Backbone conformational constraints in a microcrystalline U-15N-labeled protein by 3D dipolar-shift solid-state NMR spectroscopy.
Structural studies of uniformly labeled proteins by magic-angle spinning NMR spectroscopy have rapidly matured in recent years. Site-specific chemical shifts of several proteins have been assigned and structures determined from 2D or 3D data sets containing internuclear distance information. Here we demonstrate the application of a complementary technique for constraining protein backbone geometry using a site-resolved 3D dipolar-shift pulse sequence. The dipolar line shapes report on the relative orientations of 1H-15N[i] to 1H-15N[i+1] dipole vectors, constraining the torsion angles phi[i] and psi[i]. In addition, from the same 3D data set, several 1H-15N[i] to1H-15N[i+2] line shapes are extracted to constrain the torsion angles phi[i], psi[i], phi[i+1], and psi[i+1]. We report results for the majority of sites in the 56-residue beta1 immunoglobulin binding domain of protein G (GB1), using 3D experiments at 600 MHz 1H frequency. Excellent agreement between the SSNMR results and a new 1.14 A crystal structure illustrate the general potential of this technique for high-resolution structural refinement of solid proteins.
Franks
W Trent
WT
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Wylie
Benjamin J
BJ
Stellfox
Sara A
SA
Rienstra
Chad M
CM
D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D007136:Immunoglobulins / Q000737:chemistry / Q000378:metabolism; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D007136:Immunoglobulins; D009587:Nitrogen Isotopes; D019204:GTP-Binding Proteins