Single-molecule biophysical studies of RNA-protein interactions
Interactions between nucleic acids and proteins form the foundation of the central dogma of molecular biology. The functional importance of these biomolecules hinges on their ability to sample multiple conformations. By studying these dynamic structure-function relationships, we are able to identify the mechanisms that govern the biochemical processes arising from these nucleic acid-protein interactions.
This notion is also perfectly applicable to viruses, which can be crudely approximated as infectious sub-micrometer particles consisting of genetic material encapsulated by proteins, forming a so-called nucleocapsid. For example, assembly of the hepatitis C virus (HCV) nucleocapsid depends on interactions between the genomic RNA and numerous copies of the multifunctional core protein. Preventing these interactions represents a promising approach to inhibit HCV assembly. However, this approach has not been thoroughly explored because the structure-function relationship associated with the assembly process is not well understood.
One of the goals of our lab is to use concepts from biology, chemistry, and physics to provide a molecular description of the HCV assembly pathway by studying the structural and functional aspects responsible for RNA-induced formation of HCV nucleocapsid-like particles (NLPs).
Although there are a number of established experimental techniques that can be used to tackle this challenge, most of them are complicated by heterogeneous biological samples and asynchronous molecular behavior. The complications associated with these ensemble techniques can be avoided by studying single molecules. In particular, single-molecule FRET is an ideal experimental approach for these types of investigations because it simultaneously reports on the structural, energetic, and kinetics properties of fluorescently-labeled biomolecules. Accordingly, our lab uses these single-molecule methods, in conjunction with conventional ensemble techniques, to study a variety of nucleic acid-protein interactions.
Holmstrom ED, Holla A, Zheng W, Nettels D, Best RB, Schuler B. Accurate Transfer Efficiencies, Distance Distributions, and Ensembles of Unfolded and Intrinsically Disordered Proteins from Single-Molecule FRET. Methods Enzymol. 2018; 611: 287-325. PMID: 30471690
Holmstrom ED, Nesbitt DJ. Biophysical Insights from Temperature-Dependent Single-Molecule Förster Resonance Energy Transfer. Annu Rev Phys Chem. 2016; 67: 441-65. PMID: 27215819
Holmstrom ED, Nettels D, Schuler B. Conformational Plasticity of Hepatitis C Virus Core Protein Enables RNA-Induced Formation of Nucleocapsid-like Particles. J Mol Biol. 2018; 430(16): 2453-2467. PMID: 29045818
Vieweger M, Holmstrom ED, Nesbitt DJ. Single-Molecule FRET Reveals Three Conformations for the TLS Domain of Brome Mosaic Virus Genome. Biophys J. 2015; 109(12): 2625-2636. PMID: 26682819
Holmstrom ED, Polaski JT, Batey RT, Nesbitt DJ. Single-molecule Conformational Dynamics of a Biologically Functional Hydroxocobalamin Riboswitch. J Am Chem Soc. 2014; 136(48):16832-43. PMID: 25325398