Controlling residual dipolar couplings in high-resolution NMR of proteins by strain induced alignment in a gel.
Water-soluble biological macromolecules can be weakly aligned by dissolution in a strained, hydrated gel such as cross-linked polyacrylamide, an effect termed 'strain-induced alignment in a gel' (SAG). SAG induces nonzero nuclear magnetic dipole-dipole couplings that can be measured in high-resolution NMR spectra and used as structural constraints. The dependence of experimental 15N-1H dipolar couplings extracted from two-dimensional heteronuclear single quantum coherence (HSQC) spectra on several properties of compressed polyacrylamide, including the extent of compression, the polyacrylamide concentration, and the cross-link density, is reported for the B1 immunoglobulin binding domain of streptococcal protein G (protein G/B1, 57 residues). It is shown that the magnitude of macromolecular alignment can be widely varied by adjusting these properties, although the orientation and asymmetry of the alignment tensor are not affected significantly. The dependence of the 15N relaxation times T1 and T2 of protein G/B1 on polyacrylamide concentration are also reported. In addition, the results of 15N relaxation and HSQC experiments on the RNA binding domain of prokaryotic protein S4 from Bacillus stearothermophilus (S4 delta41, residues 43-200) in a compressed polyacrylamide gel are presented. These results demonstrate the applicability of SAG to proteins of higher molecular weight and greater complexity. A modified in-phase/anti-phase (IPAP) HSQC technique is described that suppresses natural-abundance 15N background signals from amide groups in polyacrylamide, resulting in cleaner HSQC spectra in SAG experiments. The mechanism of protein alignment in strained polyacrylamide gels is contrasted with that in liquid crystalline media.
Ishii
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Y
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, 20892-0520, USA.
Markus
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MA
Tycko
R
R
D000180:Acrylic Resins; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D005782:Gels; D001411:Geobacillus stearothermophilus; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*
D000180:Acrylic Resins; D001426:Bacterial Proteins; D005782:Gels; C034284:IgG Fc-binding protein, Streptococcus; D011506:Proteins; C016680:polyacrylamide gels