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Journal Issue - Volume 11 Issue 10 (October 2002)

Nuclear mitotic apparatus protein (NuMA) is an essential vertebrate component in organizing microtubule ends at spindle poles. The NuMA-dynactin/dynein motor multiprotein complex not only explains the transport of NuMA along spindle fibers but also is linked to the process of microtubule focusing. The interaction sites of NuMA to dynein/dynactin have not been mapped. In the yet functionally uncharacterized N terminus of NuMA, we predict a calponin-homology (CH) domain, a motif with binding...

The aggregation observed in protein conformational diseases is the outcome of significant new -sheet structure not present in the native state. Peptide model systems have been useful in studies of fibril aggregate formation. Experimentally, it was found that a short peptide AGAAAAGA is one of the most highly amyloidogenic peptides. This peptide corresponds to the Syrian hamster prion protein (ShPrP) residues 113-120. The peptide was observed to be conserved in all species for which the PrP...

Amyloid fibrils of patients treated with regular hemodialysis essentially consists of 2-microglobulin (2-m) and its truncated species N62-m lacking six residues at the amino terminus. The truncated fragment has a more flexible three-dimensional structure and constitutes an excellent candidate for the analysis of a protein in the amyloidogenic conformation. The surface topology of synthetic fibrils obtained from intact 2-m and truncated N62-m was investigated by the limited proteolysis/mass...

Replication and related processes in eukaryotic cells require replication factor C (RFC) to load a molecular clamp for DNA polymerase in an ATP-driven process, involving multiple molecular interactions. The detailed understanding of this mechanism is hindered by the lack of data regarding structure, mutual arrangement, and dynamics of the players involved. In this study, we analyzed interactions that take place during loading onto DNA of either the PCNA clamp or the Rad9-Rad1-Hus1 checkpoint...

The NarL response regulatory protein of Escherichia coli has been engineered by covalent modification with 1,10-phenanthroline (OP) to create a set of site-specific DNA-cleaving agents. This was accomplished by introducing single cysteine amino acid replacements at selected locations within the carboxy-terminal DNA-binding domain in or nearby the helix 8 to helix 9 region of the NarL protein using site-directed mutagenesis. Of 18 modified NarL-OP proteins made, 13 retained the ability to bind...

Desulfovibrio gigas desulforedoxin (Dx) consists of two identical peptides, each containing one [Fe-4S] center per monomer. Variants with different iron and zinc metal compositions arise when desulforedoxin is produced recombinantly from Escherichia coli. The three forms of the protein, the two homodimers [Fe(III)/Fe(III)]Dx and [Zn(II)/Zn(II)]Dx, and the heterodimer [Fe(III)/Zn(II)]Dx, can be separated by ion exchange chromatography on the basis of their charge differences. Once separated, the...

The folding kinetics of G-CSF were determined by trp-fluorescence and far-UV circular dichroism. Folding and unfolding was achieved by rapid dilution and mixing of the denaturant, GdnHCl. G-CSF is a four-helical bundle protein with two long loops between the first and second helices and between the third and fourth helices. The entire conformational change expected by fluorescence was observed by stopped-flow technology, but due to rapid refolding kinetics only a portion was observed by...

Inappropriate or unregulated activation of complement can contribute to pathology in inflammatory diseases. Previous studies have shown that soluble recombinant regulators of complement are effective in animal models and some human diseases. However, limitations include cost, rapid clearance, and unwanted systemic effects. To avoid some of these problems, bacterial expression of regulators has been optimized and methods for the addition of a membrane-targeting moiety to the complement regulator...