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SCOPUS: ar.j, info:eu-repo/semantics/published

The possibility of predicting the overall shape of a macromolecule in solution from its diffusional properties has gained increasing importance in the structural genomic era. Here we explore and quantify the influence that unstructured and flexible regions have on the motions of a globular protein, a situation that can occur from the presence of such regions in the natural sequence or from additional tags. 127, an immunoglobulin-like module from the muscle protein titin, whose structure and properties are well characterized, was selected for our studies. The backbone dynamics and the overall tumbling of three different constructs of 127 were investigated using N-15 NMR relaxation collected at two N-15 frequencies (60.8 and 81.1 MHz) and fluorescence depolarization spectroscopy after labeling of a reactive cysteine with an extrinsic fluorophore. Our data show that the presence of disordered tags clearly exerts a frictional drag that increases with the length of the tags, thus affecting the module tumbling in solution. We discuss the use and the limitations of current approaches to hydrodynamic calculations, especially when having to take into account local flexibility.

info:eu-repo/semantics/published

info:eu-repo/semantics/published

Adenovirus infection affects the nuclear distribution of host splicing factors. Late phase-infected cells contain discrete clusters of small nuclear ribonucleoproteins (snRNPs) that are separate from centers containing the viral 72-kilodalton DNA-binding protein (72K protein). In the present study, we demonstrate that these snRNP clusters also contain splicing factors from the SR protein family. We show that a previously described monoclonal antibody, 3C5, detects SR proteins. Furthermore, we demonstrate that late region 3 transcription occurs at a maximal rate in infected cultures in which greater than 90% of the cells contain the snRNP clusters, indicating that such cells are actively transcribing their late genes. During the onset of the late phase, the intranuclear distribution of splicing factors is very different from that seen after the late phase is established. When late viral transcription commences, cells with snRNP clusters are less prevalent than in cultures that are maintaining maximum levels of late transcription. Instead, a cell type which shows snRNPs, concentrated in foci that also contain the viral 72K DNA-binding protein is detected. This cell type disappears from cultures by 18 to 20 h after a high-multiplicity infection. These results suggest a dynamic organization of splicing factors in infected cells that can be correlated to the status of viral gene expression. Our work also provides an explanation for the differing results that have been published concerning the organization of splicing factors in the adenovirus-infected cell nucleus (L. F. Jiménez-García and D. L. Spector, Cell 73:47-59, 1993). During the present study we observed that a monoclonal antibody against the SC-35 protein, which was used by Jiménez-García and Spector to study the localization of the SC-35 splicing factor in adenovirus-infected cells, cross-reacts with the adenovirus 72K DNA-binding protein and is thus unsuitable for this type of study.