Substrate binding and conformational changes of Clostridium glutamicum diaminopimelate dehydrogenase revealed by hydrogen/deuterium exchange and electrospray mass spectrometry

Abstract

C. glutamicum meso-diaminopimelate dehydrogenase is an enzyme of the L-lysine biosynthetic pathway in bacteria. The binding of NADPH and diaminopimelate to the recombinant, overexpressed enzyme has been analyzed using hydrogen/ deuterium exchange and electrospray ionization/mass spectrometry. NADPH binding reduces the extent of deuterium exchange, as does the binding of diaminopimelate. Pepsin digestion of the deuterated enzyme and enzyme-substrate complexes coupled with liquid chromatography/mass spectrometry have allowed the identification of eight peptides whose deuterium exchange slows considerably upon the binding of the substrates. These peptides represent regions known or thought to bind NADPH and diaminopimelate. One of these peptides is located at the interdomain hinge region and is proposed to be exchangeable in the "open," catalytically inactive, conformation but nonexchangeable in the "closed," catalytically active conformation formed after NADPH and diaminopimelate binding and domain closure. Furthermore, the dimerization region has been localized by this method, and this study provides an example of detecting protein-protein interface regions using hydrogen/deuterium exchange and electrospray ionization.Keywords: amide hydrogen exchange; diaminopimelate dehydrogenase; electrospray ionization mass spectrometry; protein conformational change; substrate binding Isotopic exchange rates of amide hydrogens have been used for many years to provide information about the high-order structure, structural changes, and structural dynamics of proteins (Englander et al., 1979;Woodward et al., 1982). With the advent of electrospray ionization mass spectrometry (ESI-MS) has come the ability to investigate intact protein structures using mass spectrometry. The rates of hydrogen/deuterium (H/D) exchange have been monitored by ESI-MS to study denaturation of bovine ubiquitin and hen lysozyme (Katta & Chait, 1993), to characterize structural perturbations in proteins (Robinson et al., 1994), and to probe conformational heterogeneity and stability of apomyoglobin (Wang & Tang, 1996). These studies on intact proteins, which were carried out in solvents that are compatible with ESI-MS, do not provide high resolution information with regard to specific changes within portions of the protein.

Hydrogen/deuterium exchange studies of native rabbit MM‐CK dynamics

“…The extent of back exchange for the various proteolytic peptides ranged from 20% to 48% during quench and liquid chromatography (LC)‐MS analysis. These values are consistent with the 25% to 55% or 18% to 54% reported by Wang et al (1998a,b).…”

Enhanced correction methods for hydrogen exchange‐mass spectrometric studies of amyloid fibrils

“…The method of Zhang and Smith (1993) was the first evaluated. It has been used widely for correcting back exchange and further forward exchange of amide protons during HX studies on globular proteins (Johnson and Walsh 1994; Wang et al 1998, 1999; Anderson et al 2001; Zhang et al 2001; Tobler and Fernandez 2002). In this approach, nondeuterated and fully deuterated protein samples are analyzed using the same work‐up as the incubated (partially exchanged) sample, and the corrected deuterium content (D corr ) in the backbone amides of the target sample is then given by where m is the observed molecular mass for a partially deuterated target analyte; m 0% and m 100% are the molecular masses obtained when the procedure is applied to nondeuterated and fully deuterated analytes, respectively; and N is the number of backbone amides (N = 39 for Aβ[1–40]; Zhang and Smith 1993).…”

Hydrogen-exchange mass spectrometry for the study of intrinsic disorder in proteins

“…Mass spectrometry-detected amide exchange (HXMS) experiments were traditionally performed to study protein folding and unfolding and the measured amide proton exchange rates were used to interpret the amount of hydrogen bond 31 , however, HXMS can also be a useful indicator of solvent accessibility changes at protein surfaces due to ligand binding 32; 33; 34 and for epitope mapping of antibodies 35 . The rates of amide exchange in small peptides have been accurately measured by NMR, and these reveal subtle differences in the intrinsic rates of exchange due to sequence context 36 .…”