Next Generation Methods for Single-Molecule Force Spectroscopy on Polyproteins and Receptor-Ligand Complexes

Abstract

Single-molecule force spectroscopy with the atomic force microscope provides molecular level insights into protein function, allowing researchers to reconstruct energy landscapes and understand functional mechanisms in biology. With steadily advancing methods, this technique has greatly accelerated our understanding of force transduction, mechanical deformation, and mechanostability within single-and multi-domain polyproteins, and receptor-ligand complexes. In this focused review, we summarize the state of the art in terms of methodology and highlight recent methodological improvements for AFM-SMFS experiments, including developments in surface chemistry, considerations for protein engineering, as well as theory and algorithms for data analysis. We hope that by condensing and disseminating these methods, they can assist the community in improving data yield, reliability, and throughput and thereby enhance the information that researchers can extract from such experiments. These leading edge methods for AFM-SMFS will serve as a groundwork for researchers cognizant of its current limitations who seek to improve the technique in the future for in-depth studies of molecular biomechanics.

Quantitative determination of mechanical stability in the novel coronavirus spike protein

“…This result highlights the need to launch new rapid research not only in the development of antibodies, entry inhibitors, and antivirals as mechanism of action, but also in new therapies aimed at de-stabilizing certain key contacts responsible for the increased stability. Also, we expect to motivate nanomechanical studies via single molecule force spectroscopy 35,36 of the spike proteins with focus on the RBD in coronavirus system as well as other experiments which can be relevant to elucidate other weaknesses in the protein structure of CoV2 and connect this information with its biological role during the cell recognition process.…”

Наноиндентирование И Механические Свойства Материалов В Субмикро- И Наношкале. Недавние Результаты И Достижения (О Б З О Р)

“…Для исследования природных биологических костных и мягких тканей и искусственных материалов медицинского назначения, где обычно необходимы силы P ≫ 100 nN, используют наноиндентометры [13,17,245,246]. Для изучения биомеханики на клеточном уровне, где требуются силы 10 pN < P < 100 nN, используют AFM [247][248][249], в том числе, обеспечивающие микросекундное разрешение во времени и позволяющие осуществлять испытания в очень широком диапазоне скоростей нагружения (от 10 2 до 10 13 pN/s) [250][251][252].…”

Influence of Fluorination on Single-Molecule Unfolding and Rupture Pathways of a Mechanostable Protein Adhesion Complex

“…To ensure we only analyzed valid single-molecule interactions, we performed AFM-SMFS with fingerprint domains [52][53][54] attached to both the cantilever and surface molecules. We were concerned that TFL incorporation into fingerprint domains would change their unfolding signatures and severely reduce expression yields.…”