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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biophysical Chemistry Study Section (BBCB)
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Wehrle, Janna P
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Wadsworth Center
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LeMaster, David M; Anderson, Janet S; Wang, Limin et al. (2007) NMR and X-ray analysis of structural additivity in metal binding site-swapped hybrids of rubredoxin. BMC Struct Biol 7:81
LeMaster, David M; Anderson, Janet S; Hernandez, Griselda (2007) Spatial distribution of dielectric shielding in the interior of Pyrococcus furiosus rubredoxin as sampled in the subnanosecond timeframe by hydrogen exchange. Biophys Chem 129:43-8
LeMaster, David M; Anderson, Janet S; Hernandez, Griselda (2007) Normal carbon acid referencing for protein amide hydrogen exchange. Magn Reson Chem 45:601-4
LeMaster, David M; Hernandez, Griselda (2007) Residue cluster additivity of thermodynamic stability in the hydrophobic core of mesophile vs. hyperthermophile rubredoxins. Biophys Chem 125:483-9
LeMaster, David M; Anderson, Janet S; Hernandez, Griselda (2006) Role of native-state structure in rubredoxin native-state hydrogen exchange. Biochemistry 45:9956-63
Anderson, Janet S; LeMaster, David M; Hernandez, Griselda (2006) Electrostatic potential energy within a protein monitored by metal charge-dependent hydrogen exchange. Biophys J 91:L93-5
LeMaster, David M; Hernandez, Griselda (2006) Additivity of differential conformational dynamics in hyperthermophile/mesophile rubredoxin chimeras as monitored by hydrogen exchange. Chembiochem 7:1886-9
LeMaster, David M; Tang, Jianzhong; Paredes, Diana I et al. (2005) Contribution of the multi-turn segment in the reversible thermal stability of hyperthermophile rubredoxin: NMR thermal chemical exchange analysis of sequence hybrids. Biophys Chem 116:57-65
LeMaster, David M; Tang, Jianzhong; Paredes, Diana I et al. (2005) Enhanced thermal stability achieved without increased conformational rigidity at physiological temperatures: spatial propagation of differential flexibility in rubredoxin hybrids. Proteins 61:608-16
LeMaster, David M; Hernandez, Griselda (2005) Additivity in both thermodynamic stability and thermal transition temperature for rubredoxin chimeras via hybrid native partitioning. Structure 13:1153-63

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