Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding

Abstract

Green fluorescent protein (GFP) variants are widely used as genetically encoded fluorescent fusion tags, and there is an increasing interest in engineering their structure to develop in vivo optical sensors, such as for optogenetics and force transduction. Ensemble experiments have shown that the fluorescence of GFP is quenched upon denaturation. Here we study the dependence of fluorescence on protein structure by driving single molecules of GFP into different conformational states with optical tweezers and simultaneously probing the chromophore with fluorescence. Our results show that fluorescence is lost during the earliest events in unfolding, 3.5 ms before secondary structure is disrupted. No fluorescence is observed from the unfolding intermediates or the ensemble of compact and extended states populated during refolding. We further demonstrate that GFP can be mechanically switched between emissive and dark states. These data definitively establish that complete structural integrity is necessary to observe single-molecule fluorescence of GFP.optical tweezers | protein folding | fluorescent protein | mechanoswitch G reen fluorescent protein (GFP) is a 27-kDa β-barrel protein with an intrinsic chromophore. (1, 2) It is widely used in imaging applications that rely on its structural stability [to thermal and high-pressure unfolding (3), in fusion constructs (4), and to circular permutation (5)] or its optical response to environment, such as in biosensors for force transduction (6-9), calcium concentration (10), protease activity (11), and pH (12, 13). Understanding the relationship between protein structure and fluorescence is essential for these applications. Photophysical properties of the chromophore are sensitive to its hydrogen bonding, solvation, isomerization state, and binding-pocket structure. (1,2,14). It is known that fluorescence is quenched upon denaturation and that several intermediates have been observed in folding experiments and simulations (3,(15)(16)(17)(18)(19)(20)(21), although it has not been possible to definitively probe the fluorescence properties of partially structured intermediates in isolation from the native state. Speculation exists regarding whether single-molecule photobleaching can be reversed by unfolding and refolding the protein (22). It is not known whether tension on the native state alters fluorescence in similarity to the compressive, pressure-induced elastic effect (3) and whether its emission can be reversibly mechanically switched. In this study, we address these questions by making an explicit connection between structure and fluorescence, using single-molecule methods.Single-molecule experiments are widely used to reveal distributions in biophysical structure and kinetics that underlie ensemble-averaged properties. Single-molecule manipulation using force experiments such as optical tweezers can be used to observe states nominally at negligible concentration in a bulk distribution (23)(24)(25). Single-molecule fluorescence experiments have been used to probe the ...

Deciphering Limitations to Meet Highly Stable Bio‐Hybrid Light‐Emitting Diodes

“…Therefore, the changes in both intensity and band shape of the FP coating beyond the temperature‐induced quenching (second and third stages) are attributed to a partial denaturation of the protein backbone and/or changes in the molecular structure or electronic state of the chromophore, since the FPs are embedded in a quasi‐melted phase in the matrix network. This includes 1) small conformational changes of the β‐barrel induced by dehydration processes, 2) changes in the nature of the emitting excited state via excited‐state proton transfer (i.e., neutral, anionic, and intermediate forms), and 3) deactivation via, for example, triplets, cis / trans isomerization, etc . Other emission deactivation mechanisms such as photooxidative reddening and reversible photochromism are excluded, since the final emission should be redshifted …”

Simultaneous sensing and imaging of individual biomolecular complexes enabled by modular DNA–protein coupling

“…5a, b). In contrast, previous studies on stable proteins using the hybridization approach were limited to applying high forces (~45 pN) only briefly (~50 ms), owing to the risk of tether rupture 43 . Ligated tethers thus are useful to explore a wide range of conformational states and timescales 25,26 .…”

“…The former yields strong coupling but is practically limited to short tethers below 500 bp, in part due to the electrostatic repulsion of large DNA molecules 41 . The two-step method has been used for longer handles up to 3 kbp 43 . However, the involved hybridization interactions provide lower mechanical stability than the former direct coupling approach and cannot resist high forces for extended periods of time 43,44 .…”

“…The two-step method has been used for longer handles up to 3 kbp 43 . However, the involved hybridization interactions provide lower mechanical stability than the former direct coupling approach and cannot resist high forces for extended periods of time 43,44 .…”

Using Single-Molecule Approaches to Understand the Molecular Mechanisms of Heat-Shock Protein Chaperone Function

“…For instance, the development of novel, fluorescence-based methods to study sHsp function will enable the direct observation of sHsp-client interactions, allowing the determination of on/off rates, stoichiometries and binding affinities. By combining fluorescence and force-based singlemolecule techniques, which has been shown previously [155][156][157], fundamental aspects of sHsp chaperone function, typically inaccessible by ensemble measurements, can be interrogated.…”