Double Histidine Based EPR Measurements at Physiological Temperatures Permit Site‐Specific Elucidation of Hidden Dynamics in Enzymes

Abstract

Protein dynamics is at the heart of all cellular processes. Here, we utilize the dHis-Cu II NTA label to obtain site-specific information on dynamics for both an a-helix and b-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 a-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 sitespecific dynamics using room temperature lineshape analysis.

Going the dHis-tance: Site-Directed Cu2+ Labeling of Proteins and Nucleic Acids

“…The dHis-labeled α-helix on hGSTA1-1 and β-sheet on GB1 are shown in red and blue, respectively. Adapted with permission from ref . Copyright 2020 Wiley-VCH.…”

“…In recent work, this concept was established using three dHis mutants of the small globular protein GB1 (Figure B) in which the dHis site was placed on two different β-sheet locations and one α-helical location . CW EPR data for a β-sheet site are shown in Figure B.…”

“…Despite the much larger size of hGSTA1-1 compared to GB1 (444 residues vs 56 residues), the CW EPR line shape analysis indicated that the α9 helix fluctuates ∼20 times faster than any of the GB1 sites . Note the distinct difference in line shape between the GB1 and hGSTA1-1 CW EPR data in Figure B.…”

“…Note the distinct difference in line shape between the GB1 and hGSTA1-1 CW EPR data in Figure B. Importantly, spectral simulations also reported two dynamical modes of the helixone with a rotational correlation time of 68 ± 10 ps and the other at 119 ± 16 ps . These data were significant, as previous NMR studies could not quantify the dynamics of this helix in the unliganded state, presumably due to exchange between the two dynamical states .…”

“…Additionally, as discussed previously, a picture of such fluctuations could be translated into an estimate of the Cu 2+ g -tensor values and orientations using the program ORCA. , These g -tensor data have shed light on the phenomenon of orientational selectivity within the dHis system at the X- and Q-band and may be used to correlate orientational information from PDS data to the protein structure at the Q-band. Finally, the MD runs can be used to estimate the reorientational correlation times and order parameters for Cu 2+ . These results were crucial for establishing the use of dHis as an assay of site-specific dynamics in proteins.…”