Investigators and Program Directors

Research Interests

The proteins obtained from hyperthermophilic organisms that live at temperatures above 90°C are far more thermostable than the homologous proteins from organisms that live at more moderate temperatures (mesophiles). Detailed physical studies have not adequately rationalized the structural basis of this differential stability. To unravel this question we have chosen to study the most thermostable protein characterized to date, Pyroccocus furious (Pf) rubredoxin vs. its homolog from a mesophilic organism, Clostridium pasteuranium.

A popular postulate is that hyperthermophile proteins are less prone to unfold due to an assumed higher degree of structural rigidity. In contrast to this hypothesis, using NMR hydrogen exchange techniques, we have shown that near room temperature essentially all of the backbone hydrogen bonds of the Pf rubredoxin are transiently broken for exchange with bulk water on the millisecond timescale. Measurement of kinetics and thermodynamics of the folding transition have been complicated by the lack of thermodynamically reversible unfolding under standard conditions for most hyperthermophile proteins. We have established conditions for determining the populations and kinetics of the 25,000 s^-1 reversible unfolding transition for Pf rubredoxin near its midpoint at 144°C using NMR chemical exchange.

Using a series of hybrid rubredoxins derived from combinations of the Cp and Pf sequences which have stabilities intermediate between those of the parental proteins, we are carrying out NMR experiments to monitor conformational dynamics and stability both near the thermal transition and at lower temperatures where the native structure is strongly favored. These measurements will help us probe further the relationship between flexibility and thermostability in a systematic and quantitative manner.

Contact Information

Phone: (518) 474-4673
FAX: (518) 473-2900
E-mail: griselda@wadsworth.org