Magic angle spinning NMR experiments for structural studies of differentially enriched protein interfaces and protein assemblies.
Protein-protein interactions play vital roles in numerous biological processes. These interactions often result in formation of insoluble and noncrystalline protein assemblies. Solid-state NMR spectroscopy is rapidly emerging as a premier method for structural analysis of such systems. We introduce a family of two-dimensional magic angle spinning (MAS) NMR experiments for structural studies of differentially isotopically enriched protein assemblies. Using 1-73((13)C,(15)N)/74-108((15)N) labeled thioredoxin reassembly, we demonstrate that dipolar dephasing followed by proton-assisted heteronuclear magnetization transfer yields long-range (15)N-(13)C correlations arising exclusively from the interfaces formed by the pair of differentially enriched complementary fragments of thioredoxin. Incorporation of dipolar dephasing into the (15)N proton-driven spin diffusion and into the (1)H-(15)N FSLG-HETCOR sequences permits (1)H and (15)N resonance assignments of the 74-108((15)N) enriched C-terminal fragment of thioredoxin alone. The differential isotopic labeling scheme and the NMR experiments demonstrated here allow for structural analysis of both the interface and each interacting protein. Isotope editing of the magnetization transfers results in spectral simplification, and therefore larger protein assemblies are expected to be amenable to these experiments.
Yang
Jun
J
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Tasayco
Maria Luisa
ML
Polenova
Tatyana
T
D002247:Carbon Isotopes / Q000737:chemistry; D018360:Crystallography, X-Ray; D004058:Diffusion; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009587:Nitrogen Isotopes / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry*; D011522:Protons; D013113:Spin Labels; D013879:Thioredoxins / Q000737:chemistry*
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D011522:Protons; D013113:Spin Labels; D013879:Thioredoxins