Magic angle spinning NMR spectroscopy of thioredoxin reassemblies.
Differentially isotopically enriched 1-73((13)C,(15)N)/74-108((15)N) and 1-73((15)N)/74-108((13)C,(15)N) Escherichia coli thioredoxin reassemblies prepared by fragment complementation were investigated by high-resolution magic angle spinning solid-state NMR spectroscopy. Nearly complete resonance assignments, secondary and tertiary structure analysis are reported for 1-73((13)C,(15)N)/74-108((15)N) reassembled thioredoxin. Temperature dependence of the dipolar-assisted rotational resonance (DARR) spectra reveals the residues undergoing intermediate timescale motions at temperatures below - 15 degrees C. Analysis of the DARR intensity buildups as a function of mixing time in these reassemblies indicates that at long mixing times medium- and long-range cross-peaks do not experience dipolar truncation, suggesting that isotopic dilution is not required for gaining nontrivial distance restraints for structure calculations.
D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009038:Motion; D009587:Nitrogen Isotopes; D011487:Protein Conformation; D013696:Temperature; D013879:Thioredoxins / Q000737:chemistry*
D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; D009587:Nitrogen Isotopes; D013879:Thioredoxins