Hellman Lance M, Yin Liusong, Wang Yuan, Blevins Sydney J, Riley Timothy P, Belden Orrin S, Spear Timothy T, Nishimura Michael I, Stern Lawrence J, Baker Brian M
Department of Chemistry & Biochemistry and the Harper Cancer Research Institute, A224 Harper Hall, University of Notre Dame, Notre Dame, IN 46556, USA.
Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
J Immunol Methods. 2016 May;432:95-101. doi: 10.1016/j.jim.2016.02.016. Epub 2016 Feb 18.
Measurements of thermal stability by circular dichroism (CD) spectroscopy have been widely used to assess the binding of peptides to MHC proteins, particularly within the structural immunology community. Although thermal stability assays offer advantages over other approaches such as IC50 measurements, CD-based stability measurements are hindered by large sample requirements and low throughput. Here we demonstrate that an alternative approach based on differential scanning fluorimetry (DSF) yields results comparable to those based on CD for both class I and class II complexes. As they require much less sample, DSF-based measurements reduce demands on protein production strategies and are amenable for high throughput studies. DSF can thus not only replace CD as a means to assess peptide/MHC thermal stability, but can complement other peptide-MHC binding assays used in screening, epitope discovery, and vaccine design. Due to the physical process probed, DSF can also uncover complexities not observed with other techniques. Lastly, we show that DSF can also be used to assess peptide/MHC kinetic stability, allowing for a single experimental setup to probe both binding equilibria and kinetics.
通过圆二色性（CD）光谱法测量热稳定性已被广泛用于评估肽与MHC蛋白的结合，特别是在结构免疫学领域。尽管热稳定性测定法比其他方法（如IC50测量）具有优势，但基于CD的稳定性测量受到大样本需求和低通量的阻碍。在这里，我们证明了一种基于差示扫描荧光法（DSF）的替代方法对于I类和II类复合物产生的结果与基于CD的结果相当。由于它们需要的样品少得多，基于DSF的测量减少了对蛋白质生产策略的需求，并且适合高通量研究。因此，DSF不仅可以替代CD作为评估肽/MHC热稳定性的手段，还可以补充用于筛选、表位发现和疫苗设计的其他肽-MHC结合测定法。由于所探测的物理过程，DSF还可以揭示其他技术未观察到的复杂性。最后，我们表明DSF还可用于评估肽/MHC动力学稳定性，允许通过单一实验设置来探测结合平衡和动力学。
