Directly observed /sup 15/N NMR spectra of uniformly enriched proteins
The proteins cytochrome c/sub 2/, cytochrome c', and ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum were enriched in /sup 15/N by growth of the organism on /sup 15/NH/sub 4/Cl. The proteins were purified to homogeneity and studied by /sup 15/N NMR. Longitudinal and transverse relaxation times as well as the nuclear Overhauser effects were determined for various groups of the proteins which vary in molecular weight from 13,000 to 114,000. The values of these parameters for the amide resonances or for groups thought to be rigid were consistent with the molecular weights of the proteins. Relaxation times of the amino-terminal ..cap alpha..-amino groups and the side chain nitrogen atoms of arginine and lysine were consistent with much more rapid motion. Nitrogen atoms having bound protons were generally found to be decoupled from the protons by chemical exchange. Demonstrable /sup 1/H-/sup 15/N coupling was taken as an indication that exchange was hindered, either by hydrogen bonding interactions or by inaccessibility of the group to solvent. Histidine side chain nitrogen atoms, which experience a large chemical shift upon protonation/deprotonation, were often found to be broadened beyond detectability by chemical shift upon protonation/deprotonation, were often found to be broadened beyond detectability by chemical exchange and tautomerization. Strategies for improving sensitivity and for obtaining specific peak assignments are also discussed.
- Research Organization:
- Univ. of California, Davis
- OSTI ID:
- 5559509
- Journal Information:
- Biochemistry; (United States), Vol. 26:8
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
CYTOCHROMES
NUCLEAR MAGNETIC RESONANCE
OXYGENASES
AMMONIUM CHLORIDES
CHEMICAL SHIFT
NITROGEN 15
NMR SPECTRA
OVERHAUSER EFFECT
PHOTOSYNTHESIS
PROTONS
RELAXATION TIME
RHODOSPIRILLUM
AMMONIUM COMPOUNDS
AMMONIUM HALIDES
BACTERIA
BARYONS
CHEMICAL REACTIONS
CHLORIDES
CHLORINE COMPOUNDS
ELEMENTARY PARTICLES
ENZYMES
FERMIONS
HADRONS
HALIDES
HALOGEN COMPOUNDS
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
NITROGEN ISOTOPES
NUCLEI
NUCLEONS
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
OXIDOREDUCTASES
PHOTOCHEMICAL REACTIONS
PIGMENTS
PROTEINS
RESONANCE
SPECTRA
STABLE ISOTOPES
SYNTHESIS
550601* - Medicine- Unsealed Radionuclides in Diagnostics