Study of Protein Dynamics under Nanoconfinement by Spin-Label ESR: A Case of T4 Lysozyme Protein

Abstract

The electron spin resonance (ESR) spectra of spin-labeled proteins are sensitive to dynamics, but discrimination between the various dynamics is often difficult. Here, we report an improvement in ESR spectral sensitivity to local backbone dynamics of a protein by a methodology that performs ESR measurement when the protein is confined in the nanochannels of a mesoporous material. An extensive set of ESR data, which includes the spectra of a nitroxide-based side chain from buried and solvent-exposed sites of a T4 lysozyme (T4L) protein, were obtained over a range of temperatures, 200-300 K, to explore the dynamics of T4L under nanoconfinement. Spectra were simulated by performing theoretical fits to the data using the microscopic ordering with macroscopic disordering model. Two principle dynamic modes, which differ in mobility and ordering, are required to account for the spectra at temperatures >240 K. We show that the mobile one correlates only with the local backbone dynamics of buried sites, whereas the other reflects the difference in local hydration dynamics between the labeling sites in T4L. The assignment of the mobile component is supported by the X-ray crystallography data of T4L. Collectively, this study has demonstrated the validity of such a methodology for improving ESR sensitivity to buried sites in a protein.

Accessing local structural disruption of Bid protein during thermal denaturation by absorption-mode ESR spectroscopy

“…The expression and purification of T4L were performed as previously described in our publication. 31 …”

“…Cw-ESR is a common biophysical approach for studying protein dynamics. 24,[29][30][31][32][33] The cw-ESR spectrum of a spin-labeled protein encodes information on the overall motion of the nitroxide ring on the nanosecond time scale with contributions mainly from the internal motion of the R1 side chain, which can be modulated by interactions of the spin probe with the environment, and rotational diffusion of the protein. The spectral lineshapes obtained (Fig.…”

Slow Dynamics around a Protein and Its Coupling to Solvent

“… 27 − 30 SDSL in combination with ESR techniques is a powerful tool to explore local information on dynamics of molecular structure in an ensemble system. 31 , 32 Basically, change in the dynamic structure of solvent can be probed by a nitroxide radical doped in the solvent. As opposed to other techniques detecting the overall dynamic motions in the ensemble, the SDSL-ESR provides local information on the dynamics of spin probes and the corresponding potential energy associated with the local environment in the solvent.…”

Anti-apoptotic BCL-2 regulation by changes in dynamics of its long unstructured loop

“…To prepare cleaved Bid (cBid) and T4 lysozyme (T4L), we followed the protocols that we previously published 36,37 .…”