Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)

Graduate Group


First Advisor

Ernest J. Petersson


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.

Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Included in

Chemistry Commons