Concurrent atomic force spectroscopy

Abstract

Force-spectroscopy by atomic force microscopy (AFM) is the technique of choice to measure mechanical properties of molecules, cells, tissues and materials at the nano and micro scales. However, unavoidable calibration errors of AFM probes make it cumbersome to quantify modulation of mechanics. Here, we show that concurrent AFM force measurements enable relative mechanical characterization with an accuracy that is independent of calibration uncertainty, even when averaging data from multiple, independent experiments. Compared to traditional AFM, we estimate that concurrent strategies can measure differences in protein mechanical unfolding forces with a 6-fold improvement in accuracy or a 30-fold increase in throughput. Prompted by our results, we demonstrate widely applicable orthogonal fingerprinting strategies for concurrent single-molecule nanomechanical profiling of proteins.

Nanomechanical Phenotypes in Cardiac Myosin-Binding Protein C Mutants That Cause Hypertrophic Cardiomyopathy

“…Mechanical unfolding of a C3 domain within the polyprotein results in the extension of the polypeptide by 24–25 nm. The presence of multiple such unfolding steps fingerprints successful single-polyprotein recordings (Figure a) . We measured the force at which mechanical unfolding occurs in the 40 pN/s force ramp for hundreds of WT and mutant domains (Supplementary File S2) and built distributions of unfolding forces (Figures b, a; Supplementary Figure S9).…”

“…In these experiments, a single polyprotein is tethered between a cantilever and a moving piezo actuator, and its length is recorded while a linear increase in force is applied. Unfolding events are detected as step increases in the length of 24–25 nm . (b) Cumulative probability of unfolding with force for WT ( n = 1033 unfolding events) and R502Q domains ( n = 1254 unfolding events) during a 40 pN/s force ramp.…”

Protein haploinsufficiency drivers identify MYBPC3 variants that cause hypertrophic cardiomyopathy

“…In general, we found that expression at lower concentrations of IPTG and temperature ≤25 °C resulted in better yield of purified challenging-to-express domains, so these conditions were preferred for the expression of mutant domains. Purification of His-tagged domains was achieved by metal affinity and gel filtration chromatographies ( 75 ). Although different expression conditions were assayed, WT domains C6 and C8 could not be produced ( File S4 ), so variants targeting these domains could not be analyzed.…”

Protein haploinsufficiency drivers identifyMYBPC3mutations that cause hypertrophic cardiomyopathy

“…In general, we found that expression at lower concentrations of IPTG and temperature ≤ 25°C resulted in better yield of purification of challenging-to-express domains, so these conditions were preferred for the expression of mutant domains. Purification of His-tagged domains was achieved by metal affinity and gel filtration chromatographies 71 . WT domains C6 and C8 were refractory to recombinant expression (data not shown), so variants targeting these domains could not be analyzed.…”