Probability of observing a number of unfolding events while stretching poly-proteins

TL;DR

This paper develops an analytical framework to predict the number of unfolding events in single-molecule poly-protein stretching experiments, providing insights into experimental design and molecular binding randomness.

Contribution

It introduces a formal description of observed unfolding events, a protocol duration rule, and a method to estimate molecule counts from experimental data, revealing random binding behavior.

Findings

Poly-ubiquitin binds randomly to substrate and probe.

Observing all modules in poly-ubiquitin is highly unlikely.

The methodology improves estimation accuracy of molecule counts.

Abstract

The mechanical stretching of single poly-proteins is an emerging tool for the study of protein (un)folding, chemical catalysis and polymer physics at the single molecule level. The observed processes i.e unfolding or reduction events, are typically considered to be stochastic and by its nature are susceptible to be censored by the finite duration of the experiment. Here we develop a formal analytical and experimental description on the number of observed events under various conditions of practical interest. We provide a rule of thumb to define the experiment protocol duration. Finally we provide a methodology to accurately estimate the number of stretched molecules based on the number of observed unfolding events. Using this analysis on experimental data we conclude for the first time that poly-ubiquitin binds at a random position both to the substrate and to the pulling probe and that observing all the existing modules is the less likely event.