JILA, National Institute of Standards and Technology and University of Colorado , Boulder, Colorado 80309, United States.
Catalent Biologics-West , Emeryville, California 94608, United States.
J Am Chem Soc. 2017 Jul 26;139(29):9867-9875. doi: 10.1021/jacs.7b02958. Epub 2017 Jul 17.
Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is a powerful yet accessible means to characterize mechanical properties of biomolecules. Historically, accessibility relies upon the nonspecific adhesion of biomolecules to a surface and a cantilever and, for proteins, the integration of the target protein into a polyprotein. However, this assay results in a low yield of high-quality data, defined as the complete unfolding of the polyprotein. Additionally, nonspecific surface adhesion hinders studies of α-helical proteins, which unfold at low forces and low extensions. Here, we overcame these limitations by merging two developments: (i) a polyprotein with versatile, genetically encoded short peptide tags functionalized via a mechanically robust Hydrazino-Pictet-Spengler ligation and (ii) the efficient site-specific conjugation of biomolecules to PEG-coated surfaces. Heterobifunctional anchoring of this polyprotein construct and DNA via copper-free click chemistry to PEG-coated substrates and a strong but reversible streptavidin-biotin linkage to PEG-coated AFM tips enhanced data quality and throughput. For example, we achieved a 75-fold increase in the yield of high-quality data and repeatedly probed the same individual polyprotein to deduce its dynamic force spectrum in just 2 h. The broader utility of this polyprotein was demonstrated by measuring three diverse target proteins: an α-helical protein (calmodulin), a protein with internal cysteines (rubredoxin), and a computationally designed three-helix bundle (αD). Indeed, at low loading rates, αD represents the most mechanically labile protein yet characterized by AFM. Such efficient SMFS studies on a commercial AFM enable the rapid characterization of macromolecular folding over a broader range of proteins and a wider array of experimental conditions (pH, temperature, denaturants). Further, by integrating these enhancements with optical traps, we demonstrate how efficient bioconjugation to otherwise nonstick surfaces can benefit diverse single-molecule studies.
原子力显微镜（AFM）基础的单分子力谱（SMFS）是一种强大且易于使用的方法，可以用于表征生物分子的机械性质。从历史上看，这种方法的可及性依赖于生物分子对表面和悬臂的非特异性粘附，对于蛋白质，则依赖于目标蛋白整合到多蛋白中。然而，这种测定方法会导致高质量数据的产量低，高质量数据的定义为多蛋白的完全展开。此外，非特异性表面粘附会阻碍对α-螺旋蛋白的研究，因为α-螺旋蛋白在低力和低延伸下展开。在这里，我们通过合并两个发展来克服这些限制：（i）一种具有多功能、遗传编码的短肽标签的多蛋白，这些标签通过机械坚固的 Hydrazino-Pictet-Spengler 连接进行功能化；（ii）生物分子与聚乙二醇（PEG）涂层表面的高效定点连接。通过无铜点击化学将这种多蛋白构建体和 DNA 异双官能团锚定到 PEG 涂层的基底上，以及通过强但可还原的链霉亲和素-生物素键将其与 PEG 涂层的 AFM 尖端连接，提高了数据质量和通量。例如，我们实现了高质量数据的产量增加了 75 倍，并在短短 2 小时内重复探测相同的单个多蛋白，以推断其动态力谱。通过测量三种不同的靶蛋白：一种α-螺旋蛋白（钙调蛋白）、一种具有内部半胱氨酸的蛋白（血红素）和一种计算设计的三螺旋束（αD），证明了这种多蛋白的更广泛用途。事实上，在低加载速率下，αD 代表了迄今为止 AFM 所表征的最机械不稳定的蛋白。在商业 AFM 上进行如此高效的 SMFS 研究，可以在更广泛的蛋白质和更广泛的实验条件（pH 值、温度、变性剂）下快速表征大分子折叠。此外，通过将这些增强功能与光学陷阱相结合，我们展示了如何将生物分子有效地偶联到原本不粘的表面，从而使各种单分子研究受益。
