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Carbon-deuterium bonds as probes of dihydrofolate reductase

Academic Article
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Overview

related to degree

  • Thielges, Megan, Ph.D. in Biophysics, Scripps Research 2003 - 2009

authors

  • Thielges, Megan
  • Case, David A.
  • Romesberg, Floyd

publication date

  • May 2008

journal

  • Journal of the American Chemical Society  Journal

abstract

  • Much effort has been directed toward understanding the contributions of electrostatics and dynamics to protein function and especially to enzyme catalysis. Unfortunately, these studies have been limited by the absence of direct experimental probes. We have been developing the use of carbon-deuterium bonds as probes of proteins and now report the application of the technique to the enzyme dihydrofolate reductase, which catalyzes a hydride transfer and has served as a paradigm for biological catalysis. We observe that the stretching absorption frequency of (methyl- d 3) methionine carbon-deuterium bonds shows an approximately linear dependence on solvent dielectric. Solvent and computational studies support the empirical interpretation of the stretching frequency in terms of local polarity. To begin to explore the use of this technique to study enzyme function and mechanism, we report a preliminary analysis of (methyl- d 3) methionine residues within dihydrofolate reductase. Specifically, we characterize the IR absorptions at Met16 and Met20, within the catalytically important Met20 loop, and Met42, which is located within the hydrophobic core of the enzyme. The results confirm the sensitivity of the carbon-deuterium bonds to their local protein environment, demonstrate that dihydrofolate reductase is electrostatically and dynamically heterogeneous, and lay the foundation for the direct characterization protein electrostatics and dynamics and, potentially, their contribution to catalysis.

subject areas

  • Crystallography, X-Ray
  • Deuterium
  • Ligands
  • Methionine
  • Models, Molecular
  • NADP
  • Spectroscopy, Fourier Transform Infrared
  • Static Electricity
  • Tetrahydrofolate Dehydrogenase
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Identity

PubMed Central ID

  • PMC2748670

International Standard Serial Number (ISSN)

  • 0002-7863

Digital Object Identifier (DOI)

  • 10.1021/ja0779607

PubMed ID

  • 18412341
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Additional Document Info

start page

  • 6597

end page

  • 6603

volume

  • 130

issue

  • 20

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