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 {sup 1}H-{sup 15}N[i] to {sup 1}H-{sup 15}N[i+1] dipole vectors, constraining the torsion angles {phi}[i] and {psi}[i]. In addition, from the same 3D data set, several {sup 1}H-{sup 15}N[i] to{sup 1}H-{sup 15}N[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 {beta}1 immunoglobulin binding domain of protein G (GB1), using 3D experiments at 600 MHz {sup 1}H frequency. Excellent agreement between the SSNMR results and a new 1.14 {angstrom} crystal structure illustrate the general potential of this technique for high-resolution structural refinement of solid proteins.