Abstract
Order parameters are a useful tool for quantifying amplitudes of molecular motions. Here we measure dipolar order parameters by recoupling heteronuclear dipole-dipole couplings under fast spinning. We apply symmetry based recoupling methods to samples spinning under magic angle at 60 kHz by employing a variable flip angle compound inversion pulse. We validate the methods by measuring site-specific 15N-1H order parameters of a microcrystalline protein over a small temperature range and the same protein in a large, precipitated complex with antibody. The measurements of the order parameters in the complex are consistent with the observed protein undergoing overall motion within the assembly.
Highlights
The astounding number of structures found in the protein databank speaks to the usefulness of structural data to provide insights into the structure-function relationship in biology and biochemistry (Berman et al, 2000; Burley et al, 2021)
An ultimate example of this idea are intrinsically disordered proteins (IDP) and intrinsically disordered regions (IDR), which are involved in controlling countless processes in eukaryotic and prokaryotic organisms, e.g. biosynthetic steps in production of bioactive natural products (Jenner et al, 2018; Kosol et al, 2019; Fage et al, 2021)
We introduce an approach to generate pulse sequences with optimized recoupling at fast spinning given probe performance requirements
Summary
The astounding number of structures found in the protein databank speaks to the usefulness of structural data to provide insights into the structure-function relationship in biology and biochemistry (Berman et al, 2000; Burley et al, 2021). A series of simulations were performed where the applied field was varied from the lowest match condition up to 250 kHz for the composite R-elements to produce dipole recoupling efficiency curves The sequences R22219 (300(0),480(180)) and R14225 (460(0),640(180)) were chosen to test the robustness of the simulations approach, since the unusual flip-angles in the composite pulses change the scaling factors from almost 0 to above 0.2 This same in silico analysis can, be made under 100 kHz spinning (Supplementary Figure S4). Three recoupling sequences with good theoretical scaling factors and appropriate match conditions were chosen to validate our approach, and to determine which candidate scheme is the most promising These sequences: R2098 (115(0),295(180)); R22219 (300(0),480(180)); and R14225 (460(0),640(180)) were tested with both their π-pulse version and the numerically optimized sequence elements (Supplementary Figure S5). This provides further support for presence of a microsecond range overall motion of GB1 in the complex with IgG as proposed previously (Lamley et al, 2015a)
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