Measurement Of Molecular Conformations And Dynamics Using Single Molecule Fluorescence Techniques
The use of fluorescence spectroscopy to study biological problems has gained popularity over the past few decades. Beyond a spatial understanding provided by microscopy, fluorescence techniques like Fluorescence Correlation Spectroscopy (FCS) and fluorescence lifetime spectroscopy can also elucidate the important temporal dynamics of molecular conformations. We have applied FCS to study the conformational fluctuations in a model protein apomyoglobin. By pushing the technical limitations of FCS, we were able to observe conformational dynamics spanning two orders of magnitude in time (10[-]6 to 10[-]3 seconds). We found that the amplitude of fluctuations changes as the molecule becomes unfolded, with principal shifts of amplitudes and timescales occurring at the transition across the molten globule state. We also measured the diffusion of apomyoglobin, confirming theoretical predictions of less compaction of the molecule upon acid denaturation. We were able to observe the fluctuations in apomyoglobin using FCS due to the quenching of an N-terminal labeled Alexa488 fluorophore by contact with various amino acids. We showed that quenching can occur with up to four amino acids. We investigated the mechanisms of quenching using a combination of fluorescence intensity and lifetime measurements. We showed that quenching takes place through a combination of static and dynamic mechanisms. Our results demonstrate that care needs to be taken when making quan- titative measurements of fluorescently labeled proteins. Coupling single molecule functionality to fluorescence techniques allows researchers to discern subpopulations within ensembles. We used single molecule ¨ Forster Resonance Energy Transfer (smFRET) to measure the end-to-end distances in single stranded nucleic acids as a probe of the flexibility. We also measured the radius of gyration using small angle X-ray scattering, and extracted parameters of polymer properties that fit the data from both techniques. We observed clear differences between our model single stranded DNA (ssDNA) and RNA (ssRNA). We also observed a difference between the screening efficiency of monovalent and divalent cations. By characterizing the intrinsic differences in nucleic acids and its dependence on the ionic strength, we hope to improve our understanding of the mechanisms of RNA folding and the role of ions in the process.
Webb, Watt Wetmore
Zipfel, Warren R; Chen, Peng; Pollack, Lois
Ph. D., Applied Physics
Doctor of Philosophy
dissertation or thesis