Initial photophysical characterization of the proteorhodopsin optical proton sensor (PROPS)

Abstract

Fluorescence is not frequently used as a tool for investigating the photocycles of rhodopsins, largely because of the low quantum yield of the retinal chromophore. However, a new class of genetically encoded voltage sensors is based upon rhodopsins and their fluorescence. The first such sensor reported in the literature was the proteorhodopsin optical proton sensor (PROPS), which is capable of indicating membrane voltage changes in bacteria by means of changes in fluorescence. However, the properties of this fluorescence, such as its lifetime decay components and its origin in the protein photocycle, remain unknown. This paper reports steady-state and nanosecond time-resolved emission of this protein expressed in two strains of Escherichia coli, before and after membrane depolarization. The voltage-dependence of a particularly long lifetime component is established. Additional work to improve quantum yields and improve the general utility of PROPS is suggested.

Mammalian histones facilitate antimicrobial synergy by disrupting the bacterial proton gradient and chromosome organization

“…Cells treated with both H2A and LL-37 or with both H2A and PMB exhibited significantly higher PROPS fluorescence than cells treated with LL-37 or PMB alone, indicating that H2A further depolarizes the membrane and disrupts the PMF. We note that PROPS fluorescence is affected by the growth phase and pH 47 . All the cells were harvested at mid-exponential growth phase and were cultured in buffered medium, which minimizes potential changes in pH.…”

Raman spectroscopy of a near infrared absorbing proteorhodopsin: Similarities to the bacteriorhodopsin O photointermediate

“…In order to assess the effects of the D97N mutation on the MMAR chromophore, we measured the UV-Vis-NIR absorption of GPR-D97N:MR along with its RRS using 532-nm excitation (see Materials and Methods). The visible absorption maximum at pH 7.3 of D97N with the native retinal chromophore (A1) peaks near 550 nm [28, 50, 74] and is shifted to 632 nm for GPR-D97N:MR with a very small NIR band appearing at 790 nm (Fig 2). RRS measured using 532-nm laser excitation is expected to resonance enhance components which absorb mainly in the visible, especially those species with λ max near 532 nm.…”

“…A variety of genetically encoded voltage indicators (GEVIs) have been developed including those based on fusion of fluorescent proteins to transmembrane voltage sensing domains and on native and mutant microbial rhodopsin proton pumps [80]. For example, the first microbial rhodopsin GEVI developed is based on the GPR D97N mutant (referred to as a Proteorhodopsin Optical Proton Sensor or PROPS) which when excited by a 632-nm HeNe laser fluoresces around 735 nm [30, 74, 80]. Similar to the homologous D85N BR mutant, the substitution of an Asn for an Asp at position 97 neutralizes the retinal PSB counterion causing a red-shift in the λ max from 525 nm to 555 nm (S2 Fig) and also blocks proton transport [81].…”

Photocycle Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor QuasAr1

“…Tracking membrane potential of cells, especially neurons, using fluorescence methods is of high interest and is an active field of research (change of membrane voltage causes change of fluorescence efficiency) [1][2][3][4][5][6][7][8][9][10]. To determine membrane voltage, a variety of voltage sensitive dyes [11][12][13], genetically encoded calcium indicators (GECI) [4,14,15], and genetically encoded voltage indicators (GEVI) based on voltage sensing domains (VSD, composed of four trans-membrane helices and fused fluorescent proteins) [16][17][18][19][20] and on microbial rhodopsins (composed of seven trans-membrane α-helices with covalently bound retinal, using the intrinsic fluorescence of retinal [9,10,[21][22][23] or the modified fluorescence from attached fluorescent proteins [19,23,24] or dyes [12]) are in use. Often, Förster-type resonance energy transfer (FRET) is involved in dye or fluorescent protein connection to VSDs and rhodopins [12,25].…”