PMID:10684627
| Citation |
Fischer, CJ, Schauerte, JA, Wisser, KC, Gafni, A and Steel, DG (2000) Hydrogen exchange at the core of Escherichia coli alkaline phosphatase studied by room-temperature tryptophan phosphorescence. Biochemistry 39:1455-61 |
|---|---|
| Abstract |
The room-temperature tryptophan (Trp) phosphorescence lifetime is sensitive to details of the local environment and has been shown to increase significantly in some proteins following H-D exchange. Careful analysis of the phosphorescence lifetime distribution of Trp 109 in Escherichia coli alkaline phosphatase (AP) in solution as a function of time during the H-D exchange shows that this process corresponds to a two-state reaction resulting from the deuteration of a single, specific hydrogen in the core of the protein. The absence of a pH dependence of the exchange rate suggests that the exchange is not an EX2 process, and therefore, a certain degree of unfolding is required for exchange to occur. This discovery opens up the use of phosphorescence-detected hydrogen exchange as a sensitive tool for monitoring the local susceptibility and activation energy for exchange in proteins having a phosphorescent Trp and, for example, for studying the effects of local mutations upon that susceptibility. |
| Links | |
| Keywords |
Alkaline Phosphatase/chemistry; Buffers; Deuterium/chemistry; Escherichia coli/enzymology; Hydrogen/chemistry; Kinetics; Solutions; Spectrometry, Fluorescence; Temperature; Tryptophan/chemistry; Water/chemistry |
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