Expanding the Scope of Single Molecule FRET Spectroscopy towards Primarily Undergraduate Institutions

Abstract

Single molecule fluorescence methods provide a unique and ultrasensitive set of probes for monitoring the conformations and dynamics of protein chains within physiologically relevant contexts. In particular, single molecule fluorescence resonance energy transfer (smFRET) is capable of monitoring the distances between two fluorescent dyes with sub-nanometer spatial and nanosecond temporal resolution. Unfortunately, smFRET remains inaccessible to many biophysical researchers and most of all to students at primarily undergraduate research institutions. This is in part due to the difficulties involved in making dual-labelled protein samples but also stems from the expense and/or complexity of existing single-molecule fluorescence detection platforms. To address the first issue, we have developed a sample generation protocol suitable for making large libraries of dual-labelled proteins and ribosome-bound nascent chains (RNCs). This method consists of using a purified and reconstituted in-vitro translation system for protein expression, the incorporation of azide or alkyne-bearing non-standard amino acids, and finally click-chemistry-based dye attachment. Here, we show that this approach is simple and robust enough to be used by undergraduate students to make large libraries of dual-labelled protein samples in about a day. To enable smFRET-based screening of these libraries we have also built the first-ever confocal smFRET microscope at a primarily undergraduate institution. This instrument was built for less than $20K largely using second-hand optical components. It has a 3-axis piezo-scanning stage which enables single-molecule imaging and trajectory analysis on surface-immobilized molecules. It also has alternating laser excitation capabilities which enable digital sorting of single molecules using multi-dimensional histograms. This work marks the first implementation of smFRET as a research tool at a primarily undergraduate research institution.