RESUMEN
Recent theories of protein folding suggest that individual proteins within a large ensemble may follow different routes in conformation space from the unfolded state toward the native state and vice versa. Herein, we introduce a new type of kinetics experiment that shows how different unfolding pathways can be selected by varying the initial reaction conditions. The relaxation kinetics of the major cold shock protein of Escherichia coli (CspA) in response to a laser-induced temperature jump are exponential for small temperature jumps, indicative of folding through a two-state mechanism. However, for larger jumps, the kinetics become strongly nonexponential, implying the existence of multiple unfolding pathways. We provide evidence that both unfolding across an energy barrier and diffusive downhill unfolding can occur simultaneously in the same ensemble and provide the experimental requirements for these to be observed.
Asunto(s)
Proteínas Bacterianas/química , Pliegue de Proteína , Escherichia coli/química , Cinética , Modelos Químicos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Espectrofotometría Infrarroja , Temperatura , Termodinámica , Factores de TiempoRESUMEN
Using the haem group of myoglobin as a probe in optical experiments makes it possible to study its conformational fluctuations in real time. Results of these experiments can be directly interpreted in terms of the structure of the potential energy surface of the protein. The current view is that proteins have rough energy landscapes comprising a large number of minima which represent conformational substates, and that these substates are hierarchically organized. Here, we show that the energy landscape is characterized by a number of discrete distributions of barrier heights each representing a tier within a hierarchy of conformational substates. Furthermore, we provide evidence that the energy surface is self-similar and offer suggestions for a characterization of the protein fluctuations.