Proton NMR measurements of bacteriophage T4 lysozyme aided by 15N isotopic labeling: structural and dynamic studies of larger proteins

Journal Article · Sat Feb 28 23:00:00 EST 1987 · Proc. Natl. Acad. Sci. U.S.A.; (United States)

DOI:https://doi.org/10.1073/pnas.84.5.1244· OSTI ID:6593635

McIntosh, L P; Griffey, R H; Muchmore, D C; Nielson, C P; Redfield, A G; Dahlquist, F W

A strategy for resolution and assignment of single proton resonances in proteins of molecular mass up to at least 40 kDa is presented. This approach is based on /sup 15/N (or /sup 13/C) labeling of selected residues in a protein. The resonances from protons directly bonded to labeled atoms are detected in a two-dimensional 1H-/sup 15/N (or /sup 13/C) spectrum. The nuclear Overhauser effects from isotopically tagged protons are selectively observed in one-dimensional isotope-directed measurements. Using this approach, we have observed approximately 160 resonances from /sup 15/N-bonded protons in the backbone and sidechains of uniformly /sup 15/N-labeled T4 lysozyme (molecular mass = 18.7 kDa). Partial proton-deuterium exchange can be used to simplify the 1H-/sup 15/N spectrum of this protein. These resonances are identified by amino acid class using selective incorporation of /sup 15/N-labeled amino acids and are assigned to specific residues by mutational substitution, multiple /sup 15/N and /sup 13/C labeling, and isotope-directed nuclear Overhauser effect measurements. For example, using a phenyl(/sup 15/N)alanine-labeled lysozyme variant containing two consecutive phenylalanine residues in an alpha-helical region, we observe an isotope-directed nuclear Overhauser effect from the amide proton of Phe-66 to that of Phe-67.

• Research Organization: Univ. of Oregon, Eugene
• OSTI ID: 6593635
• Journal Information: Proc. Natl. Acad. Sci. U.S.A.; (United States), Journal Name: Proc. Natl. Acad. Sci. U.S.A.; (United States) Vol. 5; ISSN PNASA
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
62 RADIOLOGY AND NUCLEAR MEDICINE
BACTERIA
BACTERIOPHAGES
BARYONS
CARBON 13
CARBON ISOTOPES
CELL CONSTITUENTS
DEUTERIUM
ELEMENTARY PARTICLES
ENZYMES
ESCHERICHIA COLI
EVEN-ODD NUCLEI
FERMIONS
GLYCOSYL HYDROLASES
HADRONS
HYDROGEN ISOTOPES
HYDROLASES
ISOTOPE EFFECTS
ISOTOPES
LIGHT NUCLEI
LYSOZYME
MAGNETIC RESONANCE
MICROORGANISMS
MOLECULAR STRUCTURE
NITROGEN 15
NITROGEN ISOTOPES
NMR SPECTRA
NUCLEAR MAGNETIC RESONANCE
NUCLEI
NUCLEONS
O-GLYCOSYL HYDROLASES
ODD-EVEN NUCLEI
ODD-ODD NUCLEI
PARASITES
PLASMIDS
PROTONS
RESONANCE
SPECTRA
STABLE ISOTOPES
VIRUSES