Real-Time NMR Monitoring of Protein-Folding Kinetics by a Recycle Flow System for Temperature Jump

An NMR method was developed that allows for real-time monitoring of reactions (on the order of seconds) induced by a temperature jump. In a recycle flow system, heating and cooling baths were integrated, with the latter inside the NMR probe. A refolding reaction of ribonuclease A was triggered by rapid cooling and monitored by a series of NMR measurements over 12 s. Data were processed by principal component analysis, in which a factor related to the structural change with an exponential rate constant of 0.2–0.7 s–1 was successfully separated from factors related to baseline instability and/or noise. Temperature dependency of the rate constant revealed the entropy-driven formation of the transition state of the refolding reaction.