and its two isolated domains as a function of binding of Ca2+ or Mg2+. The reversible urea unfolding of either isolated apo‐domain follows a two‐state mechanism with relatively low δG°20 values of ˜2.7 (N‐domain) and ˜1.9 kcal/mol (C‐domain). The apo‐C‐domain is significantly unfolded at normal temperatures ...

Abstract The biotin holoenzyme synthetases (BHS) are essential enzymes in all organisms that catalyze post‐translational linkage of biotin to biotin‐dependent carboxylases. The primary sequences of a large number of these enzymes are now available and homologies are found among all. The glycine‐rich sequence, GRGRXG, constitutes one of the homologous regions in these enzymes and, based on its similarity to sequences found in a...

Abstract We have used the homonuclear Overhauser effect (NOE) to characterize a model protein: carbonic anhydrase B. We have obtained NOE difference spectra for this protein, centering the on‐resonance signals either at the methyl‐proton or at the water‐proton signals. The spin‐diffusion spectra obtained as a function of protein concentration and temperature provide direct evidence of much greater protein–water interaction in the...

Abstract A 38‐residue protein associated with cholesteryl ester transfer inhibition has been identified in baboons (Papio sp.). The cholesteryl ester transfer inhibitor protein (CETIP) corresponds to the N‐terminus of baboon apoC‐I. Relative to CETIP, baboon apoC‐I is a weak inhibitor of baboon cholesteryl ester transferase (CET). To study the structural features responsible for CET inhibition, CETIP was synthesized by solid‐phase methods....

Abstract Integral membrane proteins carry out some of the most important functions of living cells, yet relatively few details are known about their structures. This is due, in large part, to the difficulties associated with preparing membrane protein crystals suitable for X‐ray diffraction analysis. Mechanistic studies of membrane protein crystallization may provide insights that will aid in determining future membrane protein...

Abstract The interdomain disulfide bond present in the C‐lobe of all the transferrins was postulated to restrict the domain movement resulting in the slow rate of iron uptake and release. In the present study, the conformational stability and iron binding properties of a derivative of the isolated C‐lobe of ovotransferrin in which the interdomain disulfide bond, Cys478‐Cys671 was selectively reduced and alkylated with iodoacetamide...

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