Hydrogen exchange at the core of Escherichia coli alkaline phosphatase studied by room-temperature tryptophan phosphorescence

Biochemistry. 2000 Feb 15;39(6):1455-61. doi: 10.1021/bi991560h.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alkaline Phosphatase / chemistry*
  • Buffers
  • Deuterium / chemistry
  • Escherichia coli / enzymology*
  • Hydrogen / chemistry*
  • Kinetics
  • Solutions
  • Spectrometry, Fluorescence
  • Temperature
  • Tryptophan / chemistry*
  • Water / chemistry

Substances

  • Buffers
  • Solutions
  • Water
  • Hydrogen
  • Tryptophan
  • Deuterium
  • Alkaline Phosphatase