title: Single-molecule force spectroscopy measurements of bond elongation during a bimolecular reaction.
abstract: It is experimentally challenging to directly obtain structural information of the transition state (TS), the high-energy bottleneck en route from reactants to products, for solution-phase reactions. Here, we use single-molecule experiments as well as high-level quantum chemical calculations to probe the TS of disulfide bond reduction, a bimolecular nucleophilic substitution (S N2) reaction. We use an atomic force microscope in force-clamp mode to apply mechanical forces to a protein disulfide bond and obtain force-dependent rate constants of the disulfide bond reduction initiated by a variety of nucleophiles. We measure distances to the TS or bond elongation (Delta x), along a 1-D reaction coordinate imposed by mechanical force, of 0.31 +/- 0.05 and 0.44 +/- 0.03 A for thiol-initiated and phosphine-initiated disulfide bond reductions, respectively. These results are in agreement with quantum chemical calculations, which show that the disulfide bond at the TS is longer in phosphine-initiated reduction than in thiol-initiated reduction. We also investigate the effect of solvent environment on the TS geometry by incorporating glycerol into the aqueous solution. In this case, the Delta x value for the phosphine-initiated reduction is decreased to 0.28 +/- 0.04 A whereas it remains unchanged for thiol-initiated reduction, providing a direct test of theoretical calculations of the role of solvent molecules in the reduction TS of an S N2 reaction. These results demonstrate that single-molecule force spectroscopy represents a novel experimental tool to study mechanochemistry and directly probe the sub-ångström changes in TS structure of solution-phase reactions. Furthermore, this single-molecule method opens new doors to gain molecular level understanding of chemical reactivity when combined with quantum chemical calculations.
authors.affiliation: Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
authors.forename: Sri Rama
authors.initials: SR
authors.lastname: Koti Ainavarapu
authors.forename: Arun P
authors.initials: AP
authors.lastname: Wiita
authors.forename: Lorna
authors.initials: L
authors.lastname: Dougan
authors.forename: Einar
authors.initials: E
authors.lastname: Uggerud
authors.forename: Julio M
authors.initials: JM
authors.lastname: Fernandez
mesh_terms: D064211:Connectin; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry; D007700:Kinetics; D018625:Microscopy, Atomic Force / Q000379:methods*; D008956:Models, Chemical; D009124:Muscle Proteins / Q000737:chemistry; D010720:Phosphines / Q000737:chemistry; D015202:Protein Engineering; D011494:Protein Kinases / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D011789:Quantum Theory; D019163:Reducing Agents / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D064211:Connectin; D004220:Disulfides; D009124:Muscle Proteins; D010720:Phosphines; D011506:Proteins; D019163:Reducing Agents; D011494:Protein Kinases; D005978:Glutathione; D003545:Cysteine