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.
Best
Robert B
RB
Department of Chemistry, Medical Research Council Centre for Protein Engineering, Lensfield Road, Cambridge CB2 1EW, United Kingdom.
Fowler
Susan B
SB
Toca-Herrera
Jose L
JL
Clarke
Jane
J
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D064211:Connectin; D066298:In Vitro Techniques; D018625:Microscopy, Atomic Force / Q000379:methods*; D009010:Monte Carlo Method; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics; D017354:Point Mutation; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D064211:Connectin; D009124:Muscle Proteins; D011506:Proteins; D011994:Recombinant Proteins; D011494:Protein Kinases