PNAS
(Ed.)
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 Escherichia coli 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 Ni+1 to Ψi. With selective 15N and 13C enrichment of Ile, only the unique I60-I61
pair was expected to be detected in 13C’-15N
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.
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