Methods for study of protein dynamics and protein-protein interaction in protein-ubiquitination by electron paramagnetic resonance spectroscopy.
Ubiquitination is a post-translation modification whereby the C-terminal end of ubiquitin (Ub) is covalently attached to the amino group of a lysine in a target protein. Additional ubiquitin groups are added using Ub-Ub linkages to form a polyubiquitin chain. A 26S protease complex specifically binds polyubiquitinated proteins and degrades them in an ATP-dependent manner. The target lysine in the substrate protein resides in a domain that is recognized by the ubiquitination machinery in a temporally and spatially controlled manner. The accessibility and the molecular dynamics of the target domain for each protein substrate is expected to be distinctive and this article is intended to facilitate investigations in this uncharted research area of ubiquitination mediated protein turnover by means of site-directed spin labeling. Examples illustrate the methodology of electron paramagnetic resonance data acquisition and interpretation in terms of secondary and tertiary structure resolution of proteins and protein complexes. Analysis of the spin labeled side chain mobility, its solvent accessibility, the polarity of the spin label micro-environment and distances between spin labels allow to model protein domains or protein-protein interaction sites and their conformational changes with a spatial resolution at the level of the backbone fold. The structural changes accompanying protein function or protein-protein interaction can be monitored in the millisecond time range. These features make site-directed spin labeling an attractive approach for the study of protein--ubiquitin interaction and protein ubiquitination.
Steinhoff
Heinz-Jürgen
HJ
Fachbereich Physik, Universität Osnabrück, Barbarastrasse 7, D-49069 Osnabrück, Germany. hsteinho@uni-osnabrueck.de
D003198:Computer Simulation; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D007700:Kinetics; D046911:Macromolecular Substances; D008956:Models, Chemical; D008958:Models, Molecular; D015394:Molecular Structure; D009589:Nitrogen Oxides / Q000737:chemistry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D025604:Rhodopsins, Microbial / Q000737:chemistry / Q000378:metabolism; D012997:Solvents / Q000737:chemistry; D014452:Ubiquitins / Q000378:metabolism*
D046911:Macromolecular Substances; D009589:Nitrogen Oxides; D011506:Proteins; D025604:Rhodopsins, Microbial; D012997:Solvents; D014452:Ubiquitins