title: Hydrogen exchange at the core of Escherichia coli alkaline phosphatase studied by room-temperature tryptophan phosphorescence.
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.
authors.affiliation: Institute of Gerontology, Applied Physics Program, Department of Biological Chemistry, Department of Physics, University of Michigan, Ann Arbor, Michigan 48109-2007, USA.
authors.forename: C J
authors.initials: CJ
authors.lastname: Fischer
authors.forename: J A
authors.initials: JA
authors.lastname: Schauerte
authors.forename: K C
authors.initials: KC
authors.lastname: Wisser
authors.forename: A
authors.initials: A
authors.lastname: Gafni
authors.forename: D G
authors.initials: DG
authors.lastname: Steel
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry*; D002021:Buffers; D003903:Deuterium / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D006859:Hydrogen / Q000737:chemistry*; D007700:Kinetics; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry*; D014867:Water / Q000737:chemistry
chemical_list: D002021:Buffers; D012996:Solutions; D014867:Water; D006859:Hydrogen; D014364:Tryptophan; D003903:Deuterium; D000469:Alkaline Phosphatase