Heterogeneity of protein substates visualized by spin-label EPR.

ABSTRACT: The energy landscape of proteins is characterized by a hierarchy of substates, which give rise to conformational heterogeneity at low temperatures. In multiply spin-labeled membranous Na,K-ATPase, this heterogeneous population of conformations is manifest by strong inhomogeneous broadening of the electron paramagnetic resonance (EPR) line shapes and nonexponential spin-echo decays, which undergo a transition to homogeneous broadening and exponential relaxation at higher temperatures (previous study). In this study, we apply these EPR methods to small water-soluble proteins, of the type for which the existence of conformational substates is well established. Both ?-helical and ?-sheet aqueous proteins that are spin-labeled on a single cysteine residue display spin-echo decays with a single phase-memory time T2M and conventional EPR line shapes with predominantly homogeneous broadening, over a broad range of temperatures from 77 K to ? 250 K or higher. Above ? 200 K, the residual inhomogeneous broadening is reduced almost to zero. In contrast, both the proteins and the spin label alone, when in a glycerol-water mixture below the glass transition, display heterogeneity in spin-echo phase-memory time and a stronger inhomogeneous broadening of the conventional line shapes, similar to multiply spin-labeled membranous Na,K-ATPase below 200 K. Above 200 K (or the glass transition), a single phase-memory time and predominantly homogeneous broadening are found in both spin-label systems. The results are discussed in terms of solvent-mediated protein transitions, the ability of single spin-label sites to detect conformational heterogeneity, and the desirability of exploring multiple sites for proteins with the size and complexity of the Na,K-ATPase.

SUBMITTER: Guzzi R 

PROVIDER: S-EPMC3945086 | biostudies-other | 2014 Feb

REPOSITORIES: biostudies-other