Structural Biophysics Laboratory, Center for Cancer Research, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.
Biochemistry. 2012 Jul 31;51(30):6017-27. doi: 10.1021/bi300455t. Epub 2012 Jul 16.
To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and (19)F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan ((5F)W). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that (5F)W incorporation lowered binding affinity for the HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when (5F)W was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. (19)F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each (5F)W in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody-antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody-antigen complexes with altered function that may not be discernible by other biophysical techniques.
为了更全面地理解抗体成熟过程中导致结合亲和力变化的分子机制，我们探索了使用定点氟标记和（19）氟核磁共振（NMR）的方法。我们构建了一系列源自亲和成熟的抗鸡卵清溶菌酶（HEL）小鼠 IgG1 的单链抗体（scFv），其中包含完全或个别取代色氨酸残基为 5-氟色氨酸（（5F）W）的 scFv。我们使用了一系列生物物理技术来深入了解氟取代对整体蛋白质结构和抗原结合的影响。SPR 测量表明，（5F）W 的掺入降低了与 HEL 抗原的结合亲和力。取代的影响程度取决于残基，当（5F）W 取代结合界面附近的残基时，亲和力降低最大。相比之下，与 HEL 结合的相应晶体结构与未取代的抗体基本相同。（19）F NMR 分析表明，在与抗原结合之前，游离氟化蛋白中信号严重重叠，这表明在复合物中每个（5F）W 的化学环境非常不同。初步弛豫分析表明，在抗体-抗原复合物中存在化学交换，这是 X 射线晶体学无法观察到的。这些数据表明，氟 NMR 可以成为一种非常有用的工具，用于辨别具有改变功能的 scFv 抗体-抗原复合物中的结构变化，这些变化可能无法通过其他生物物理技术辨别。
