JE-TROSY: combined J- and TROSY-spectroscopy for the measurement of one-bond couplings in macromolecules.
With the application of RDCs in high-resolution NMR studies of macromolecules, there has been an interest in the development of accurate, sensitive methods for measuring 15N-1H and 13C-1H one-bond coupling constants. Most methods for determining these couplings are based on the measurement of the displacement between cross-peak components in J-coupled spectra. However, for large macromolecules and macromolecular complexes, these methods are often unreliable since differential relaxation can significantly broaden one of the multiplet components (i.e., the anti-TROSY component) and thereby make accurate determination of its position difficult. To overcome this problem, a J-evolved transverse relaxation optimized (JE-TROSY) method is presented for the determination of one-bond couplings that involves J-evolution of the sharpest cross-peak multiplet component selected in a TROSY experiment. Couplings are measured from the displacement of the TROSY component in the additional J-evolution dimension relative to a zero frequency origin. The JE-TROSY method is demonstrated on uniformly labeled 15N, 13C-labeled RNA and peptide samples, as well as with an RNA-protein complex, in which the protein is uniformly 15N, 13C-labeled. In all cases, resolved, sensitive spectra are obtained from which heteronuclear one-bond J-couplings could be accurately and easily measured.
Luy
Burkhard
B
Center for Advanced Research in Biotechnology of the University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, MD 20850, USA.
Marino
John P
JP
D000465:Algorithms; D001426:Bacterial Proteins / Q000737:chemistry*; D001704:Biopolymers / Q000737:chemistry*; D002247:Carbon Isotopes; D006860:Hydrogen Bonding; D046911:Macromolecular Substances; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry*; D011485:Protein Binding; D011522:Protons; D012313:RNA / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012815:Signal Processing, Computer-Assisted; D013113:Spin Labels
D001426:Bacterial Proteins; D001704:Biopolymers; D002247:Carbon Isotopes; D046911:Macromolecular Substances; D009587:Nitrogen Isotopes; D009841:Oligonucleotides; D011522:Protons; D016601:RNA-Binding Proteins; C042833:ROM protein, Bacteria; D013113:Spin Labels; D012313:RNA