Solid-State NMR of a Protein in a Precipitated Complex with a Full-Length Antibody

Citations Over TimeTop 10% of 2014 papers

Abstract

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.

Related Papers

• → 27Al Solid-state NMR Structural Studies of Hydrotalcite Compounds Calcined at Different Temperatures(2009)21 cited
• → Recent developments in solid–state magic–angle spinning, nuclear magnetic resonance of fully and significantly isotopically labelled peptides and proteins(2004)16 cited
• → Characterization of V2O5-AlPO4 catalysts by 51V and 1H magic-angle spinning solid-state nuclear magnetic resonance spectroscopy(1995)7 cited
• → Magic‐Angle Spinning Solid‐State Nuclear Magnetic Resonance: Application to Structural Biology(2009)2 cited
• → High resolution solid state NMR of quadrupolar nuclei(1989)1 cited