Contribution of protein conformational heterogeneity to NMR lineshapes at cryogenic temperatures.

While low-temperature Nuclear Magnetic Resonance (NMR) holds great promise for the analysis of unstable samples and for sensitizing NMR detection, spectral broadening in frozen protein samples is a common experimental challenge. One hypothesis explaining the additional linewidth is that a variety of conformations are in rapid equilibrium at room temperature and become frozen, creating an inhomogeneous distribution at cryogenic temperatures. Here, we investigate conformational heterogeneity by measuring the backbone torsion angle (Ψ) in Dihydrofolate Reductase (DHFR) at 105 K. Motivated by the particularly broad N chemical shift distribution in this and other examples, we modified an established NCCN Ψ experiment to correlate the chemical shift of N to Ψ. With selective N and C enrichment of Ile, only the unique I60-I61 pair was expected to be detected in C'-N correlation spectrum. For this unique amide, we detected three different conformation basins based on dispersed chemical shifts. Backbone torsion angles Ψ were determined for each basin: 114 ± 7° for the major peak and 150 ± 8° and 164 ± 16° for the minor peaks as contrasted with 118° for the X-ray crystal structure (and 118° to 130° for various previously reported structures). These studies support the hypothesis that inhomogeneous distributions of protein backbone torsion angles contribute to the lineshape broadening in low-temperature NMR spectra.