Solid-state NMR of a protein in a precipitated complex with a full-length antibody.
NMR spectroscopy is a prime technique for characterizing atomic-resolution structures and dynamics of biomolecular complexes but for such systems faces challenges of sensitivity and spectral resolution. We demonstrate that the application of (1)H-detected experiments at magic-angle spinning frequencies of >50 kHz enables the recording, in a matter of minutes to hours, of solid-state NMR spectra suitable for quantitative analysis of protein complexes present in quantities as small as a few nanomoles (tens of micrograms for the observed component). This approach enables direct structure determination and quantitative dynamics measurements in domains of protein complexes with masses of hundreds of kilodaltons. Protein-protein interaction interfaces can be mapped out by comparison of the chemical shifts of proteins within solid-state complexes with those of the same constituent proteins free in solution. We employed this methodology to characterize a >300 kDa complex of GB1 with full-length human immunoglobulin, where we found that sample preparation by simple precipitation yields spectra of exceptional quality, a feature that is likely to be shared with some other precipitating complexes. Finally, we investigated extensions of our methodology to spinning frequencies of up to 100 kHz.
Department of Chemistry, University of Warwick , Gibbet Hill Road, Coventry CV4 7AL, U.K.
Jonathan M
JM
Lamley
Dinu
D
Iuga
Carl
C
Öster
Hans-Juergen
HJ
Sass
Marco
M
Rogowski
Andres
A
Oss
Jaan
J
Past
Andres
A
Reinhold
Stephan
S
Grzesiek
Ago
A
Samoson
Józef R
JR
Lewandowski
D000936:Antigen-Antibody Complex / Q000737:chemistry* / Q000276:immunology; D011232:Chemical Precipitation; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry* / Q000276:immunology*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011506:Proteins / Q000737:chemistry* / Q000276:immunology*
D000936:Antigen-Antibody Complex; D007136:Immunoglobulins; D011506:Proteins