Chiao-Yu Tseng

Mechano-chemistry of the enzyme Guanylate Kinase

We address the coupling of mechanics and chemistry in an enzyme through equilibrium experiments where we mechanically deform the enzyme and measure the effect on the chemical reaction it catalyzes. We use the DNA molecular spring method to exert stresses at three different specific locations on the enzyme Guanylate Kinase, and for each case determine the changes in substrates binding affinities and catalytic rate. We find that the enzyme kinetics parameters can be affected separately, depending on where the mechanical stress is applied. For one configuration the applied stress mainly affects the catalytic rate kcat, for another it mainly affects the binding affinity of the substrate GMP. These experiments show that a stress applied by pulling two residues on the surface of the protein generally results in a strain propagating into the structure.

The elastic energy of sharply bent nicked DNA

We obtain measurements of the elastic energy of short (18–30 bp) molecules of ds DNA constrained into a sharply bent conformation, using a thermodynamic method with the DNA in solution. We consider the case where there is one nick in the ds DNA, and find that the system develops a kink at a critical torque τc≈27 pN×nm. In this regime the elastic energy is linear in the end-to-end distance (EED). For smaller torques the DNA is smoothly bent and described by the worm-like–chain energy, which is also approximately linear in the EED, but with a different slope. Thus we access both the high and low elastic energy regimes, and the transition between the two.

Mechanical control of Renilla luciferase.

We report experiments where the activity of the enzyme luciferase from Renilla reniformis is controlled through a DNA spring attached to the enzyme. In the wake of previous work on kinases, these results establish that mechanical stress applied through the DNA springs is indeed a general method for the artificial control of enzymes, and for the quantitative study of mechano-chemical coupling in these molecules. We also show proof of concept of the luciferase construct as a sensitive molecular probe, detecting a specific DNA target sequence in an easy, one-step, homogeneous assay, as well as SNP detection without melting curve analysis.

Asymmetric Effect of Mechanical Stress on the Forward and Reverse Reaction Catalyzed by an Enzyme

The concept of modulating enzymatic activity by exerting a mechanical stress on the enzyme has been established in previous work. Mechanical perturbation is also a tool for probing conformational motion accompanying the enzymatic cycle. Here we report measurements of the forward and reverse kinetics of the enzyme Guanylate Kinase from yeast (Saccharomyces cerevisiae). The enzyme is held in a state of stress using the DNA spring method. The observation that mechanical stress has different effects on the forward and reverse reaction kinetics suggests that forward and reverse reactions follow different paths, on average, in the enzymes conformational space. Comparing the kinetics of the stressed and unstressed enzyme we also show that the maximum speed of the enzyme is comparable to the predictions of the relaxation model of enzyme action, where we use the independently determined dissipation coefficient for the enzymes conformational motion. The present experiments provide a mean to explore enzyme kinetics beyond the static energy landscape picture of transition state theory.

Equilibrium softening of an enzyme explored with the DNA spring

We explore enzyme mechanics using a system of two mechanically coupled biomolecules. Measurements of the mechanical modulation of enzymatic activity in a Luciferase—DNA chimera are presented. These are molecules where the enzyme is deformed by the action of a DNA spring. The response of the enzyme for different states of stress is examined. It is found that small changes in the stress cause large changes in activity. This nonlinear behavior is qualitatively interpreted as arising from a soft regime of the enzyme beyond linear elasticity. This soft regime may enable large conformational motion in enzymes.