Enzyme-DNA chimeras: Construction, allostery, applications.

We describe the operational principle, synthesis, and applications of the enzyme-DNA chimeras. These are supramolecular constructions where a DNA spring is coupled to an enzyme and introduces artificial allosteric control of the enzyme. This method is universal and can be applied to various enzymes and proteins. In addition, this method is versatile as the stresses applied by the DNA spring on the enzymes can be fine-tuned semi-continuously and thus their enzymatic activities can be modulated gradually. We give detailed protocols for the synthesis of these molecules. Summarizing our experience with different enzymes, we explain their use for fundamental studies of conformational plasticity, as well as the potential as molecular probes.

Dissipative dynamics of enzymes.

We explore enzyme conformational dynamics at sub-Å resolution, specifically, temperature effects. The ensemble-averaged mechanical response of the folded enzyme is viscoelastic in the whole temperature range between the warm and cold denaturation transitions. The dissipation parameter γ of the viscoelastic description decreases by a factor of 2 as the temperature is raised from 10 to 45 °C; the elastic parameter K shows a similar decrease. Thus, when probed dynamically, the enzyme softens for increasing temperature. Equilibrium mechanical experiments with the DNA spring (and a different enzyme) also show, qualitatively, a small softening for increasing temperature.

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 enzyme's 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 [Formula: see text] for the enzyme's conformational motion. The present experiments provide a mean to explore enzyme kinetics beyond the static energy landscape picture of transition state theory.

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.

Elastic energy of protein-DNA chimeras.

We present experimental measurements of the equilibrium elastic energy of protein-DNA chimeras, for two different sets of attachment points of the DNA "molecular spring" on the surface of the protein. Combining these with measurements of the enzyme's activity under stress and a mechanical model of the system, we determine how the elastic energy is partitioned between the DNA and the protein. The analysis shows that the protein is mechanically stiffer than the DNA spring.