Protein Folding Intermediates Characterized by Pulsed Hydrogen Exchange

Abstract

Publisher Summary This chapter reviews the protein-folding intermediates characterized by pulsed hydrogen exchange. Protein backbone amide hydrogens exchange with solvent in a reaction which is acid, base, and water catalyzed. The rate of exchange varies linearly with ‘H + ’ and ‘OH - ’ and thus, pH can be used to modulate the intrinsic exchange rate over many orders of magnitude. The minimum exchange rate for most residues occurs between pH 3 and 4. The kinetics by which amides accrue protection can be determined by performing a series of exchange experiments in which the refolding time is varied while the pulse conditions are kept constant. The Cα-NH cross-peak integrals for a variety of amides at each refolding time are then converted to proton occupancy and are fit to one or more exponential phases. The presence of labeling after long refolding times indicates either a very slow refolding phase or an incomplete quench. The strength of amide protection within a folding intermediate can be examined by varying the labeling pulse intensity at a fixed refolding time.