Date of Award
2015
Degree Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Graduate Group
Chemistry
First Advisor
Ernest J. Petersson
Abstract
The development of new methods that provide mechanistic information on the
structural dynamics of proteins represents a significant challenge in the field of
biochemistry. Fluorescence spectroscopy is a highly sensitive technique that is ideally
suited for monitoring protein movement in situ. However, the most commonly used
fluorophores generally yield poor structural resolution, due to their relatively large size
compared to the protein of interest. Research in our laboratory has demonstrated that a
thioamide, a single atom-substitution of the peptide backbone, is capable of quenching a
wide array of fluorophores in a distance-dependent fashion. We have shown that
thioamide quenching of tyrosine and tryptophan can be used to monitor biological
interactions, such as ligand binding to the protein Calmodulin (CaM). To expand the
utility of the thioamide group as a spectroscopic probe, our laboratory has developed
semi-synthesis techniques for its installation into full-length proteins. Having validated
thioamide quenching of intrinsic protein fluorescence in our model system, we then
applied this technique to monitoring the misfolding of the amyloidogenic protein α-
synuclein (αS), implicated in the pathogenesis of Parkinsonâ??s Disease. In order to
determine which of our spectroscopic pairs best behaves in accordance with our theoretical models, we also examined thioamide quenching of Cnf using our CaM model
system. For intramolecular studies with Cnf/thioamide FRET pairs, we combined
unnatural amino acid mutagenesis with native chemical ligation to access double-labeled αS using a minimum of chemical synthesis. We have also shown that we can combine unnatural amino acid mutagenesis with expressed protein ligation at methionine to incorporate the probe pair in an entirely traceless manner. Using thioamide/Cnf FRET of our constructs, we were able to monitor conformational changes of monomeric αS with unprecedented structural resolution. In addition to our work using thioamides, we have also developed efficient strategies for producing variants of singly and doubly-labeled αS containing red-shifted fluorophores for fluorescence polarization (FP) and other FRET based assays, respectively. Most recently, we have shown that we can use site specifically labeled αS in conjunction with FP to glean mechanistic insight into the processes of aggregation and disaggregation. Ultimately, these labeled constructs will allow us to study these processes in vivo.
Recommended Citation
Wissner, Rebecca Felice, "Minimally Perturbing Fluorescence Probe Pairs for the Study of Protein Folding and Misfolding" (2015). Publicly Accessible Penn Dissertations. 1164.
https://repository.upenn.edu/edissertations/1164