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.

Effect of Heavy Water on Protein Flexibility

“…During the lifetime transition the phosphorescence decay is not exponential and can be fitted reasonably well in terms of two components having the lifetime of the protein in water and in D 2 O, respectively. Both the kinetics of the process and the magnitude of the lifetime components are in accord with the results reported before by Fischer et al (2000). The authors have proposed that H-D exchange at the indole ring nitrogen is responsible for the increase of but provided no evidence of concomitant ring deuteration.…”

“…2 refer to the averaged lifetime ( av ϭ ␣ 1 1 ϩ ␣ 2 2 ). Lastly, a comparison with previous studies, shows that both H and D are substantially longer for LADH (Saviotti, 1975;Kishner et al, 1979;Vanderkooi et al, 1987) but are similar, under equivalent experimental conditions, in the case of AP (Fischer et al, 2000). The shorter lifetimes reported previously for LADH presumably reflects incomplete sample deoxygenation.…”

“…The phosphorescence lifetime is a local probe reporting on the structure about the indole ring and, therefore, provides a limited, site specific picture of conformational dynamics. Moreover, there is the possibility, even if remote, that be affected by D 2 O induced changes in protein conformation that bring intramolecular quenching residues (Cys, His, and Tyr) within interaction distance of Trp or that, as suggested recently by Fischer et al (2000) for AP, H/D exchange at the ring nitrogen play a role in the increase in D 2 O. An independent monitor of protein flexibility is the permeability of the macromolecule to small solutes like acrylamide that quench the phosphorescence emission (Cioni and Strambini, 1998).…”

“…In brief, the magnitude of the intrinsic room-temperature phosphorescence lifetime ( 0 ) of Trp reports on the local flexibility of the protein matrix around the chromophore (Strambini and Gonnelli, 1995;Gonnelli and Strambini, 1995), whereas the bimolecular rate constant (k q ), derived from quenching of protein phosphorescence by acrylamide, relates to the diffusion of the solute through the protein fold to the chromophore' site and is correlated to the structural flexibility of the macromolecule (Cioni and Strambini, 1998). The influence of D 2 O on the phosphorescence of alcohol dehydrogenase (Saviotti, 1975;Kishner et al, 1979;Vanderkooi et al, 1987) and alkaline phosphatase (Schlyer et al, 1996;Fischer et al, 2000) have reported an increase of 0 in D 2 O, but the lengthening of the lifetime was not unanimously attributed to a decreased protein flexibility. The present report examines the influence of D 2 O on both 0 and k q on multiple protein systems and across a wide temperature range.…”

Accessibility of oxygen with respect to the heme pocket in horseradish peroxidase

“…Reviews by Vanderkooi18 and Subramanian et al19 illustrate the versatility of room‐temperature tryptophan phosphorescence in monitoring protein dynamics and flexibility. The penetration of different phosphorescent quenchers in protein matrices has been studied by Calhoun et al,20 and protein dynamics have been studied by either monitoring the quenching of tryptophan phosphorescence by oxygen and acrylamide as a function of temperature and solvent viscosity,21, 22 or measuring the time‐dependent change in the tryptophan phosphorescence lifetime as a function of the deuterium exchange 23–25…”