Determination of macromolecular folding and structure by synchrotron X-rayradiolysis techniques

Radiolysis of water by synchrotron X-rays generates oxygen-containing radic als that undergo reactions with solvent accessible sites of macromolecules inducing stable covalent modifications or cleavage on millisecond time scal es. The extent and site of these reactions are determined by gel electropho resis and mass spectrometry analysis. These data are used to construct a hi gh-resolution map of solvent accessibility at individual reactive sites. Th e experiments can be performed in a time-resolved manner to provide kinetic rate constants for dynamic events occurring at individual sites within mac romolecules or can provide equilibrium parameters of binding and thermodyna mics of folding processes. The application of this synchrotron radiolysis t echnique to the study of lysozyme protein structure and the equilibrium ure a induced unfolding of apomyoglobin are described. The Mg2+-induced folding of Tetrahymena thermophila group I ribozyme shows the capability of the me thod to study kinetics of folding. (C) 2001 Academic Press.