Bimanes 22. Flexible fluorescent molecules. Solvent effects on the photophysical properties of syn-bimanes (1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-diones)

In this study, we address the mechanism of visual arrestin release from light-activated rhodopsin using fluorescently labeled arrestin mutants. We find that two mutants, I72C and S251C, when labeled with the small, solvent-sensitive fluorophore monobromobimane, exhibit spectral changes only upon binding light-activated, phosphorylated rhodopsin. Our analysis indicates that these changes are probably due to a burying of the probes at these sites in the rhodopsin-arrestin or phospholipid-arrestin interface. Using a fluorescence approach based on this observation, we demonstrate that arrestin and retinal release are linked and are described by similar activation energies. However, at physiological temperatures, we find that arrestin slows the rate of retinal release ϳ2-fold and abolishes the pH dependence of retinal release. Using fluorescence, EPR, and biochemical approaches, we also find intriguing evidence that arrestin binds to a post-Meta II photodecay product, possibly Meta III. We speculate that arrestin regulates levels of free retinal in the rod cell to help limit the formation of damaging oxidative retinal adducts. Such adducts may contribute to diseases like atrophic age-related macular degeneration (AMD). Thus, arrestin may serve to both attenuate rhodopsin signaling and protect the cell from excessive retinal levels under bright light conditions.The visual photoreceptor rhodopsin is perhaps the best model system for understanding the mechanisms used in Gprotein-coupled receptor (GPCR) 1 signaling, as detailed information exists on the structures and dynamic interactions of the protein constituents (1). Visual activation begins with absorption of light by the 11-cis-retinal chromophore in rhodopsin. The photoactivated form of rhodopsin, Rho* or "Meta II," interacts with and activates the G-protein transducin, which exchanges nucleotide and then diffuses to interact with downstream effectors. Signaling by Rho* is terminated by slow thermal decay and the release of retinal. Alternatively, signaling can be quickly terminated by a process that begins with phosphorylation of rhodopsin's C-terminal tail through the action of rhodopsin kinase (2, 3). The phosphorylated Rho* is then bound by arrestin, which stops signaling by physically occluding the G-protein binding site (4, 5).In the present study, we address how these two inactivation mechanisms are related and, specifically, what governs arrestin release from rhodopsin. Arrestin is known to bind to phosphorylated Meta II, a form in which the photolyzed chromophore all-trans-retinal is still attached to the receptor by a deprotonated Schiff base. Arrestin does not bind phosphorylated opsin, but all-trans retinal added exogenously can stimulate arrestin binding to phosphorylated opsin (6, 7). Early studies showed indirectly that retinal release and arrestin release are probably interrelated events (7,8). However, how these processes are linked or whether arrestin binds other photointermediates of rhodopsin (such as the storage form Meta III) is still unknown...

Section: Resultsmentioning

confidence: 95%

Dynamics of Arrestin-Rhodopsin Interactions

Sommer

Smith

Farrens

2005

“…This approach takes advantage of the short-range quenching of bimane fluorescence by tryptophan, a process that involves collisional contact between the bimane excited state and the indole ring of tryptophan (22,23). Bimane fluorescence displays some variation with environmental parameters (19,24), but its efficient quenching by tryptophan can provide a measure of short-range interactions in proteins (25). The substrate binding domain of HscA was selectively labeled with monobromobimane, and the ability of IscU-derived peptides having tryptophan at the N or C terminus (W-97 and W-105) to quench bimane fluorescence was measured.…”

Section: Resultsmentioning

confidence: 99%

Preferential Substrate Binding Orientation by the Molecular Chaperone HscA

Tapley

Vickery

2004

HscA, a specialized bacterial hsp70-class chaperone, interacts with the iron-sulfur cluster assembly protein IscU by recognizing a conserved LPPVK sequence motif at positions 99 -103. We have used a site-directed fluorescence labeling and quenching strategy to determine whether HscA binds to IscU in a preferred orientation. HscA was selectively labeled on opposite sides of the substrate binding domain with the fluorescent probe bimane, and the ability of LPPVK-containing peptides having tryptophan at the N or C terminus to quench bimane fluorescence was measured. Quenching was highly dependent on the position of tryptophan in the peptide and the location of bimane on HscA implying a strong directional preference for peptide binding. Similar experiments showed that full-length IscU binds in the same orientation as IscU-derived peptides and that binding orientation is unaffected by the co-chaperone HscB. The preferred orientation of the HscA-IscU complex is the reverse of that previously described for peptide complexes of Escherichia coli DnaK and rat Hsc70 substrate binding domain fragments establishing that hsp70 isoforms can bind peptide/polypeptide substrates in different orientations.

“…Our experience in using the mBBr reagent to label Cys residues within the colicin E1 channel domain indicates that the product Cysbimane is a small and effective probe for evaluating protein structure and also for membrane protein topology (33,42). This probe is relatively nonperturbing to protein structure and is sufficiently ambivalent to allow for its partitioning into the membrane bilayer at varying depths and consequently in a wide range of dielectric constants.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, the reduced Q F values of these mutant proteins suggest the presence of intrinsic fluorescence quenching mechanism(s) within the vicinity of the tethered fluorophores at these helix 1 sites. In earlier studies, Kosower et al (33) and Farrens and co-workers (43) showed that a proximal aromatic residue, Trp, and to a lesser extent Tyr strongly quenched bimane fluorescence (32,33). The colicin E1 p190H 6 channel domain consists of three Trp residues (Trp-424, Trp-460, and Trp-495; Fig.…”

Section: Surface Orientation and Membrane Disposition Of Helix 1-mentioning

confidence: 97%

Scanning the Membrane-bound Conformation of Helix 1 in the Colicin E1 Channel Domain by Site-directed Fluorescence Labeling

Musse

Wang

deLeon

et al. 2006

Helix 1 of the membrane-associated closed state of the colicin E1 channel domain was studied by site-directed fluorescence labeling where bimane was covalently attached to a single cysteine residue in each mutant protein. A number of fluorescence properties of the tethered bimane fluorophore were measured in the membranebound state of the channel domain, including fluorescence emission maximum, fluorescence quantum yield, fluorescence anisotropy, membrane bilayer penetration depth, surface accessibility, and apparent polarity. The data show that helix 1 is an amphipathic ␣-helix that is situated parallel to the membrane surface. A least squares fit of the various data sets to a harmonic function indicated that the periodicity and angular frequency for helix 1 are typical for an amphipathic ␣-helix (3.7 ؎ 0.1 residues per turn and 97 ؎ 3.0°, respectively) that is partially bathing into the membrane bilayer. Dual fluorescence quencher analysis also revealed that helix 1 is peripherally membrane-associated, with one face of the helix dipping into the lipid bilayer and the other face projecting toward the solvent. Finally, our data suggest that the helical boundaries of helix 1, at least at the C-terminal region, remain unaffected upon binding to the surface of the membrane in support of a toroidal pore model for this colicin.The colicins are a family of antimicrobial proteins that are secreted by Escherichia coli strains under environmental stress, because of nutrient depletion or overcrowding, and these proteins often target sensitive bacterial strains (1). The lethal actions of colicins against their target cells are manifested in a number of different modes that include the following: (i) formation of depolarizing ion channels in the cytoplasmic membrane, (ii) inhibition of protein and peptidoglycan synthesis, and (iii) degradation of cellular nucleic acids (1-7). In this context, the bacterial machinery responsible for colicin biological activity feature important mechanisms that are fundamental to various biological processes. These mechanisms include protein receptor binding, membrane translocation, membrane binding and protein unfolding, membrane insertion, voltage-gated ion channel formation, catalysis, and inhibition of enzymes.Colicin E1 is a member of the channel-forming subfamily of colicins and is secreted by E. coli that harbors the naturally occurring colE1 plasmid; the whole colicin consists of three functional segments, the translocation, receptor-binding domains, and channel-forming domains. Initially, the receptor-binding domain (8) interacts with the vitamin B 12 receptor of target cells (9). Following receptor recognition, the translocation domain associates with the tolA gene product, which permits the translocation of colicin E1 across the outer membrane and into the periplasm (10). In the periplasm, the channel domain undergoes a conformational change to an insertion-competent state and then inserts spontaneously into the cytoplasmic membrane of the host cell, forming an ion channel. The cha...