Publication details
NMR study of a single-stranded DNA binding to the C-terminal domain of the protease from murine intracisternal A-type particle
| Authors | |
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| Year of publication | 2008 |
| Type | Conference abstract |
| MU Faculty or unit | |
| Citation | |
| Description | Murine intracisternal A-type particles are endogenous betaretroviruses which assemble and bud at the membranes of the mouse endoplasmic reticulum where they accumulate. They do not undergo maturation which involves proteolytic processing of viral proteins. Proteases play a crucial role in the retroviral replication cycle but the mechanism of their regulation remains unclear. The C-terminal domain of the retroviral proteases, rich in glycines, has been proposed to bind RNA. Biochemical tests showed that the C-terminal domain binds single-stranded oligonucleotides (both RNA and DNA) without inhibiting the proteolytic activity. The protocols of the overexpression on minimal media have been optimized and C-13, N-15, H-2 labeled full-length protease samples were prepared. A set of triple resonance experiments have been measured. Backbone assignment covering 95 % of the sequence has been achieved. Interactions between the protease and a single-stranded DNA 20-mer have been studied by titrating the protease sample with the DNA oligomer. The titration was monitored in 2D HN-HSQC and 1D proton NMR spectra. Results revealed stoichiometry of 2 protease molecules per oligonucleotide at low DNA concentrations. Signs of aggregation were observed as the DNA concentration increased. Residues mostly effected by the DNA binding were identified in the positively charged fragment between Ala 117 and Lys 127. |
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