High kinetic resolution of protein folding events
One of the oldest ways of inducing rapid protein folding is to mix a solution of unfolded protein with buffers that favor folding (Fig. 5.1). The reaction is followed by an optical probe, for example, ultraviolet—visible or infrared absorption, circular dichroism (CD), scattering, or fluorescence. In particular, CD detection in combination with rapid mixing is an exquisitely sensitive probe of conformational changes (Luchins and Beychok, 1978; Pflumm et al., 1986; Kuwajima et al., 1987, 1993, 1996; Eltive et al., 1992; Kalnin and Kuwajima, 1995; Arai and Kuwajima, 1996). Kinetic resolution of molecular dimensions became possible by advances in X-ray scattering (Semisotnov et al., 1996) and dynamic light scattering (Gast et al., 1997). H/D exchange kinetics is frequently followed by nuclear magnetic resonance (NMR) spectrometry to obtain information about local and global folding events (see Sects. 7.1 and 8.1). Mass spectrometry detection of H/D exchange requires significantly smaller quantities of protein (Miranker et al., 1996a, b). Real-time NMR spectroscopy with kinetic resolution has significantly advanced into the millisecond time range (Frieden et al., 1993; Balbach et al., 1995; Hoeltzli and Frieden, 1995).
KeywordsNuclear Magnetic Resonance Sound Velocity Dielectric Relaxation Flash Photolysis Mercury Cadmium Telluride
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