Protein dynamics is at the heart of all cellular processes. Here, we utilize the dHis‐CuIINTA 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.
Protein dynamics is at the heart of all cellular processes. Here, we utilize the dHis‐CuIINTA 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.
more » « less- NSF-PAR ID:
- 10204799
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Angewandte Chemie
- Volume:
- 132
- Issue:
- 51
- ISSN:
- 0044-8249
- Page Range / eLocation ID:
- p. 23240-23244
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Electron paramagnetic resonance (EPR) based distance measurements have been exploited to measure protein–protein docking, protein–DNA interactions, substrate binding and metal coordination sites. Here, we use EPR to locate a native paramagnetic metal binding site in a protein with less than 2 Å resolution. We employ a rigid Cu 2+ binding motif, the double histidine (dHis) motif, in conjunction with double electron electron resonance (DEER) spectroscopy. Specifically, we utilize a multilateration approach to elucidate the native Cu 2+ binding site in the immunoglobulin binding domain of protein G. Notably, multilateration performed with the dHis motif required only the minimum number of four distance constraints, whereas comparable studies using flexible nitroxide-based spin labels require many more for similar precision. This methodology demonstrates a significant increase in the efficiency of structural determinations via EPR distance measurements using the dHis motif.more » « less
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