Sequential 1H and 15N NMR assignments and secondary structure of a recombinant anti-digoxin antibody VL domain.
A uniformly 15N-labeled recombinant light-chain variable (VL) domain from the anti-digoxin antibody 26-10 has been investigated by heteronuclear two-dimensional (2D) and three-dimensional (3D) NMR spectroscopy. Complementary homonuclear 2D NMR studies of the unlabeled VL domain were also performed. Sequence-specific assignments for 97% of the main-chain and 70% of the side-chain proton resonances have been obtained. Patterns of nuclear Overhauser effects observed in 2D NOESY, 3D NOESY-HSQC, and 3D NOESY-TOCSY-HSQC spectra afford a detailed characterization of the VL domain secondary structure in solution. The observed secondary structure--a nine-stranded antiparallel beta-barrel--corresponds to that observed crystallographically for VL domains involved in quaternary associations. The locations of slowly exchanging amide protons have been discerned from a 2D TOCSY spectrum recorded after dissolving the protein in 2H2O. Strands B, C, E, and F are found to be particularly stable. The possible consequences of these results for domain-domain interactions are discussed.
Constantine
K L
KL
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543.
Goldfarb
V
V
Wittekind
M
M
Anthony
J
J
Ng
S C
SC
Mueller
L
L
D000595:Amino Acid Sequence; D000937:Antigen-Antibody Reactions; D004077:Digoxin / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007136:Immunoglobulins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009587:Nitrogen Isotopes; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D007136:Immunoglobulins; D009587:Nitrogen Isotopes; D011522:Protons; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D004077:Digoxin