Structural and dynamic properties of the Fv fragment and the single-chain Fv fragment of an antibody in solution investigated by heteronuclear three-dimensional NMR spectroscopy.
Fv fragments, heterodimers of the variable light (VL) and variable heavy chain (VH) domains, are the smallest functional antibody units with molecular masses of approximately 26 kDa. The structural and dynamic properties of the Fv fragment and the corresponding single-chain Fv fragment (scFv: VH-linker-VL, 252 amino acids) of the phosphorylcholine-binding antibody McPC603 in the presence of hapten have been studied in solution by heteronuclear multidimensional NMR spectroscopy. Both 15N TOCSY-HMQC and triple-resonance experiments (HNCA and HN(CA)H, with 15N-13C-labeled protein) gave poor spectra, due to short T2 relaxation times for most of the backbone 1H, 15N, and 13C alpha atoms. The assignment procedure therefore relied upon the combination of amino acid and domain (VL) specifically labeled spectra and the 3D NOESY-HMQC spectrum of the uniformly 15N labeled Fv and scFv fragments. Approximately 80% of the 15N and 1H backbone and 60% of the 1H side-chain resonances have been assigned. Short- and long-range NOEs were used to determine the extent of beta-sheet structure and were compared to the X-ray crystallographic data. The 1H-15N NOE data indicate that the scFv backbone has a well-defined structure of limited conformational flexibility. However, the linker of the scFv fragment exhibits substantial fast internal motion (on the picosecond to nanosecond time scale) compared with the overall rotational correlation time of the whole molecule. Several residues in the CDRs, in turns, or at the C-terminal end of the protein have smaller NOEs, reflecting some degree of rapid motion in the protein backbone.
Freund
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C
Max Planck Institute for Biochemistry, Martinsried, FRG.
Ross
A
A
Plückthun
A
A
Holak
T A
TA
D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D012996:Solutions
D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D046911:Macromolecular Substances; D012996:Solutions