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Abstract A streamlined one-day protocol is described to produce isotopically methyl-labeled protein with high levels of deuterium for NMR studies. Using this protocol, the D₂O and²H-glucose content of the media and protonation level of ILV labeling precursors (ketobutyrate and ketovalerate) were varied. The relaxation rate of the multiple-quantum (MQ) state that is present during the HMQC-TROSY pulse sequence was measured for different labeling schemes and this rate was used to predict upper limits of molecular weights for various labeling schemes. The use of deuterated solvents (D₂O) or deuterated glucose is not required to obtain¹H–¹³C correlated NMR spectra of a 50 kDa homodimeric protein that are suitable for assignment by mutagenesis. High quality spectra of 100–150 kDa proteins, suitable for most applications, can be obtained without the use of deuterated glucose. The proton on the β-position of ketovalerate appears to undergo partial exchange with deuterium under the growth conditions used in this study. 

Abstract The catalytic activity of human glutathione S‐transferase A1‐1 (hGSTA1‐1), a homodimeric detoxification enzyme, is dependent on the conformational dynamics of a key C‐terminal helix α9 in each monomer. However, the structural details of how the two monomers interact upon binding of substrates is not well understood and the structure of the ligand‐free state of the hGSTA1‐1 homodimer has not been resolved. Here, we used a combination of electron paramagnetic resonance (EPR) distance measurements and weighted ensemble (WE) simulations to characterize the conformational ensemble of the ligand‐free state at the atomic level. EPR measurements reveal a broad distance distribution between a pair of Cu(II) labels in the ligand‐free state that gradually shifts and narrows as a function of increasing ligand concentration. These shifts suggest changes in the relative positioning of the two α9 helices upon ligand binding. WE simulations generated unbiased pathways for the seconds‐timescale transition between alternate states of the enzyme, leading to the generation of atomically detailed structures of the ligand‐free state. Notably, the simulations provide direct observations of negative cooperativity between the monomers of hGSTA1‐1, which involve the mutually exclusive docking of α9 in each monomer as a lid over the active site. We identify key interactions between residues that lead to this negative cooperativity. Negative cooperativity may be essential for interaction of hGSTA1‐1 with a wide variety of toxic substrates and their subsequent neutralization. More broadly, this work demonstrates the power of integrating EPR distances with WE rare‐events sampling strategy to gain mechanistic information on protein function at the atomic level. 

Abstract Protein dynamics is at the heart of all cellular processes. Here, we utilize the dHis‐Cu^IINTA label to obtain site‐specific information on dynamics for both an α‐helix and β‐sheet site of GB1, the immunoglobulin binding domain of protein G. Spectral features found in our CW‐EPR measurements were consistent with the overall rigid nature of GB1 and with predictions from molecular dynamics simulations. Using this information, we show the potential of this approach to elucidate the role of dynamics in substrate binding of a functionally necessary α‐helix in human glutathione transferase A1‐1 (hGSTA1‐1). We observe two dynamical modes for the helix. The addition of the inhibitor GS‐Met and GS‐Hex resulted in hGSTA1‐1 to favor the more rigid active state conformation, while the faster mode potentially aids the search for substrates. Together the results illustrate the remarkable potential of the dHis‐based labelling approach to measure site‐specific dynamics using room temperature lineshape analysis.