Directly measuring single molecule heterogeneity using force spectroscopy

TL;DR

This paper introduces a theoretical method to detect and quantify functional heterogeneity in single molecule experiments, revealing widespread heterogeneity and providing bounds on conformational switching times.

Contribution

It presents a novel analysis procedure for rupture force distributions to measure heterogeneity and interconversion rates from AFM and optical tweezer data.

Findings

Heterogeneity found in 5 out of 10 datasets analyzed.

Interconversion rates are slower than 10 s$^{-1}$ in observed systems.

Predicted cross-over regime could enable precise measurement of slow conformational switching.

Abstract

One of the most intriguing results of single molecule experiments on proteins and nucleic acids is the discovery of functional heterogeneity: the observation that complex cellular machines exhibit multiple, biologically active conformations. The structural differences between these conformations may be subtle, but each distinct state can be remarkably long-lived, with random interconversions between states occurring only at macroscopic timescales, fractions of a second or longer. Though we now have proof of functional heterogeneity in a handful of systems---enzymes, motors, adhesion complexes---identifying and measuring it remains a formidable challenge. Here we show that evidence of this phenomenon is more widespread than previously known, encoded in data collected from some of the most well-established single molecule techniques: AFM or optical tweezer pulling experiments. We present a theoretical procedure for analyzing distributions of rupture/unfolding forces recorded at different pulling speeds. This results in a single parameter, quantifying the degree of heterogeneity, and also leads to bounds on the equilibration and conformational interconversion timescales. Surveying ten published datasets, we find heterogeneity in five of them, all with interconversion rates slower than 10 s$^{-1}$. Moreover, we identify two systems where additional data at realizable pulling velocities is likely to find a theoretically predicted, but so far unobserved cross-over regime between heterogeneous and non-heterogeneous behavior. The significance of this regime is that it will allow far more precise estimates of the slow conformational switching times, one of the least understood aspects of functional heterogeneity.