Flexibility vs Thermal Stability in Rubredoxin Proteins

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DESCRIPTION (provided by applicant): The recent characterizations of organisms that live at temperatures above 90oC, so-called hyperthermophiles, have provided an ever-increasing set of proteins which are far more thermostable than the homologous proteins obtained from organisms living at typical ambient temperatures (i.e. mesophiles). With the massive genomic sequence information now available, enzyme-based industrial processes and pharmacological therapies utilizing bioactive proteins are positioned for greatly expanded development. Physical studies of the hyperthermophile proteins are stimulated by the expectation that detailed comparison to the homologous mesophile proteins will provide insight into how increased thermal stability can be systematically engineered into proteins. Unfortunately, the physical studies to date have made it clear that the structural bases of thermostabilization are subtle in detail. Furthermore, hyperthermophile enzymes are uniformly found to be far less active than their mesophile counterparts when tested under the same ambient conditions. Both thermal stability and reduced catalytic activities of hyperthermophile proteins are commonly ascribed in increased conformational rigidity. Understanding the relationship between flexibility in the native state and global stability requires determination of the timeframe and magnitude of motions which differ between mesophile and hyperthermophile protein as well as an approach to systematic alteration of these effects. The rubredoxins from Pyrococcus furiosus and Clostridium pasteurianum offer a good model system in which the amide exchange for all backbone amides can be monitored over a wide range of pH, temperature and denaturant in a fashion which distinguishes conformational transitions both faster and slower than 1 sec(-1). Dioxygen paramagnetic relaxation measurements provide a time averaged measure of the spatial distribution of O2 throughout the protein structure. Furthermore, the spatial distribution of the sequence variations between the two rubredoxins provides a systematic path of interconversion which preserves native-like interactions.