CDA Award Year 2004

Abstract

BOISBOUVIER Jérôme (FRANCE)
Institut de Biologie Structurale Jean-Pierre Ebel - CNRS / CEA - GRENOBLE - FRANCE
Quaternary structure study of human kinetochore using liquid crystal and cross-correlated NMR

The purpose of this proposal is to develop a general method, which will allow for solving of the quaternary structure of large physiologically relevant complexes by NMR spectroscopy. Using novel selective labeling strategies with cross-correlated and liquid crystal NMR technologies, intermolecular distance and orientation information will be measured in biomolecular systems beyond 100 kDa. Such long-range information, inaccessible using standard NMR methods, will be used to determine the relative position of each subunit within the active complex. The methodology described above will be applied to study the quaternary structure of human kinetochore, an essential multiprotein complex that assembles on centromeric DNA. Relative position and orientation of each subunit of a prekinetochore will be first determined in order to obtain a structural basis for mechanisms involved in genetic stability during mitosis. Then this prekinetochore fold will be used to monitor its interaction with identified specific mitotic factors in order to build a quaternary structure model of functional human kinetochore. These studies will allow us to obtain information on the mechanism by which kinetochores attach to spindle microtubules and to know how this kinetochore-microtubules attachment is regulated. This innovative strategy will permit to exceed the size limitations of standard NMR structural studies, and will complement existing X-ray crystallography and cryo-electron microscopic methods. This new tool for structural biology will open a new field of applications for biologists.