Contribution of protein conformational heterogeneity to NMR lineshapes at cryogenic temperatures

• 成果类型: Article
• 署名作者: Yi, Xu; Fritzsching, Keith J.; Rogawski, Rivkah; Xu, Yunyao; McDermott, Ann E.
• 署名单位: Columbia University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-9147
• DOI: 10.1073/pnas.2301053120
• 发表日期: 2024-02-20
• 关键词: solid-state nmr; dynamic nuclear-polarization; dihydrofolate-reductase; cross-polarization; amyloid fibrils; sh3 domain; angle; assignment; polypeptide; peptides

摘要

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 (psi) 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 psi experiment to correlate the chemical shift of Ni+1 to psi i. 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 psi were determined for each basin: 114 +/- 7 degrees for the major peak and 150 +/- 8 degrees and 164 +/- 16 degrees for the minor peaks as contrasted with 118 degrees for the X - ray crystal structure (and 118 degrees to 130 degrees 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.