Single-molecule force spectroscopy measurements of bond elongation during a bimolecular reaction.
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.
Koti Ainavarapu
Sri Rama
SR
Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
Wiita
Arun P
AP
Dougan
Lorna
L
Uggerud
Einar
E
Fernandez
Julio M
JM
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
D064211:Connectin; D004220:Disulfides; D009124:Muscle Proteins; D010720:Phosphines; D011506:Proteins; D019163:Reducing Agents; D011494:Protein Kinases; D005978:Glutathione; D003545:Cysteine