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Journal Issue - Volume 13 Issue 3 (March 2004)

Abstract ClpB is a member of the bacterial protein‐disaggregating chaperone machinery and belongs to the AAA+ superfamily of ATPases associated with various cellular activities. The mechanism of ClpB‐assisted reactivation of strongly aggregated proteins is unknown and the oligomeric state of ClpB has been under discussion. Sedimentation equilibrium and sedimentation velocity show that, under physiological ionic strength in the absence of...

Abstract Phosphagen kinases catalyze the reversible transfer of a phosphate between ATP and guanidino substrates, a reaction that is central to cellular energy homeostasis. Members of this conserved family include creatine and arginine kinases and have similar reaction mechanisms, but they have distinct specificities for different guanidino substrates. There has not been a full structural rationalization of specificity, but two...

Abstract Spontaneous conformational transition of the prion protein from an α‐helical isoform to a β‐sheet‐rich isoform underlies the pathogenesis of sporadic prion diseases. To study the rate‐limiting steps of spontaneous conversion, the formation of amyloid fibrils by the recombinant human PrP C‐terminal fragment spanning residues 90–231 (recPrP) was monitored in the presence of urea. The kinetics of spontaneous fibril formation...

Abstract The generation of cytotoxic T lymphocyte (CTL) epitopes from an antigenic sequence involves number of intracellular processes, including production of peptide fragments by proteasome and transport of peptides to endoplasmic reticulum through transporter associated with antigen processing (TAP). In this study, 409 peptides that bind to human TAP transporter with varying affinity were analyzed to explore the selectivity and...

Abstract The rapidly evolving subsets of a protein are often evident in multiple sequence alignments as poorly defined, gap‐containing regions. We investigated the 3D context of these regions observed in 28 protein structures containing a GTP‐binding domain assumed to be homologous to the transforming factor p21‐RAS. The phylogenetic depth of this data set is such that it is possible to observe lineages sharing a common protein core...

Abstract 53BP1 interacts with the DNA‐binding core domain of the tumor suppressor p53 and enhances p53‐mediated transcriptional activation. The p53‐binding region of 53BP1 maps to the C‐terminal BRCT domains, which are homologous to those found in the breast cancer protein BRCA1 and in other proteins involved in DNA repair. Here we compare the thermodynamic behavior of the BRCT domains of 53BP1 and BRCA1 and examine their ability to...

Abstract The Caenorhabditis elegans SEM‐5 SH3 domains recognize proline‐rich peptide segments with modest affinity. We developed a bivalent peptide ligand that contains a naturally occurring proline‐rich binding sequence, tethered by a glycine linker to a disulfide‐closed loop segment containing six variable residues. The glycine linker allows the loop segment to explore regions of greatest diversity in sequence and structure of the...

Abstract Using only hard‐sphere repulsion, we investigated short polyalanyl chains for the presence of sterically imposed conformational constraints beyond the dipeptide level. We found that a central residue in a helical peptide cannot adopt dihedral angles from strand regions without encountering a steric collision. Consequently, an α‐helical segment followed by a β‐strand segment must be connected by an intervening linker. This...

Abstract The potential for using paramagnetic lanthanide ions to partially align troponin C in solution as a tool for the structure determination of bound troponin I peptides has been investigated. A prerequisite for these studies is an understanding of the order of lanthanide ion occupancy in the metal binding sites of the protein. Two‐dimensional {1H, 15N} HSQC NMR spectroscopy has been used to examine the binding order of Ce3+, Tb3+, and...

Abstract A rapid unidirectional method for cloning PCR‐amplified cDNA fragments into virtually any fusion protein expression vector is described. The method, termed PRESAT‐vector cloning, is based on a T‐vector technique that does not require restriction endonuclease digestion of the PCR product. Subsequently, we applied a novel ORF selection method of the ligated plasmid products. This second step involves restriction endonuclease...

Abstract aIF2β is the archaeal homolog of eIF2β, a member of the eIF2 heterotrimeric complex, implicated in the delivery of Met‐tRNAiMet to the 40S ribosomal subunit. We have determined the solution structure of the intact β‐subunit of aIF2 from Methanobacterium thermoautotrophicum. aIF2β is composed of an unfolded N terminus, a mixed α/β core domain and a C‐terminal zinc finger. NMR data shows the two folded domains display restricted mobility with...

Abstract RNase PH is a member of the family of phosphorolytic 3′ → 5′ exoribonucleases that also includes polynucleotide phosphorylase (PNPase). RNase PH is involved in the maturation of tRNA precursors and especially important for removal of nucleotide residues near the CCA acceptor end of the mature tRNAs. Wild‐type and triple mutant R68Q‐R73Q‐R76Q RNase PH from Bacillus subtilis have been crystallized and the structures determined by X‐ray...

Abstract Deamidation is a prevalent modification of crystallin proteins in the vertebrate lens. The effect of specific sites of deamidation on crystallin stability in vivo is not known. Using mass spectrometry, a previously unreported deamidation in βB1‐crystallin was identified at Gln146. Another deamidation was investigated at Asn157. It was determined that whole soluble βB1 contained 13%–17% deamidation at Gln146 and Asn157....

Abstract Gram‐positive pathogens synthesize isopentenyl diphosphate, the five‐carbon precursor of isoprenoids, via the mevalonate pathway. The enzymes of this pathway are essential for the survival of these organisms, and thus may represent possible targets for drug design. To extend our investigation of the mevalonate pathway in Enterococcus faecalis, we PCR‐amplified and cloned into pET‐28b the mvaK1 gene thought to encode mevalonate kinase,...

Abstract α1‐Antitrypsin is the most abundant protease inhibitor in plasma and is the archetype of the serine protease inhibitor superfamily. Genetic variants of human α1‐antitrypsin are associated with early‐onset emphysema and liver cirrhosis. However, the detailed molecular mechanism for the pathogenicity of most variant α1‐antitrypsin molecules is not known. Here we examined the structural basis of a dozen deficient α1‐antitrypsin variants....

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