Eric Merkley

Graduate Student
University of Washington
Department of Biochemistry

edm4@u.washington.edu

Research | top

Thermophilic proteins are isolated from organisms that grow at high temperature, or thermophiles. They have higher denaturation temperatures, increased thermodynamic stability, and higher temperatures of optimal enzymatic activity than proteins from organisms that grow at moderate temperatures (mesophiles). Many thermophilic proteins are inactive below 40 ºC. Several authors have explained this observation by the concept of "corresponding states." They propose that thermophilic and mesophilic enzymes require a similar degree of flexibility or dynamics in order to function and that thermophilic proteins reach this state at higher temperatures than mesophilic enzymes. Thus, the "corresponding states hypothesis" is really two hypotheses in one: first, that thermophilic proteins are more rigid than mesophilic proteins; and second, that flxibility is required for catalysis. My molecular dynamics studies with thermophilic NADH oxidase and a mesophilic homologue indicate that while the thermophile maintains it's native conformation at higher temperatures, the thermophile and the mesophile have a similar degree of flexibility by several global measures.

To make the connection between simulation and experiment, I am carrying out fluorescence measurements on purified NADH oxidase. Currently, I am examining the temperature dependence of the fluorescence of the enzyme's flavin cofactor as a probe of structural changes in the active site. The tryptophan fluorescence of NADH oxidase is known to change in low concentrations of urea (Zoldak, G.; Sutak, R.; Antalik, M.; Sprinzl, M.; Sedlak, E. 2003, Eur. J. Biochem., 4887-4897). The enzyme's activity is enhanced under these conditions, suggesting changes in active site structure in the presence of the denaturant. High temperatures (66-75 ºC) also increase catalytic activity, so any temperature-dependent structural changes in the active site are of great interest.

Publications | top

• Merkley, E. D.; Bernard, B.; Daggett, V., Conformational Changes Below the Tm: Molecular Dynamics Studies of the Thermal Pretransition of Ribonuclease A. Biochemistry, 47, 880-892, 2008. [DOI]
• Merkley, E.D.; Parson, W.W.; Daggett, V., Computational Studies of the Flexibility of Mesophilic and Thermophilic Enzymes of the Nitroreductase Fold Family. Manuscript it preparation, 2008.

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  Daggett Group
  Department of Bioengineering
  University of Washington
  Foege Building N310B, Box 355061
  1705 NE Pacific Street
  Seattle, WA 98195-5061