Journal
BIOCHEMISTRY
Volume 50, Issue 38, Pages 8251-8260Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi200640s
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Funding
- NIH [R01 GM025765, R01 GM074134, T32 GM08295]
- NSF [MCB0446395, CHE-0715448, BSF 2007256]
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The technique of hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) has been applied to a mesophilic (E. coli) dihydrofolate reductase under conditions that allow direct comparison to a thermophilic (B. stearothermophilus) ortholog Ec-DHFR and Bs-DHFR, respectively. The analysis of hydrogen deuterium exchange patterns within proteolytically derived peptides allows spatial resolution, while requiring a series of controls to compare orthologous proteins with only ca. 40% sequence identity. These controls include the determination of primary structure effects on intrinsic rate constants for HDX as well as the use of existing 3-dimensional structures to evaluate the distance of each backbone amide hydrogen to the protein surface. Only a single peptide from the Ec-DHFR is found to be substantially more flexible than the Bs-DHFR at 25 C in a region located within the protein interior at the intersection of the cofactor and substrate-binding sites. The surrounding regions of the enzyme are either unchanged or more flexible in the thermophilic DHFR from B. stearothermophilus. The region with increased flexibility in Ec-DHFR corresponds to one of two regions previously proposed to control the enthalpic barrier for hydride transfer in Bs-DHFR [Oyeyemi et al. (2010) Proc. Natl. Acad. Sci. USA. 107, 10074].
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