Van Patten William J, Walder Robert, Adhikari Ayush, Okoniewski Stephen R, Ravichandran Rashmi, Tinberg Christine E, Baker David, Perkins Thomas T
JILA, National Institute of Standards and Technology and the University of Colorado, Department of Physics and Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, 440 UCB, Boulder, CO, 80309-0440, USA.
University of Washington, Seattle, Department of Biochemistry, Institute for Protein Design and Howard Hughes Medical Institute, Seattle, Washington, 98195, USA.
Chemphyschem. 2018 Jan 5;19(1):19-23. doi: 10.1002/cphc.201701147. Epub 2017 Dec 4.
Quantifying the energy landscape underlying protein-ligand interactions leads to an enhanced understanding of molecular recognition. A powerful yet accessible single-molecule technique is atomic force microscopy (AFM)-based force spectroscopy, which generally yields the zero-force dissociation rate constant (k ) and the distance to the transition state (Δx ). Here, we introduce an enhanced AFM assay and apply it to probe the computationally designed protein DIG10.3 binding to its target ligand, digoxigenin. Enhanced data quality enabled an analysis that yielded the height of the transition state (ΔG =6.3±0.2 kcal mol ) and the shape of the energy barrier at the transition state (linear-cubic) in addition to the traditional parameters [k (=4±0.1×10 s ) and Δx (=8.3±0.1 Å)]. We expect this automated and relatively rapid assay to provide a more complete energy landscape description of protein-ligand interactions and, more broadly, the diverse systems studied by AFM-based force spectroscopy.
对蛋白质-配体相互作用的能量景观进行量化，有助于加深对分子识别的理解。一种强大且易于使用的单分子技术是基于原子力显微镜（AFM）的力谱技术，该技术通常可得出零力解离速率常数（k）和到过渡态的距离（Δx）。在此，我们介绍一种改进的AFM检测方法，并将其应用于探测通过计算设计的蛋白质DIG10.3与其靶配体地高辛的结合。更高的数据质量使得分析不仅能够得出传统参数[k（=4±0.1×10⁻⁵ s⁻¹）和Δx（=8.3±0.1 Å）]，还能得出过渡态的高度（ΔG = 6.3±0.2 kcal mol⁻¹）以及过渡态能量屏障的形状（线性-立方）。我们期望这种自动化且相对快速的检测方法能够更完整地描述蛋白质-配体相互作用的能量景观，更广泛地说，能够更完整地描述基于AFM的力谱技术所研究的各种系统。
