Large protein complexes with extreme rotational correlation times investigated in solution by magic-angle-spinning NMR spectroscopy.

We show that large protein complexes can be investigated in solution using magic-angle-spinning (MAS) NMR spectroscopy without the need for sample crystallization or precipitation. In order to efficiently average anisotropic interactions with MAS, the rotational diffusion of the molecule has to be suppressed. This can be readily achieved by lowering the sample temperature and by adding glycerol to the protein solution. The approach is demonstrated using the human small heat shock protein (sHSP) alphaB-Crystallin, which forms oligomeric assemblies of approximately 600 kDa. We suggest this scheme as an approach for overcoming size limitations imposed by overall tumbling in solution-state NMR investigations of large protein complexes.

[1]  R. Tycko,et al.  Molecular structure of amyloid fibrils: insights from solid-state NMR , 2006, Quarterly Reviews of Biophysics.

[2]  W. D. de Jong,et al.  Structural and functional similarities of bovine alpha-crystallin and mouse small heat-shock protein. A family of chaperones. , 1993, The Journal of biological chemistry.

[3]  Johannes Buchner,et al.  The eye lens chaperone α-crystallin forms defined globular assemblies , 2009, Proceedings of the National Academy of Sciences.

[4]  J. Horwitz.  Alpha-crystallin can function as a molecular chaperone. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[5]  P. Stewart,et al.  The small heat-shock protein, αb-crystallin, has a variable quaternary structure , 1998 .

[6]  Kurt Wüthrich,et al.  NMR analysis of a 900K GroEL–GroES complex , 2002, Nature.

[7]  Lewis E. Kay,et al.  Quantitative dynamics and binding studies of the 20S proteasome by NMR , 2007, Nature.

[8]  R. Tycko,et al.  Probing site-specific conformational distributions in protein folding with solid-state NMR. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[9]  A. Wand,et al.  High-resolution NMR of encapsulated proteins dissolved in low-viscosity fluids. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[10]  R. Riek,et al.  Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[11]  P. Schurtenberger,et al.  Structural properties of polydisperse biopolymer solutions: A light scattering study of bovine α‐crystallin , 1991, Biopolymers.

[12]  R. Klevit,et al.  alphaB-crystallin: a hybrid solid-state/solution-state NMR investigation reveals structural aspects of the heterogeneous oligomer. , 2009, Journal of molecular biology.

[13]  Carol V Robinson,et al.  Phosphorylation of αB-Crystallin Alters Chaperone Function through Loss of Dimeric Substructure* , 2004, Journal of Biological Chemistry.

[14]  B. Reif,et al.  αB‐crystallin competes with Alzheimer's disease β‐amyloid peptide for peptide–peptide interactions and induces oxidation of Abeta‐Met35 , 2006 .

[15]  B. Reif,et al.  Ultrahigh resolution in proton solid-state NMR spectroscopy at high levels of deuteration. , 2006, Angewandte Chemie.

[16]  T. Ban,et al.  αB-crystallin, a small heat-shock protein, prevents the amyloid fibril growth of an amyloid β-peptide and β2-microglobulin , 2005 .

[17]  A. McDermott,et al.  Solid-state NMR on a type III antifreeze protein in the presence of ice. , 2008, Journal of the American Chemical Society.

[18]  M. Delaye,et al.  Short-range order of crystallin proteins accounts for eye lens transparency , 1983, Nature.

[19]  W. W. Jong,et al.  The Molecular Chaperone aB-crystallin Enhances Amyloid Neurotoxicity , 1999 .

[20]  A. McDermott.  Structure and dynamics of membrane proteins by magic angle spinning solid-state NMR. , 2009, Annual review of biophysics.