Hydrogen-Deuterium Exchange to Study Conformational

HYDROGEN-DEUTERIUM EXCHANGE TO STUDY CONFORMATIONAL STATES OF PROTEINS 

The mechanism of amide-hydrogen exchange in proteins is well nnderstood. The exchange could be catalyzed by H+ and OH ions both. The rate constant for hydrogen exchange (fcex) is the snm of the rates for the acid- ( h) and base-catalyzed ( oh) reactions 

Two extreme situations are encountered. In one, the protein folding is much faster than the isotopic exchange rate (i.e., kz), as at a neutral pH and 

Experimental Measurements of Amide Hydrogen Isotopic Exchange 

Equilibrium experiments. The conformational changes are monitored as a fnnction of pH, the concentration of denaturant, and temperature. Measurements are made only after equilibrium has been established. 

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Coenzyme dissociation is rate limiting for both enzymes [341,4 3). Furthermore, no kinetic evidence has been obtained for an isomerization of the binary YADH-NADH complex, in contrast to the corresponding LADH complex 303). Hydrogen-deuterium exchange studies (464), however, indicate a conformational change upon coenzyme binding. 

I. D. Figueroa and D. H. Russell, Matrix-assisted laser desorption/ionization hydrogen/deuterium exchange studies to probe peptide conformational changes, J. Am. Soc. Mass Spectrom. 10, 719-731 (1999). 

Several experimental techniques have have been used to study conformational flexibility in protein molecules. Spectroscopic methods such as NMR, ESR, Mossbauer, and fluorescence techniques provide direct and detailed studies of internal motions in proteins. Hydrogen-deuterium (or tritium) exchange methods are particularly convenient for kinetic studies. Immuno-chemistry may be also a very helpful method to study conformational fluctuations. These results are discussed in the second part of this volume. 

Physico-chemical properties. In the fifties and sixties, several studies on the conformation of ACTH in solution were carried out. Among the used techniques were ORD, CD, fluorescence depolarization studies and kinetics of deuterium hydrogen exchange (for a review see ref. 2). The results pointed to a highly flexible random coil in solution however, Eisinger (40) found that the distance between Tyr and Trp [in ACTH-(1-24)] as measured by excitation spectroscopy, was in better agreement with some form of loop or helical structure. In addition. Squire and Bewley noted 11-15% helical content, located in the N-terminal 1-11 part of the molecule, when measuring the ORD of ACTH at pH 8.1 (41) (a random coil was found at neutral and acidic pH values, 2). 

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