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Journal Issue - Volume 14 Issue 1 (January 2005)

Abstract Amyloid‐related diseases are often ascribed to protein “misfolding.” Yet in the absence of high‐resolution structures for mature fibrils or intermediates, the connection between the mechanism of amyloid formation and protein folding remains tenuous. The simplistic view of amyloid fibrillogenesis as a homogeneous self‐assembly process is being increasingly challenged by observations that amyloids interact with a variety of...

Abstract We identified key residues from the structural alignment of families of protein domains from SCOP which we represented in the form of sparse protein signatures. A signature‐generating algorithm (SigGen) was developed and used to automatically identify key residues based on several structural and sequence‐based criteria. The capacity of the signatures to detect related sequences from the SWISSPROT database was assessed by...

Abstract pH‐dependent conformational changes are known to occur in dimeric procaspase‐3, and they have been shown to affect the rate of automaturation. We studied the equilibrium unfolding of procaspase‐3(C163S) as a function of pH (between pH 8.5 and pH 4) in order to examine these changes in the context of folding and stability. The data show that the procaspase dimer undergoes a pH‐dependent dissociation below pH 5, so that the...

Abstract Carbamoyl phosphate synthetase synchronizes the utilization of two ATP molecules at duplicated ATP‐grasp folds to catalyze carbamoyl phosphate formation. To define the dedicated functional role played by each of the two ATP sites, we have carried out pulse/labeling studies using the synthetases from Aquifex aeolicus and Methanococcus jannaschii, hyperthermophilic organisms that encode the two ATP‐grasp folds on separate subunits. These...

Abstract Disulfide bonding of lens crystallins contributes to the aggregation and insolubilization of these proteins that leads to cataract. A high concentration of reduced glutathione is believed to be key in preventing oxidation of crystallin sulfhydryls to form disulfide bonds. This protective role is decreased in aged lenses because of lower glutathione levels, especially in the nucleus. We recently found that human...

Abstract Plasminogen activator inhibitor‐1 (PAI‐1) belongs to the serine protease inhibitor (serpin) protein family, which has a common tertiary structure consisting of three β‐sheets and several α‐helices. Despite the similarity of its structure with those of other serpins, PAI‐1 is unique in its conformational lability, which allows the conversion of the metastable active form to a more stable latent conformation under...

Abstract Covalent modification is an important strategy for introducing new functions into proteins. As engineered proteins become more sophisticated, it is often desirable to introduce multiple, modifications involving several different functionalities in a site‐specific manner. Such orthogonal labeling schemes require independent labeling of differentially reactive nucleophilic amino acid side chains. We have developed two...

Abstract Extended proteins such as calmodulin and troponin C have two globular terminal domains linked by a central region that is exposed to water and often acts as a function‐regulating element. The mechanisms that stabilize the tertiary structure of extended proteins appear to differ greatly from those of globular proteins. Identifying such differences in physical properties of amino acid sequences between extended proteins and...

Abstract Amide hydrogen‐deuterium exchange has proven to be a powerful tool for detecting and characterizing high‐energy conformations in protein ensembles. Since interactions with ligands can modulate these high‐energy conformations, hydrogen exchange appears to be an ideal experimental probe of the physical mechanisms underlying processes like allosteric regulation. The chemical mechanism of hydrogen exchange, however, can...

Abstract The α‐lactalbumins form stable molten globule states under a range of conditions, with the low pH form being the best characterized. The stability of the molten globule varies among different members of this family, but the origin of the stability difference is not clear. We compare the folding and stability of α‐subdomain constructs of human and bovine α‐lactalbumin. Previous studies have demonstrated that the isolated...

Abstract The crystal structure of the vitamin B6‐dependent enzyme phosphoserine aminotransferase from the obligatory alkaliphile Bacillus alcalophilus has been determined at 1.08 Å resolution. The model was refined to an R‐factor of 11.7% (Rfree = 13.9%). The enzyme displays a narrow pH optimum of enzymatic activity at pH 9.0. The final structure was compared to the previously reported structure of the mesophilic phosphoserine aminotransferase from...

Abstract In aqueous solution, the ensemble of conformations sampled by peptides and unfolded proteins is largely determined by their interaction with water. It has been a long‐standing goal to capture these solute‐water energetics accurately and efficiently in calculations. Historically, accessible surface area (ASA) has been used to estimate these energies, but this method breaks down when applied to amphipathic peptides and...

Abstract Mammalian telomeres consist of long tandem arrays of double‐stranded telomeric TTAGGG repeats packaged by the telomeric DNA‐binding proteins TRF1 and TRF2. Both contain a similar C‐terminal Myb domain that mediates sequence‐specific binding to telomeric DNA. In a DNA complex of TRF1, only the single Myb‐like domain consisting of three helices can bind specifically to double‐stranded telomeric DNA. TRF2 also binds to...

Abstract Phosphopeptide‐binding domains, including the FHA, SH2, WW, WD40, MH2, and Polo‐box domains, as well as the 14‐3‐3 proteins, exert control functions in important processes such as cell growth, division, differentiation, and apoptosis. Structures and mechanisms of phosphopeptide binding are generally diverse, revealing few general principles. A computational method for analysis of phosphopeptide‐binding domains was therefore...

Abstract The mammalian mitochondrial NADP‐dependent isocitrate dehydrogenase is a citric acid cycle enzyme and an important contributor to cellular defense against oxidative stress. The Mn2+‐isocitrate complex of the porcine enzyme was recently crystallized; its structure indicates that Ser95, Asn97, and Thr78 are within hydrogen‐bonding distance of the γ‐carboxylate of enzyme‐bound isocitrate. We used site‐directed mutagenesis to replace each...

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