Abstract
Solid state NMR is a powerful method to obtain information on the structure and dynamics of protein complexes that, due to solubility and size limitations, cannot be achieved by other methods. Here, we present an approach that allows the quantification of microsecond conformational exchange in large protein complexes by using a paramagnetic agent to accelerate 15N R1ρ relaxation dispersion measurements and overcome sensitivity limitations. The method is validated on crystalline GB1 and then applied to a >300 kDa precipitated complex of GB1 with full length human immunoglobulin G (IgG). The addition of a paramagnetic agent increased the signal to noise ratio per time unit by a factor of 5, which allowed full relaxation dispersion curves to be recorded on a sample containing less than 50 μg of labelled material in 5 and 10 days on 850 and 700 MHz spectrometers, respectively. We discover a similar exchange process across the β-sheet in GB1 in crystals and in complex with IgG. However, the slow motion observed for a number of residues in the α-helix of crystalline GB1 is not detected in the complex.
Highlights
Higher concentrations (2–5 mM) of Gd(DTPA-BMA) gave similar signal to noise ratios (SNRs) per unit time for GB1:immunoglobulin G (IgG) corresponding to an increase of approximately 5 times compared to a sample without Gd(DTPA-BMA)
The results of 15N R1ρ relaxation dispersion experiments suggest that, in crystalline GB1 and in GB1 in the complex with IgG, slow conformational exchange occurs for nearly identical sets of residues in the β-sheet
Extrapolated to room temperature, this exchange process would occur on the order of hundreds of nanoseconds, which is too fast to induce relaxation dispersion in the here considered regime
Summary
Solid state NMR is a powerful method to obtain information on the structure and dynamics of protein complexes that, due to solubility and size limitations, cannot be achieved by other methods. Many motions implicated in these processes occur on microsecond or slower time scale and can be probed using chemical exchange based methods such as relaxation dispersion[1,2] Applying these methods to slowly tumbling proteins and protein complexes above a few tens of kDa in solution becomes increasingly difficult due to the enhanced T2 relaxation resulting in size dependent broadening of NMR lines. The strong spinning frequency dependence of 15N R1ρ14 suggested the presence of extensive slow μs motions of GB1 in the complex with IgG that are not present in GB1 crystals[3] Since methods such as R1ρ relaxation dispersion[15] rely on recording numerous 2D (or 3D) spectra to measure R1ρ relaxation rates at several different spin lock fields, they require unpractically long experimental times for large complexes (estimated to be on the order of one to two months of experimental time for our samples). We applied the validated technique to obtain site specific information on microsecond conformational exchange of GB1 in the >300 kDa complex with IgG
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