Rotamer libraries of spin labelled cysteines for protein studies.
Studies of structure and dynamics of proteins using site-directed spin labelling rely on explicit modelling of spin label conformations. The large computational effort associated with such modelling with molecular dynamics (MD) simulations can be avoided by a rotamer library approach based on a coarse-grained representation of the conformational space of the spin label. We show here that libraries of about 200 rotamers, obtained by iterative projection of a long MD trajectory of the free spin label onto a set of canonical dihedral angles, provide a representation of the underlying trajectory adequate for EPR distance measurements. Rotamer analysis was performed on selected X-ray structures of spin labelled T4 lysozyme mutants to characterize the spin label rotamer ensemble on a single protein site. Furthermore, predictions based on the rotamer library approach are shown to be in nearly quantitative agreement with electron paramagnetic resonance (EPR) distance data on the Na(+)/H(+) antiporter NhaA and on the light-harvesting complex LHCII whose structures are known from independent cryo electron microscopy and X-ray studies, respectively. Suggestions for the selection of labelling sites in proteins are given, limitations of the approach discussed, and requirements for further development are outlined.
Laboratory of Physical Chemistry, ETH Zürich, Wolfgang-Pauli-Str. 10, 8093 Zürich, Switzerland.
Yevhen
Y
Polyhach
Enrica
E
Bordignon
Gunnar
G
Jeschke
D020285:Cryoelectron Microscopy; D003545:Cysteine / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D008958:Models, Molecular; D008968:Molecular Conformation; D009113:Muramidase / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry*; D019151:Peptide Library; D013113:Spin Labels
D010446:Peptide Fragments; D019151:Peptide Library; D013113:Spin Labels; D009113:Muramidase; D003545:Cysteine