ABSTRACT
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.
Subject(s)
GTP-Binding Proteins/chemistry , GTP-Binding Proteins/metabolism , Immunoglobulins/chemistry , Immunoglobulins/metabolism , Nitrogen Isotopes , Nuclear Magnetic Resonance, Biomolecular/methods , Protein Conformation , Protein Structure, TertiaryABSTRACT
Heating due to high power 1H decoupling limits the experimental lifetime of protein samples for solid-state NMR (SSNMR). Sample deterioration can be minimized by lowering the experimental salt concentration, temperature or decoupling fields; however, these approaches may compromise biological relevance and/or spectroscopic resolution and sensitivity. The desire to apply sophisticated multiple pulse experiments to proteins therefore motivates the development of probes that utilize the RF power more efficiently to generate a high ratio of magnetic to electric field in the sample. Here a novel scroll coil resonator structure is presented and compared to a traditional solenoid. The scroll coil is demonstrated to be more tolerant of high sample salt concentrations and cause less RF-induced sample heating. With it, the viable experimental lifetime of a microcrystalline ubiquitin sample has been extended by more than an order of magnitude. The higher B1 homogeneity and permissible decoupling fields enhance polarization transfer efficiency in 15N-13C correlation experiments employed for protein chemical shift assignments and structure determination.