Department of Physics, Indian Institute of Science Education and Research Pune, Dr. Homi Bhabha Road, Pashan, Pune 411008, India.
Department of Chemistry, Indian Institute of Science Education and Research Pune, Dr. Homi Bhabha Road, Pashan, Pune 411008, India.
Nanotechnology. 2021 Feb 19;32(8):085103. doi: 10.1088/1361-6528/abc5f3.
We measured viscoelasticity of two nanoscale systems, single protein molecules and molecular layers of water confined between solid walls. In order to quantify the viscoelastic response of these nanoscale systems in liquid environment, the measurements are performed using two types of atomic force microscopes (AFMs), which employ different detection schemes to measure the cantilever response. We used a deflection detection scheme, available in commercial AFMs, that measures cantilever bending and a fibre-interferometer based detection which measures cantilever displacement. The hydrodynamics of the cantilever is modelled using Euler-Bernoulli equation with appropriate boundary conditions which accommodate both detection schemes. In a direct contradiction with many reports in the literature, the dissipation coefficient of a single octomer of titin I27 is found to be immeasurably low. The upper bound on the dissipation coefficient is 5 × 10 kg s, which is much lower than the reported values. The entropic stiffness of single unfolded domains of protein measured using both methods is in the range of 10 mN m. We show that in a conventional deflection detection measurement, the phase of the bending signal can be a primary source of artefacts in the dissipation estimates. It is recognized that the measurement of cantilever displacement, which has negligibly small phase lag due to hydrodynamics of the cantilever at low excitation frequencies, is better suited for ensuring artefact-free measurement of viscoelasticity compared to the measurement of the cantilever bending. Further, it was possible to measure dissipation in molecular layers of water confined between the tip and the substrate using fibre interferometer based AFM with similar experimental parameters. It confirms that the dissipation coefficient of a single I27 is below the detection limit of AFM. The results shed light on the discrepancy observed in the measured diffusional dynamics of protein collapse measured using Force spectroscopic techniques and single-molecule optical techniques.
我们测量了两个纳米系统的粘弹性，即单个蛋白质分子和被固体壁限制的水分子分子层。为了量化这些纳米系统在液体环境中的粘弹性响应，使用了两种原子力显微镜（AFM）进行测量，这两种显微镜采用不同的检测方案来测量悬臂梁的响应。我们使用了一种商业 AFM 中可用的挠度检测方案，该方案测量悬臂梁的弯曲，以及一种基于光纤干涉仪的检测方案，该方案测量悬臂梁的位移。使用适当的边界条件，通过欧拉-伯努利方程对悬臂梁的流体动力学进行建模，这些边界条件适用于两种检测方案。与文献中的许多报道直接矛盾的是，我们发现单个 titin I27 八聚物的耗散系数低得无法测量。耗散系数的上限为 5×10 kg s，远低于报道的值。使用两种方法测量的单个未折叠蛋白质域的熵弹性在 10 mN m 的范围内。我们表明，在传统的挠度检测测量中，弯曲信号的相位可能是耗散估计中伪影的主要来源。人们认识到，与测量悬臂梁弯曲相比，测量悬臂梁位移（由于悬臂梁的流体动力学，在低激励频率下，相位滞后可忽略不计）更适合确保粘弹性的无伪影测量。此外，使用基于光纤干涉仪的 AFM 可以在类似的实验参数下测量限制在尖端和基底之间的水分子层中的耗散。这证实了单个 I27 的耗散系数低于 AFM 的检测极限。研究结果阐明了使用力谱技术和单分子光学技术测量蛋白质折叠的扩散动力学时观察到的差异。
