UCLA

Proteins are compact but flexible molecules; their functions are dependent upon a delicate balance between structural stability and flexibility. Enzymes couple chemical reactions with mechanical motions. More specifically, substrate binding in enzymes drives conformational motion which is an integral part of the catalytic cycle. Understanding the mechanics of the large molecular deformations is the topic of this thesis. Our approach is to use a DNA molecular spring to mechanically stretch an enzyme and measure its enzymatic response. A large part of our work was done on Guanylate Kinase (GK), which showed a decrease in enzymatic activity in response to the mechanical perturbation provided by the DNA spring. A DNA spring is a general tool which can be used to control the activity of an enzyme, and more generally, to explore the mechanical properties of a protein. In addition, the enzyme-DNA chimeras may be developed into interesting biosensors. These are the subjects upon which we will elaborate in this dissertation.

First, we use the DNA spring to exert stresses at three different specific locations on GK, and for each case, determine the changes in substrate binding affinities and catalytic rate. We find that the enzyme's kinetic parameters can be affected separately, depending on where the mechanical stress is applied. For one configuration the applied stress mainly affects the catalytic rate ${k}_{cat}$, for another it mainly affects the binding affinity of the substrate GMP. The anisotropic response of GK shows that the mechanical stress does not only bias the population of the folded and unfolded states of the enzyme. Instead, it is a way to access many intermediate states in the equilibrium situation. This result also leads us to start thinking about continuum mechanics models for our chimera system, which motivates our later work.

Next, we apply the DNA molecular spring to an enzyme from an entirely different class: Renilla Luciferase (RLuc). The successful mechanical control on RLuc shows that the DNA spring is indeed a general tool to manipulate enzymatic activities. We also show proof of concept of the RLuc-DNA chimera as a molecular probe, where the presence of a DNA target sequence can be detected by the decrease in luminescence intensity of RLuc in a one-step solution assay. This probe is also capable of detecting a single-base-pair mismatch on the DNA spring without melting curve analysis. The defect in the DNA spring, caused by the DNA mismatch, results in decreased stress applied by the spring, which leads to a discernible change in enzymatic activity. Inherent in the RLuc-DNA chimera is a reporter system capable of detecting a small change in the stress applied to the enzyme.

In a chimera, the protein and the DNA are mechanically coupled, where the DNA is bent and the protein is mechanically deformed. The DNA is a nonlinear molecule which softens beyond the elastic regime. Using this knowledge, we are able to capture some feature of the mechanics of the protein. Measurements of the response of RLuc for different states of stress suggest that the protein also undergoes a softening transition beyond a few \AA \ deformation. In addition, we find that the sensitivity of the chimera to small changes in the stiffness of the DNA spring, which we observed in both the GK and RLuc chimeras, arises from the mechanical nonlinearities of both the DNA spring and the protein.