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 Zn2+ 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 ZnCl2 in SMFS assay buffer increased the stability of most structural segments. This effect was not mimicked by CaCl2, CdCl2, or CoCl2. Thus, Zn2+ stabilizes the structure of rhodopsin in a specific manner.

FIGURE 1. Single-molecule force spectroscopy on ROS disc membranes

F-D curves were recorded from disc membranes in SMFS assay buffer (A and B) or in SMFS assay buffer supplemented with 200 μm ZnCl₂ (C and D). Curves obtained from samples in SMFS assay buffer devoid of ZnCl₂ were those reported previously (9). Only curves that exhibited lengths corresponding to the unfolding of a rhodopsin polypeptide chain with an intact Cys¹¹⁰–Cys¹⁸⁷ bond (≈65 nm) were analyzed (A and C), and a selection of F-D curves is shown for each condition tested (B and D). Several F-D curves were superimposed (B, n = 42; D, n = 34) to highlight peaks that occurred with high frequency (black) and those that occurred with lower frequency (gray shaded). Each peak in the individual F-D curves was fit with the WLC model to reveal the number of amino acid residues stretched between the probe and the membrane surface. Average values are shown above each fit, and the color of each fit corresponds to the coloring of stable structural segments in Fig. 2.

FIGURE 2. Secondary structure of rhodopsin mapped with stable structural segments

Values from WLC model fitting (Table 1, Fig. 1) were used to estimate the location of stable structural segments in the secondary structure of rhodopsin. Each stable structural segment and its name is colored differently. Arrows indicate the start and end of each structural segment, and the corresponding amino acid residue number is indicated. Numbers in brackets are the values obtained from WLC model fits, which indicate the number of amino acid residues stretched above the membrane. Zn²⁺-binding sites observed in crystal structures of rhodopsin are shown in white. Amino acid residues that coordinate Zn²⁺ at those sites are as follows: ${\mathrm{Zn}}_1^{2+}$, Asn¹⁵¹ and His¹⁵²; ${\mathrm{Zn}}_2^{2+}$, Glu¹²² and His²¹¹; ${\mathrm{Zn}}_3^{2+}$, Glu²⁰¹ and Gln²⁷⁹; ${\mathrm{Zn}}_4^{2+}$, Glu¹⁹⁷ and His¹⁹⁵.

FIGURE 3. Specificity of ZnCl₂ effects on rhodopsin

A, F-D curves were collected from disc membranes in SMFS assay buffer supplemented with 200 μm ZnCl₂, CaCl₂, CdCl₂, or CoCl₂. The average force required to unfold each stable structural segment in rhodopsin observed in the presence of bivalent metal ions is shown relative to the average force observed in the absence of added bivalent metal ions. B, F-D curves were collected from disc membranes in SMFS assay buffer (25 mm MgCl₂), in SMFS assay buffer devoid of MgCl₂ (0 mm MgCl₂), or in SMFS assay buffer devoid of MgCl₂ and supplemented with 1 mm EDTA (0 mm MgCl₂, 1 mm EDTA). The average force obtained from each condition is shown. Error bars represent the standard deviation. Values represented in the figure are given in Table 1.

FIGURE 4. Effect of different ZnCl₂ concentrations on unfolding forces

The average force required to unfold each stable structural segment was recorded at increasing concentrations of ZnCl₂ (0–400 μm) added to SMFS assay buffer (A–C). The color of the curves corresponds to the structural segments highlighted in D–F. Symbols placed at added [ZnCl₂] of less than 0 μm represent data from F-D curves collected in SFMS assay buffer devoid of MgCl₂ and in the presence of 1 mm EDTA. Error bars represent the standard deviation. Values represented in the figure are given in Table 1.

FIGURE 5. Preservation of the Cys¹¹⁰–Cys¹⁸⁷ disulfide bond by nonspecific electrostatic effects

Ratios of F-D curves corresponding to the unfolding of rhodopsin in the presence (65 nm) and absence (95 nm) of the Cys¹¹⁰–Cys¹⁸⁷ disulfide bond are shown. F-D curves were obtained from samples either in SMFS assay buffer supplemented with bivalent metal ions or in SMFS assay buffer without added MgCl₂ and supplemented with 1 mm EDTA. The number of curves used to calculate the ratio is as follows: 1 mm EDTA, 264; 0 μm ZnCl₂, 274; 10 μm ZnCl₂, 187; 25 μm ZnCl₂, 202; 50 μm ZnCl₂, 222; 100 μm ZnCl₂, 315; 200 μm ZnCl₂, 114; 400 μm ZnCl₂, 133; 200 μm CaCl₂, 89; 200 μm CdCl₂, 88; 200 μm CoCl₂, 85.

FIGURE 6. Side view of a rhodopsin dimer model

A, the position of Zn²⁺ in binding sites observed in crystal structures of rhodopsin are highlighted as purple spheres. Helices of rhodopsin are colored as follows: H-I in blue, H-II in blue-green, H-III in green, H-IV in green-yellow, H-V in yellow, H-VI in orange, H-VII and H-8 in red Amino acid residues that coordinate Zn²⁺ are shown in ball-and-stick representations. The figure was drawn using MolMol (57). B–D, the electrostatic potential is shown for a rhodopsin dimer without bound Zn²⁺ (B), with 8 bound Zn²⁺ (sites 1–4) (C), and with 6 bound Zn²⁺ (sites 2–4) (D). The electrostatic potential is colored follows: blue, positive; red, negative; white, neutral. Green and red dashed ellipses indicate favorable and unfa vorable electrostatic interactions, respectively. B–D were drawn using Yasara (version 6.2 Yasara Biosciences, Graz, Austria).