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.

Keywords: AFM; molecular biomechanics; molecular engineering; protein stability and folding; single-molecule biophysics.

FIGURE 1

Experimental configurations and pulling protocols of AFM-SMFS. (A) AFM measurement based on non-specific adsorption of proteins. (B) Immobilization of proteins using non-covalent interactions including His:Ni and biotin:avidin. (C) Immobilization of proteins using elastic linkers and covalent bonds. (D) Covalent immobilization of proteins of interest and fingerprint domains using a variety of reactions and peptide tags. (E) A free diffusion system allows continuous exchange of ligand molecules on the cantilever. (F) Different pulling protocols used in AFM-SMFS.

FIGURE 2

Overview of SMFS data processing by contour length transformation and molecular fingerprinting. (A) Top: a typical force vs. extension trace for stretching a multi-domain polyprotein assembled through a mechanically stable receptor-ligand complex. Red shows unfolding and stretching of two low-force marker domains [ddFLN4 (Schwaiger et al., 2004)], followed by unfolding and stretching of a mid-stability marker domain [CBM (Stahl et al., 2012)], followed by rupture of the mechano-stable receptor-ligand complex [SdrG:Fgβ (Milles et al., 2018)]. Bottom: Assembly of a contour length histogram following transformation into contour length space using an elasticity model of choice. Distances between peaks of the contour length histogram are used to make domain assignments to unfolding events in the data trace. (B) Four polymer elasticity models were used to transform the data from panel A. WLC, worm-like chain; FRC, freely-rotating chain; FJC, freely-jointed chain; QM-FRC, quantum mechanical freely rotating chain. For data traces that span a range of forces from <0.1 nN to >1 nN, the QM-FRC model is preferred. (C) Transformation equations of the various non-linear elasticity models and examples of model performance on test data showing stretching of unfolded CBM and rupture of the SdrG:Fgβ complex. The two curves in each plot show two separate fitting regimes below and above 150 pN.

FIGURE 3

Surface chemistry, linkers, and site-specific immobilization methods for SMFS. (A) Overview of cantilever and glass preparation for AFM-SMFS. Chemical functionalization of the substrate surface by gold-coating or aminosilanization is followed by passivation and attachment of a suitable flexible linker (typically PEG or ELP) containing a functional end group. Target molecules can be further immobilized site-specifically by several strategies: Enzymatic ligation using (B) LPXTG tag/GGG tag/Sortase A, (C) NGL tag/GL tag/OaAEP1, and (D) ybbR tag/CoA/SFP; Enzymatic self-labeling using (E) HaloTag with chloroalkane derivatives or (F) SNAP tag with benzyl group of benzylguanine; Spontaneous isopeptide bonds formation using (G) SpyTag/SpyCatcher, SnoopTag/Snoop catcher, and isopeptag/Pilin-C systems; Non-canonical amino acids incorporated by (H) amber suppression with (I) p-azidophenylalanine (pN₃F) for click reactions with alkyne or DBCO compounds or (J) p-acetylphenylalanine (pAcF) for oxide formation with an aminooxy group.