Theses Doctoral

Solid-State NMR Lineshape Broadening at Cryogenic Temperatures

Yi, Xu

Solid-state NMR measurement at cryogenic temperature shows significant potential for biological analysis due to its advantages for sample stability and detection sensitivity. However, at low temperature lineshape broadening and low spectral resolution are commonly observed and limit the applications for complex protein systems. Here, we explored the hypotheses for the underlying mechanisms of broad linewidths at low temperatures by studying E. coli Dihydrofolate reductase (DHFR). Our results support the hypothesis that conformational heterogeneity is a major source of linebroadening. We measured the protein backbone torsion angle (Ψ) at 105 K.

In a selectively enriched protein sample with only one amide 13 C’- 15 N correlation expected, we identified three different conformations with distinct N chemical shift values accounting for the dramatic broadening observed in low temperature NMR spectra. We presume that backbone torsion angle fluctuates among the conformers on picosecond timescale at room temperature and are ‘frozen out’ giving rise to static heterogeneity at cryogenic temperatures. MD simulations support this hypothesis. QM/MM predicted chemical shifts based on snapshots from a MD simulation show excellent agreement with our data in that the average agrees well with the room temperature shift and the distribution agrees well with the low temperature spectral lineshape.

On the other hand, our data suggest that there is no relationship between the μs - ms motions at room temperature and the lineshape broadening at low temperature. Resonance assignments of the apoenzyme in solution and associated liganded states were accomplished to identify the conformational transition in chemical exchange. We analyzed the 15 N relaxation dispersion profile of each residue at room temperature in solution; the rates appear to be organized in functional groups that exchange in a concerted fashion, with shift differences related to ligated-vs-unligated changes. The chemical shift changes associated with μs - ms exchange phenomena (and with ligation) are about an order of magnitude too small to explain the low temperature lineshapes, and also have no correlation with the low temperature lineshapes.


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More About This Work

Academic Units
Thesis Advisors
Gonzalez, Ruben L.
Ph.D., Columbia University
Published Here
February 8, 2023