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

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.

Structure of fully protonated proteins by proton-detected magic-angle spinning NMR

“…Uniformly 13 C, 15 N-labeled GB1 and AP205CP samples were expressed in Escherichia coli, purified, and precipitated as described previously (26,52). A full description is provided in Supporting Information.…”

“…For GB1, 1 H N , 13 Cα, 13 C′, and 15 N backbone resonances were first assigned using both 13 C-based interresidue matching (27), amide 15 N matching (48), and automated analysis using the UNIO-MATCH algorithm (60,61). These assignments were then extended by measurement of the Hα shifts using a (H)NCAHA spectrum (46).…”

“…High-resolution proton-detected methods were first demonstrated with modest spinning frequencies by today's standards (∼10 kHz) and relied on a reduction of 1 H-1 H couplings by high levels of dilution with deuterium, typically perdeuteration, and complete (8, 9) or partial (10-12) protonation at exchangeable sites. The need for narrow proton resonances without such extreme levels of deuteration has motivated a continuous technological development, resulting in a dramatic increase in the available spinning frequency (13)(14)(15)(16)(17)(18)(19)(20).At MAS frequencies of 40-60 kHz, deuteration and 100% reprotonation at exchangeable sites, primarily amide protons, result in resolved and sensitive spectra, similar in quality to the case of higher dilution levels and lower spinning frequencies (21-23). This opens the way to rapid sequential assignment of backbone resonances (24-27), as well as to the unambiguous measurement of detailed structural and dynamical parameters (28-32).…”

“…High-resolution proton-detected methods were first demonstrated with modest spinning frequencies by today's standards (∼10 kHz) and relied on a reduction of 1 H-1 H couplings by high levels of dilution with deuterium, typically perdeuteration, and complete (8,9) or partial (10)(11)(12) protonation at exchangeable sites. The need for narrow proton resonances without such extreme levels of deuteration has motivated a continuous technological development, resulting in a dramatic increase in the available spinning frequency (13)(14)(15)(16)(17)(18)(19)(20).…”

“…This opens the way to rapid sequential assignment of backbone resonances (24)(25)(26)(27), as well as to the unambiguous measurement of detailed structural and dynamical parameters (28)(29)(30)(31)(32). A further increase in the MAS frequency to 100 kHz allows resonance assignment (20), a structure determination of a model protein (16), and interaction studies (15) with as little as 0.5 mg of sample. However, a high deuteration level severely limits observation of side-chain signals, which are essential for the determination of a protein structure at high resolution.…”