The need to study proteins in living cells to acquire accurate information pertaining to their spatiotemporal dynamics drives the development and improvement of tools for visualization. Unfortunately, there does not exist a ‘one size fits all’ imaging strategy, leading to the continuous pursuit of new labeling and optical methods, with the goal of developing more widely applicable approaches that minimally disrupt the biological systems studied.
Tetratricopeptide repeat affinity proteins (TRAPs) are unique in their ability to recognize and bind to short peptide sequences that consist of about five amino acids. These binding reactions are reversible, and the pairs can be engineered to have a variety of potential binding affinities. Specific TRAP-peptide pairs can be selected for, that present with no cross-reactivity, and are capable of being utilized in live-cell systems, such as the bacteria Escherichia coli and budding yeast Saccharomyces cerevisiae. Capitalizing on these features, we turn the TRAP-peptide pairs into a versatile tag-probe imaging strategy for in vivo protein studies. The TRAPs are designated as probes labeled with a fluorophore, while the peptides are genetically fused to the C-terminus of the proteins of interest. We conduct a proof-of-principle experiment in E. coli demonstrating the TRAP-peptide pair’s utility as an imaging strategy, and later implement the system in budding yeast where we show that our reversibly-binding tag-probe system properly and reliably localizes Pma1 to the plasma membrane, unlike direct-fusions of Pma1 with fluorescent proteins.
Using fluorescence recovery after photobleaching experiments (FRAP), we characterize our tag-probe system’s binding kinetics, and proceed to use single particle tracking photoactivated localization microscopy (sptPALM) measurements under total internal reflection fluorescence (TIRF) illumination to analyze the behavior of Pma1. Remarkably, we find significant differences in the diffusional dynamics of Pma1, imaged using our novel labeling methodology, and Pma1, directly fused to a fluorescent protein.
Thesis Advisor: Simon Mochrie (simon.mochrie@yale.edu)