⁷⁷Se NMR Probes the Protein Environment of Selenomethionine

Abstract

Sulfur is critical for the correct structure and proper function of proteins. Yet, lacking a sensitive enough isotope, nuclear magnetic resonance (NMR) experiments are unable to deliver for sulfur in proteins the usual wealth of chemical, dynamic, and structural information. This limitation can be circumvented by substituting sulfur with selenium, which has similar physicochemical properties and minimal impact on protein structures but possesses an NMR compatible isotope (77Se). Here we exploit the sensitivity of 77Se NMR to the nucleus’ chemical milieu and use selenomethionine as a probe for its proteinaceous environment. However, such selenium NMR spectra of proteins currently resist a reliable interpretation because systematic connections between variations of system variables and changes in 77Se NMR parameters are still lacking. To start narrowing this knowledge gap, we report here on a biological 77Se magnetic resonance data bank based on a systematically designed library of GB1 variants in which a single selenomethionine was introduced at different locations within the protein. We recorded the resulting isotropic 77Se chemical shifts and relaxation times for six GB1 variants by solution-state 77Se NMR. For four of the GB1 variants we were also able to determine the chemical shift anisotropy tensor of SeM by solid-state 77Se NMR. To enable interpretation of the NMR data, the structures of five of the GB1 variants were solved by X-ray crystallography to a resolution of 1.2 Å, allowing us to unambiguously determine the conformation of the selenomethionine. Finally, we combine our solution- and solid-state NMR data with the structural information to arrive at general insights regarding the execution and interpretation of 77Se NMR experiments that exploit selenomethionine to probe proteins.

Selenium‐NMR Spectroscopy in Organic Synthesis: From Structural Characterization Toward New Investigations

“…This large shift dispersion overcomes the limitations of 1 H and 13 C nuclides and makes it useful as a probe for a great amount of applications. To exemplify this high 77 Se NMR chemical shift sensitivity, very recently, Rozovsky and co‐workers explored the selenomethionine amino acid residue as an active probe to provide data about the protein environment [61] . In this case, the interpretation is complex especially compared to small organic molecules, however, the authors have employed density functional theory (DFT) to aid the discernment of 77 Se NMR anisotropic effect.…”

“…To exemplify this high 77 Se NMR chemical shift sensitivity, very recently, Rozovsky and co-workers explored the selenomethionine amino acid residue as an active probe to provide data about the protein environment. [61] In this case, the interpretation is complex especially compared to small organic molecules, however, the authors have employed density functional theory (DFT) to aid the discernment of 77 Se NMR anisotropic effect. The natural abundancy of 77 Se NMR experiments (magnetic field of 11.74 T) in the biological samples was sensitive to protein variants and small changes on the temperature, affording new perspectives for 77 Se NMR spectroscopy.…”

Taming the Biological Activity of Pd(II) and Pt(II) Complexes with Triazolato “Protective” Groups: 1H, 77Se Nuclear Magnetic Resonance and X-ray Crystallographic Model Studies with Selenocysteine to Elucidate Differential Thioredoxin Reductase Inhibition

“…The exchange of S by Se, on the other hand, only leads to a chemical shift difference of 33 ppm. In addition to the 195 Pt NMR, the selenocysteine itself also offers an inherent spectroscopic handle since 77 Se is an I = 1/2 nucleus with a natural abundancy of 7.6% and a sensitivity comparable to 13 C without enrichment . For N -acetyl- l -selenocystine methyl ester 5 with a Se–Se bond, the 77 Se NMR signal is observed at 297.47 ppm (Figure S17), which shifts to −66.93 ppm in reduced selenol 6 (Figure B).…”

Exploring Sulfur Sites in Proteins via Triple-Resonance 1H-Detected 77Se NMR

“…Instead, we examine the use of 77 Se (7.6% natural abundance) as a substitute for the natural abundance S in proteins. Early work − illustrated the sensitivity of the 77 Se chemical shift to the environment and even illustrated the potential of seeing signals from proteins. , Recent work has described the potential utilization of 77 Se direct detection NMR as a means of exploring S sites in soluble proteins and revealed the sensitivity of 77 Se to environment and dynamics . Additionally, interest in selenoglycosides has led to some innovative 1 H-detected NMR experiments on relatively small molecular systems.…”

“…For the case of detecting or monitoring 77 Se sites, there are no directly bonded, nonexchangeable protons to facilitate such double-or triple-resonance methodologies. Examples of 1 H-detected multiple bond detection have been reported, which have been collected with an HMBC-type experiment and a clever extension of HSQC spectroscopy referred to as HSQCMB, , both of which rely on two-bond 2 J 1H–77Se . Each method requires a long time (∼1/2J = 35–50 ms) for both the forward and reverse coherence transfers; hence, the applications are limited to rather small molecular systems with long T2 relaxation times.…”