Biomedical subjects

H Borochov-Neori

Publications and source records attributed to H Borochov-Neori.

3 recordsLinked to original sources

PubMed1989

Rhodopsin-G-protein interactions monitored by resonance energy transfer.

Resonance energy transfer measurements were implemented to monitor the specific interactions between G-protein and rhodopsin in phospholipid vesicles reconstituted with the purified proteins. Fluorescently labeled G-protein was extracted from bleached rod outer segments (ROS) reacted with several sulfhydryl reagents: N-(1-pyrenyl)maleimide (P), monobromobimane (B), 7-(diethylamino)-3-(4-maleimidylphenyl)-4-methylcoumarin (C), and N-(4-anilino-1-naphthyl)maleimide (A). Limited labeling of ROS, resulting in the modification of less than a single -SH residue per G-protein molecule and less than 0.2 residue per rhodopsin, did not impair the specific in situ interactions between rhodopsin and G-protein. This was demonstrated by preservation of their light-activated tight association and Gpp(NH)p binding and their fast dissociation with excess GTP. The distribution of fluorescent label among the three subunits of G-protein revealed a highly reactive -SH group in the gamma subunit accessible to labeling when G-protein was bound specifically to bleached rhodopsin. Recombination of purified fluorescent derivatives of G-protein with purified rhodopsin reconstituted in lipid vesicles restored the light-activated Gpp(NH)p binding to a level comparable to that measured with unlabeled G-protein. Similar observations were obtained with ROS depleted of peripheral proteins. Likewise, modification of up to two -SH groups per rhodopsin molecule with the fluorescent reagents did not affect the functional recombination of G-protein with rhodopsin in reconstituted lipid vesicles or in depleted ROS. Interactions between rhodopsin and G-protein were monitored by resonance energy transfer measurements, with the following fluorescent conjugates as donor/acceptor couples: P-rhodopsin/C-G-protein, P-rhodopsin/B-G-protein, and P-G-protein/C-rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1983

Rhodopsin in reconstituted phospholipid vesicles. 1. Structural parameters and light-induced conformational changes detected by resonance energy transfer and fluorescence quenching.

The structure of purified rhodopsin was investigated by steady-state resonance energy transfer and fluorescence quenching techniques: (1) Fluorescence parameters and relative distances between rhodopsin sites labeled with fluorescent probes and the endogenous chromophore 11-cis-retinal were measured in micellar detergent solution and in reconstituted phospholipid vesicles. (2) The accessibility of the labeled rhodopsin sites in reconstituted vesicles to N-methyl- and N-benzylpicolinium was studied in the dark and subsequent to rhodopsin bleaching. Fluorescent-labeled rhodopsin was affinity purified in octyl glucoside from rod outer segments which were previously reacted with either the sulfhydryl-specific reagents, pyrenylmaleimide or monobromobimane, or reagents specific to amino groups, dansyl chloride or fluorescein isothiocyanate. The purified protein was recombined with phospholipids, and vesicles were formed by detergent dialysis. All four fluorophores appear to react greater than or equal to 30 A away from the endogenous chromophore as estimated from the efficiency of energy transfer and presumably probe rhodopsin domains exposed at the membrane surface. The maximal fraction of quenchable fluorescence and the mean quenching constant were determined in dark and bleached vesicles: bleaching did not affect the quenching of the fluorophores attached to sulfhydryl groups but markedly decreased the quenching constants of the fluorophores coupled to amino groups. The apparent collisional rate constant decreased by 20- and 4-fold for dansyl and fluorescein, respectively. The results suggest that bleaching reduced the accessibility of these sites which, in turn, may reflect light-induced displacements of rhodopsin domains at the membrane surface. Such structural changes may regulate rhodopsin-rhodopsin as well as rhodopsin-enzyme interactions.

Chemical Phenomena↗

PubMed1983

Rhodopsin in reconstituted phospholipid vesicles. 2. Rhodopsin-rhodopsin interactions detected by resonance energy transfer.

The interactions between rhodopsin molecules in a micellar detergent solution (octyl glucoside) and in reconstituted phospholipid vesicles were studied in the dark and after bleaching. Resonance energy transfer measurements were used to monitor the proximity between rhodopsin monomers conjugated with a fluorescent donor or a fluorescent acceptor. Reactive sulfhydryl groups of rhodopsin were labeled with pyrenylmaleimide (donor) or monobromobimane (acceptor), whereas amino groups were labeled with dansyl chloride (donor) or fluorescein isothiocyanate (acceptor). The results suggest that in the micellar solution rhodopsin was monomeric in the dark and aggregated after bleaching. If the aggregate were to be a dimer, the labeled sulfhydryl groups of the monomers would be approximately 40 A apart, while the labeled amino groups would be at least 68 A distant from each other. Rhodopsin reconstituted in phospholipid vesicles appeared aggregated both in the dark and after bleaching. The proximity between the sulfhydryl groups of the monomers was not influenced by illumination. In contrast, the labeled amino groups seemed to be largely separated in the dark and closer to each other once the vesicles were bleached. If the aggregate were to be a dimer, the labeled sulfhydryl groups would be approximately 40 A apart both in the dark and after bleaching, whereas the labeled amino groups would be greater than 60 A apart in the dark and approximately 44 A from each other after bleaching. These findings are discussed in the context of rhodopsin structure, its ability to regenerate after bleaching, and the light-induced events initiated by rhodopsin photoexcitation.

Chemical Phenomena↗