Stabilizing Effect of Zn2+ in Native Bovine Rhodopsin

Abstract

Single-molecule force spectroscopy (SMFS) is a powerful tool to dissect molecular interactions that govern the stability and function of proteins. We applied SMFS to understand the effect of Zn 2؉ on the molecular interactions underlying the structure of rhodopsin. Force-distance curves obtained from SMFS assays revealed the strength and location of molecular interactions that stabilize structural segments within this receptor. The inclusion of ZnCl 2 in SMFS assay buffer increased the stability of most structural segments. This effect was not mimicked by CaCl 2 , CdCl 2 , or CoCl 2 . Thus, Zn 2؉ stabilizes the structure of rhodopsin in a specific manner.Zinc is the second most abundant trace element found in the human body (1) and occurs in high concentrations in the retina (2). Zinc deficiency in humans can lead to vision-related disorders such as poor dark adaptation, night blindness, and retinal degeneration (3). The role of Zn 2ϩ in vision is likely related, in part, to rhodopsin, the light receptor that initiates phototransduction in the rod outer segments (ROS) 5 of retinal photoreceptor cells (4). Rhodopsin can directly associate with this bivalent metal ion (5, 6), and Zn 2ϩ increases the level of rhodopsin phosphorylation (7). Furthermore, changes are observed in the pattern of thermal bleaching and regeneration of rhodopsin by 11-cis-retinal in the presence and absence of Zn 2ϩ (6,8). Therefore, Zn 2ϩ appears to affect the structure and function of rhodopsin.Rhodopsin in native ROS disc membranes has been previously characterized by single-molecule force spectroscopy (SMFS) to reveal the molecular interactions that stabilize the dark state of the receptor (9). The receptor is organized into several stable structural segments that present barriers to unfolding. Forces required to unfold these segments provide a direct measure of the molecular interactions stabilizing the protein in a particular region. SMFS has also been used to characterize the molecular interactions underlying the stability of a few other membrane proteins including bacteriorhodopsin and halorhodopsin from Halobacterium salinarium (10, 11), the sodium/proton antiporter NhaA from Escherichia coli (12), and human aquaporin-1 (13). The sensitivity of SMFS assays enables one to probe the effects of environmental factors like temperature, pH, ion concentration, and oligomeric assembly on the molecular interactions stabilizing a protein (12, 14 -16).In the current study, we use SMFS to monitor the effect of Zn 2ϩ on molecular interactions stabilizing dark-state rhodopsin. Force-distance (F-D) curves obtained from native bovine ROS disc membranes in the presence of Zn 2ϩ revealed that the location of stable structural segments in rhodopsin is unaffected by the inclusion of this bivalent metal ion. However, forces required to unfold these segments in the presence of Zn 2ϩ were significantly increased. Thus, Zn 2ϩ appears to strengthen the molecular interactions that stabilize the native structure of rhodopsin. EXPERIMENTAL PROCEDURESROS Disc...

Stabilization of Membrane Proteins: the Case of G‐Protein‐Coupled Receptors

“…This assay is based on the heat quantity necessary to promote the cis→all trans isomerization process of the retinal chromophore as a consequence of interhelical movements resulting from temperature increase. Recently, a stabilizing effect was reported for zinc ions on Rho in membranes immobilized on a surface [56]. The apparent discrepancy between our results and the ones from the single-molecule force spectroscopy study [56] can be explained if we take into account that our thermal bleaching kinetics determined in detergent solution may be also reflecting a specific effect of the zinc chloride salt used in the experiment on the heat conductivity of the solution.…”

“…Recently, a stabilizing effect was reported for zinc ions on Rho in membranes immobilized on a surface [56]. The apparent discrepancy between our results and the ones from the single-molecule force spectroscopy study [56] can be explained if we take into account that our thermal bleaching kinetics determined in detergent solution may be also reflecting a specific effect of the zinc chloride salt used in the experiment on the heat conductivity of the solution. Therefore, our thermal bleaching results suggest zinc specific effects on the structure of the receptor by measuring the hydrolysis of the chromophore from the protein.…”

“…An interesting case is that of the effect of zinc ions on Rho stability [33,55,56]. We previously reported a decrease in the thermal stability caused by zinc ions -measured as an increase in the rate of thermally-induced Schiff base hydrolysis (of purified Rho solubilized in the neutral DM detergent) [33].…”

Point Mutations in Membrane Proteins Reshape Energy Landscape and Populate Different Unfolding Pathways

“…Since each unfolding step has a certain probability to be taken, this is also the case for the unfolding pathway. 9 Experiments on bacteriorhodopsin (BR), 10,11 NhaA 12 and bovine rhodopsin 13,14 have shown that small changes in the environment can alter interactions stabilizing the structural segments within membrane proteins. We have recently shown that inter-and intramolecular interactions contribute differently towards stabilizing these structural segments.…”

Cholesterol increases kinetic, energetic, and mechanical stability of the human β2-adrenergic receptor

“…Measuring the kinetic response of the individual structural elements by dynamic SMFS (DFS) allows quantification of their unfolding free energy, kinetic stability, conformational variability, and mechanical flexibility (33). Both SMFS and DFS have been applied to characterize these parameters of various membrane proteins and follow how they change on changing their functional state or physiological environment (34)(35)(36)(37)(38)(39).…”