Ultrasensitive measurements of microbial rhodopsin photocycles using photochromic FRET

Abstract

Microbial rhodopsins are an important class of light-activated transmembrane proteins whose function is typically studied on bulk samples. Herein, we apply photochromic fluorescence resonance energy transfer to investigate the dynamics of these proteins with sensitivity approaching the single-molecule limit. The brightness of a covalently linked organic fluorophore is modulated by changes in the absorption spectrum of the endogenous retinal chromophore that occur as the molecule undergoes a light-activated photocycle. We studied the photocycles of blue-absorbing proteorhodopsin and sensory rhodopsin II (SRII). Clusters of 2-3 molecules of SRII clearly showed a light-induced photocycle. Single molecules of SRII showed a photocycle upon signal averaging over several illumination cycles.

Figure 1

Photochromic FRET probes the photocycle of BPR. a) Ground state and O intermediate absorption spectra of BPR (solid and dotted lines respectively). Emission spectrum of Alexa 594 (dashed line). b) Transition from O to ground state in BPR photocycle. (i) Transient absorption of BPR in DDM probed at 610 nm. Photochromic FRET of (ii) BPR-Alexa 594 (iii) BPR-Atto 647N (iv) BPR-Alexa 594 without retinal.

Figure 2

Photochromic FRET measurements of the photocycle in SRII. a) Unphotolyzed state and O state absorption spectra of SRII (solid and dotted lines respectively). Emission spectrum of Alexa 546 (dashed line). b) Transition from M to ground state in SRII photocycle. (i) Transient absorption of SRII in DDM probed at 580 nm. Transient fluorescence of SRII mutants labeled with the dyes and at the locations indicated in the cartoon: (ii) SRII-Alexa 594 (iii) SRII-Alexa 546 (on the extracelullar side) (iv) SRII-Alexa 555 (v) SR-Alexa 546.

Figure 3

Transient fluorescence of SRII^S153C-Alexa 546 induced by a 50 ms flash of excitation (diamonds) or step function excitation (squares). Both datasets were fit to the three-state model (Eq. 1) with the same parameters (solid lines).

Figure 4

Dynamics of SRII in immobilized lipid vesicles. Ensemble-averaged transient fluorescence of SRII^S153C-Alexa 546 with retinal (top) and without retinal (bottom). A single beam (1 mW, 532 nm) served as both pump and probe.

Figure 5

Dependence of photochromic FRET signal of SRII^S153C-Alexa 546 on (a) rest time between pump pulses and (b) pump power.

Figure 6

Single-molecule dynamics of SRII in single lipid vesicles. (a) A field of immobilized lipid vesicles containing SRII^S153C-Alexa 546. A vesicle containing a single molecule of SRII is circled. (b) Fluorescence intensity traces of a single active SRII molecule (left) and a single inactive SRII molecule (right) during 10 consecutive pump cycles. Inset: single-step photobleaching recorded at high power at the end of the experiment. (c) Transient fluorescence signal averaged over 10 pump cycles and 9 molecules. (d) Autocorrelation functions of the fluorescence intensity from a single active molecule (left) and a single inactive molecule (right).