title: Can non-mechanical proteins withstand force? Stretching barnase by atomic force microscopy and molecular dynamics simulation.
abstract: Atomic force microscopy (AFM) experiments have provided intriguing insights into the mechanical unfolding of proteins such as titin I27 from muscle, but will the same be possible for proteins that are not physiologically required to resist force? We report the results of AFM experiments on the forced unfolding of barnase in a chimeric construct with I27. Both modules are independently folded and stable in this construct and have the same thermodynamic and kinetic properties as the isolated proteins. I27 can be identified in the AFM traces based on its previous characterization, and distinct, irregular low-force peaks are observed for barnase. Molecular dynamics simulations of barnase unfolding also show that it unfolds at lower forces than proteins with mechanical function. The unfolding pathway involves the unraveling of the protein from the termini, with much more native-like secondary and tertiary structure being retained in the transition state than is observed in simulations of thermal unfolding or experimentally, using chemical denaturant. Our results suggest that proteins that are not selected for tensile strength may not resist force in the same way as those that are, and that proteins with similar unfolding rates in solution need not have comparable unfolding properties under force.
authors.affiliation: Department of Chemistry, University of Cambridge, MRC Centre for Protein Engineering, Cambridge CB2 1EW, United Kingdom.
authors.forename: R B
authors.initials: RB
authors.lastname: Best
authors.forename: B
authors.initials: B
authors.lastname: Li
authors.forename: A
authors.initials: A
authors.lastname: Steward
authors.forename: V
authors.initials: V
authors.lastname: Daggett
authors.forename: J
authors.initials: J
authors.lastname: Clarke
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D001696:Biomechanical Phenomena; D064211:Connectin; D004795:Enzyme Stability; D018625:Microscopy, Atomic Force / Q000295:instrumentation*; D008958:Models, Molecular; D018716:Molecular Mimicry; D009124:Muscle Proteins / Q000737:chemistry*; D009132:Muscles / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012260:Ribonucleases / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D064211:Connectin; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease