Nanorheology of Biological Macromolecules

Mechanical properties are essential to biological macromolecules due to the fact that they are tightly coupled to the conformation, dynamics, and function of the macromolecules. In addition, in live systems many macromolecules are exposed to mechanical stresses. It is therefore of great interest and importance to understand the relation between the response of the macromolecules and the applied mechanical stresses, which is referred to as the nanorheology of the biological macromolecules and is the focus of this dissertation. More specifically, the dissertation mainly focuses on (1) what the mechanical properties of folded globular proteins are (e.g., elasticity, viscosity and/or both) and (2) how the native proteins respond to mechanical stresses.;In order to study the nanorheology of globular proteins, two novel methods are developed to exert mechanical forces on proteins. One is to use AC electric fields while the other is through DNA molecular springs.;We introduce a new nanorheology method to measure the mechanical properties globular proteins in their folded states. Gold nanoparticles, tethered to a gold surface by the protein, are driven by an AC electric field while their displacement is synchronously detected by evanescent wave scattering, yielding the mechanical response function of the macromolecular sample in the frequency domain. We apply this technique to explore the mechanical properties of a globular protein in the frequency range 10 Hz--10 kHz and identify different mechanical regimes. With this method, we first confirm that proteins are elastic if the deformations are small enough, and then discover that the folded state of the protein behaves like a viscoelastic solid. For increasing deformations of the proteins, we observe three different regimes: linear elasticity, then a reversible regime of viscoelasticity, and finally a irreversible regime. The first elastic regime is sensitive to the change in the elasticity of the protein due to the binding of its substrate. The second regime, which has the signature of a viscoelastic solid, gives access to the internal viscosity of the folded state of the protein. In addition, the transition of the protein rheology, from elasticity to viscoelasticity, has also been studied. We find that in both regimes, the protein deformations are linear to the applied forces, but with different proportionalities.;The other technique makes use of DNA molecular springs, which have recently been used to control the activity of enzymes and ribozymes. In this approach, the mechanical stress exerted by the molecular spring alters the enzyme's conformation and thus the enzymatic activity. In this dissertation we describe a method alternative to the previous one to attach DNA molecular springs to proteins, where two separate DNA arms are coupled to the protein and subsequently ligated. We report certain non-mechanical effects associated with the DNA spring observed in some chimeras with specific DNA sequences and the nucleotide binding enzyme guanylate kinase. We show how mechanical and non-mechanical effects of the DNA spring can be distinguished. This is important if one wants to use the protein-DNA chimeras to quantitatively study the response of the enzyme to mechanical perturbations.