HYDROGEN EXCHANGE AND METAL BINDING OF PROTEINS MONITORED BY
TIME-RESOLVED ROOM TEMPERATURE PHOSPHORESCENCE
P. Wolanin, C.J. Fischer, A. Gafni, D.G. Steel
This poster was presented at the 1999 Biophysical Society Meeting in
Baltimore, MD
Abstract
This work extends our previous studies of the effect of hydrogen/deuterium
(H/D) exchange on the room temperature phosphorescence (RTP) lifetime from
the core tryptophan (W109) of E. Coli alkaline phosphatase (AP) [Schlyer,
et al, Biochem. Biophys. Res. Comm., 223, 1996, 670-674]. The
kinetics of the exchange can be observed through an increase in the RTP
lifetime that we believe occurs due to deuteration of the indole-ring enamine
. We believe that the exchange at W109 is governed by EX1 kinetics, a hypothesis
supported by the absence of a pD dependent exchange rate. This work
shows that RTP detected H/D exchange can be monitored in other proteins,
including liver alcohol dehydrogenase (LADH) and glucose-6-phosphate dehydrogenase
(G6PDH). In addition, we explore the use of measurements of RTP lifetime
and H/D exchange rates to probe the effects of different states of metal
binding in AP on the protein's flexibility where metal binding to apo AP
changes the energy of stabilization and the core flexibility.
Our initial hypothesis was that H/D exchange rates would
monotonically increase in proteins as their RTP lifetimes decreased.
Our data shows this to be true when comparing the native conformations
of AP, LADH, and G6PDH. This is consistent with the fact that long RTP
lifetimes are only observed from tryptophans in a rigid environment, usually
deeply buried in the protein, and that this lifetime reflects the protein's
flexibility. However, it appears that AP with only Zn bound exchanges very
rapidly, despite having a RTP lifetime of about 1.1 seconds.
Supported by NIA grant AGO9761, ONR Grant
Introduction