Robert B Best — Page 2

PubMed2002

A simple method for probing the mechanical unfolding pathway of proteins in detail.

Atomic force microscopy is an exciting new single-molecule technique to add to the toolbox of protein (un)folding methods. However, detailed analysis of the unfolding of proteins on application of force has, to date, relied on protein molecular dynamics simulations or a qualitative interpretation of mutant data. Here we describe how protein engineering Phi value analysis can be adapted to characterize the transition states for mechanical unfolding of proteins. Single-molecule studies also have an advantage over bulk experiments, in that partial Phi values arising from partial structure in the transition state can be clearly distinguished from those averaged over alternate pathways. We show that unfolding rate constants derived in the standard way by using Monte Carlo simulations are not reliable because of the errors involved. However, it is possible to circumvent these problems, providing the unfolding mechanism is not changed by mutation, either by a modification of the Monte Carlo procedure or by comparing mutant and wild-type data directly. The applicability of the method is tested on simulated data sets and experimental data for mutants of titin I27.

Animals↗

PubMed2002

What can atomic force microscopy tell us about protein folding?

Force spectroscopy has emerged as a new tool to study protein folding, in which force replaces the chemical denaturant used in traditional folding experiments. This new technique complements older methods and allows a range of new questions to be investigated. What sort of protein is able to resist mechanical unfolding, and to what extent is mechanical stability dictated by fold or function? What is the effect of force on the unfolding energy surface? Do proteins unfold by the same pathway in mechanical and chemical denaturation experiments? Answers to these are starting to emerge based on a combination of experimental and computational approaches. We present some of the forced unfolding experiments to date and simple methods for characterizing the unfolding potential from the results. Several studies have also begun a more fine-grained description of mechanical unfolding, for example by invoking intermediates to explain features seen in unfolding traces and by using mutagenesis to try to localize the origin of mechanical stability. We propose further experimental approaches to this goal using the protein engineering method to characterize transition states, similar to those used in conventional folding experiments. However, it is likely that a high-resolution picture of mechanical unfolding will only emerge through a combined interpretation of careful experimental work and computer simulation.

Microscopy, Atomic Force↗