Freund C, Ross A, Plückthun A, Holak T A
Max Planck Institute for Biochemistry, Martinsried, FRG.
Biochemistry. 1994 Mar 22;33(11):3296-303. doi: 10.1021/bi00177a021.
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.
Fv片段是可变轻链（VL）和可变重链（VH）结构域的异二聚体，是最小的功能性抗体单元，分子量约为26 kDa。已通过异核多维核磁共振光谱法在溶液中研究了磷酰胆碱结合抗体McPC603的Fv片段以及相应的单链Fv片段（scFv：VH-连接子-VL，252个氨基酸）在存在半抗原的情况下的结构和动力学性质。由于大多数主链1H、15N和13Cα原子的T2弛豫时间较短，15N TOCSY-HMQC和三共振实验（HNCA和HN(CA)H，使用15N-13C标记的蛋白质）都给出了较差的光谱。因此，归属过程依赖于氨基酸和结构域（VL）特异性标记光谱以及均匀15N标记的Fv和scFv片段的3D NOESY-HMQC光谱的组合。已归属了约80%的15N和1H主链共振以及60%的1H侧链共振。短程和长程NOE用于确定β-折叠结构的程度，并与X射线晶体学数据进行比较。1H-15N NOE数据表明，scFv主链具有定义明确的结构，构象灵活性有限。然而，与整个分子的整体旋转相关时间相比，scFv片段的连接子表现出大量快速的内部运动（在皮秒到纳秒时间尺度上）。反过来，蛋白质互补决定区（CDR）中的几个残基或蛋白质C末端的残基具有较小的NOE，反映了蛋白质主链中一定程度的快速运动。
