title: 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 α-helical and β-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.
authors: Dipartimento di Fisica, Laboratorio di Biofisica Molecolare, Università della Calabria, 87036 Arcavacata di Rende, CS, Italy.
authors: Rita
authors: R
authors: Guzzi
authors: Dipartimento di Fisica, Laboratorio di Biofisica Molecolare, Università della Calabria, 87036 Arcavacata di Rende, CS, Italy.
authors: Rosa
authors: R
authors: Bartucci
authors: Max-Planck-Institut für biophysikalische Chemie, 37070 Göttingen, Germany. Electronic address: dmarsh@gwdg.de.
authors: Derek
authors: D
authors: Marsh
mesh_terms: D000818:Animals; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D007782:Lactoglobulins / Q000737:chemistry*; D012709:Serum Albumin / Q000737:chemistry*
chemical_list: D006454:Hemoglobins; D007782:Lactoglobulins; D012709:Serum Albumin