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.
Two-dimensional (15)N chemical shift/(1)H chemical shift and three-dimensional (1)H-(15)N dipolar coupling/(15)N chemical shift/(1)H 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 (1)H detection at 600MHz under 50kHz magic angle spinning using ∼0.5mg of perdeuterated and uniformly (15)N-labeled protein in which the exchangeable amide sites are partially or completely back-exchanged (reprotonated). Notably, the heteronuclear (1)H-(15)N dipolar coupling frequency dimension is shown to select among (15)N 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.
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92039-0307, USA.
Sang Ho
SH
Park
Department of Chemistry, University of California, Riverside, CA 92521, USA.
Chen
C
Yang
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92039-0307, USA. Electronic address: sopella@ucsd.edu.
Stanley J
SJ
Opella
Department of Chemistry, University of California, Riverside, CA 92521, USA.
Leonard J
LJ
Mueller
D000465:Algorithms; D025561:Bacteriophage Pf1 / Q000737:chemistry; D036022:Capsid Proteins / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry; D003903:Deuterium; D005998:Glycine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D011522:Protons
D036022:Capsid Proteins; D004279:DNA, Viral; D009587:Nitrogen Isotopes; D011506:Proteins; D011522:Protons; C071283:coat protein, Pseudomonas phage Pf1; D003903:Deuterium; D005998:Glycine
Fast MAS; Perdeuteration; Pf1 bacteriophage; Proton detection; Separated local field spectroscopy; Three-dimensional NMR; Two-dimensional NMR; Variable contact time cross-polarization