Resolution and measurement of heteronuclear dipolar couplings of a noncrystalline protein immobilized in a biological supramolecular assembly by proton-detected MAS solid-state NMR spectroscopy

摘要

Two-dimensional ¹⁵N chemical shift/¹H chemical shift and three-dimensional ¹H-¹⁵N dipolar coupling^/15N chemical shift/¹H chemical shift MAS solid-state NMR correlation spectra of the filamentous bacteriophage Pf1 major coat protein show single-site resolution in noncrystalline, intact-phage preparations. The high sensitivity and resolution result from ¹H detection at 600 MHz under 50 kHz magic angle spinning using ~ 0.5 mg of perdeuterated and uniformly ¹⁵N-labeled protein in which the exchangeable amide sites are partially or completely back-exchanged (reprotonated). Notably, the heteronuclear ¹H-¹⁵N dipolar coupling frequency dimension is shown to select among ¹⁵N resonances, which will be useful in structural studies of larger proteins where the resonances exhibit a high degree of overlap in multidimensional chemical shift correlation spectra.