The energy landscape of proteins is characterised by a hierarchy of substates, which give rise to conformational heterogeneity at low temperatures. In multiply spin-labelled membranous Na,K-ATPase, this heterogeneous population of conformations is manifest by strong inhomogeneous broadening of the electron paramagnetic resonance (EPR) line shapes and non-exponential spin-echo decays, which undergo a transition to homogeneous broadening and exponential relaxation at higher temperatures (Guzzi, et al. Biochemistry 48:8343-8354, 2009). Here, 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-labelled 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 ca. 250 K or higher. Above ca. 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-labelled 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.
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|Titolo:||Heterogeneity of protein substates visualised by spin-label EPR|
|Data di pubblicazione:||2014|
|Citazione:||Heterogeneity of protein substates visualised by spin-label EPR / Guzzi, Rita; Bartucci, Rosa; Marsh, D.. - In: BIOPHYSICAL JOURNAL. - ISSN 0006-3495. - 106:3(2014), pp. 716-722.|
|Appare nelle tipologie:||1.1 Articolo in rivista|