The studies of enzymes from extreme sources have gained significance due to their increasing potential applications. The proteins from halophiles (salt loving) have adapted to challenging environmental conditions and require salt for their structure and function. How halophilic proteins adapt to a hypersaline environment is still an intriguing question. It is important to mimic the environmental conditions of the sample under investigation with experimental techniques. In this study, structure and dynamic features of a halophilic enzyme have been investigated under high salt conditions. The acquisition of NMR data on high salt samples has always been problematic. We have devised a simple and elegant approach for obtaining NMR data for a protein in a high salt buffer that allows for virtually complete 1H, 13C, and 15N assignments. These data were then used to calculate the NMR derived structure of Haloferax volcanii dihydrofolate reductase in 3.5 M NaCl. Structure calculations showed that this protein folds in a similar manner as investigated in the crystal structures of Haloferax volcanii dihydrofolate reductase and Escherichia coli dihydrofolate reductase. To understand the effect of salt on flexibility as well as activity, NMR relaxation studies at 3.5 M and 1.0 M salt concentration were carried out. NMR dynamics of this enzyme revealed that the loss of activity as the salt concentration is lowered is due to lose in the inherent flexibility across the backbone, particularly in the catalytic loops.
| Identifer | oai:union.ndltd.org:MSSTATE/oai:scholarsjunction.msstate.edu:td-4293 |
| Date | 11 August 2007 |
| Creators | Binbuga, Bulent |
| Publisher | Scholars Junction |
| Source Sets | Mississippi State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
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