Physicochemical consequences of the perdeuteriation of glutathione S‐transferase from S. japonicum

Abstract

Glutathione S-transferase (GST) from Schistosoma japonicum has been prepared in both normal protiated (pGST) and fully deuteriated (dGST) form by recombinant DNA technology. Electrospray mass spectrometry showed that the level of deuteriation in dGST was 96% and was homogeneous across the sample. This result is attributed to the use of a deuterium-tolerant host Escherichia coli strain in the preparation of the protein. 10 heteroatom-bound deuteriums (in addition to the carbon-bound deuteriums) were resistant to exchange when dGST was incubated in protiated buffer. The physicochemical and biological properties of the two proteins were compared. dGST was relatively less stable to heat denaturation and to proteolytic cleavage than was pGST. The midpoint transition temperature for pGST was 54.9°C, whereas that for dGST was 51.0°C. Static light-scattering measurements revealed that the association behavior of dGST is also different from that of pGST. The perdeuteriated enzyme shows a tendency to associate into dimers of the fundamental dimer. This is in contrast with results that have been obtained for other perdeuteriated proteins in which perdeuteriation has been shown to promote dissociation of aggregates. dGST showed a similar K m to pGST; similar results had been obtained previously with bacterial alkaline phosphatase. However, whereas the alkaline phosphatase showed a reduced rate of catalysis on deuteriation, dGST exhibited a slightly higher rate of catalysis than pGST. It is clear that the bulk substitution of deuterium for protium has significant effects on the properties of proteins. Until many more examples have been studied, it will be difficult to predict these effects for any given protein. Nevertheless, deuteriation represents an intriguing method of preparing functional analogs of recombinant proteins.

Species-Specific and Cross-Reactive IgG1 Antibody Binding to Viral Capsid Protein 1 (VP1) Antigens of Human Rhinovirus Species A, B and C

“…The CD spectrum of the GST dimer showed predominantly α-helical structure analogous to published data [28]. The CD spectra of HRV-A34, HRV-B14 and HRV-C3 VP1 demonstrated the presence of well-folded secondary structure, with broad minima between 208–220 nm indicative of mixed β-sheet and α-helical structure.…”

Impact of Deuteration on the Assembly Kinetics of Transthyretin Monitored by Native Mass Spectrometry and Implications for Amyloidoses

“…These observations are in accordance with previous studies where deuterated proteins have been found to be less stable to heat denaturation 15, 16, 17. Hattori et al 18.…”

“…There is relatively little information available on the effect of deuterium substitution on macromolecules 9, 15, 16, 17, 20. While this study shows consistent and reproducible effects of protein and solvent deuteration on TTR tetramer stability, isotope effects such as these are generally rather subtle and unpredictable.…”

“…Moreover, smaller side chains have lower steric hindrance for non‐polar amino acid side chains. This leads to weak intermolecular and intramolecular hydrophobic interactions 17, 19…”

Deuteration of Escherichia coli Enzyme INtr alters its stability

“…Since the carbon source (glucose) was not deuterated, the expected level of deuteration was in the vicinity of 90% rather than 100% [11]. For the wild-type form of EI Ntr , the predominant form isolated was both phosphorylated at the active site and gluconoylated at the site of the His tag [12] (calculated mass of the H form = 66,969.8)(Table I).…”