Mapping Proximity Within Proteins Using Fluorescence Spectroscopy: New Advances In Distance-dependent Tryptophan-induced Fluorescence Quenching

We previously showed that the dramatic quenching of bimane fluorescence by Trp can be exploited to study protein structure, dynamics and protein-protein interactions. Here we report our efforts to extend this approach to four other probes: monobromotrimethylammoniobimane (qBBr), lucifer yellow (LY), BODIPY 507/535 (BDPY), and Atto-655 (Atto). We attached each probe to a unique reactive cysteine at four different sites on T4 lysozyme, and measured the effect of a nearby Trp on the fluorescence intensity and lifetime. We also used a relatively novel yet simple approach to calculate the relative fraction of each probe forming a static, non-fluorescent complex with the Trp.The different probes show markedly different sensitivities to quenching by the Trp, most likely due to their differences in size, rotational flexibility, length of attachment linker, and efficiencies of quenching photochemistry. Together, our data can be used to approximate the distance-dependent “sphere of quenching” for each probe. The smaller probes (bimanes and BODIPY) show substantial quenching and static-complex formation only when they are very close to the Trp (< 10A away). Thus, these probes are best suited for measuring short-range intramolecular distances or conformational changes in proteins. In contrast, the larger probes (lucifer yellow and Atto-655) show substantial quenching and static-complex formation with the Trp over longer distances. Thus, these latter probes are less selective for monitoring local changes, but are useful for monitoring longer-range distances within a protein, or inter-molecular distances between proteins. We anticipate the broad excitation and emission range covered by these four probes (380 nm - 650 nm), combined with the ability to determine the amount of static complex formation with Trp, should expand the use of Trp-induced fluorescence quenching as a method for studying protein structure and dynamics.