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Biochemistry 2010, 49, 10412–10420 DOI: 10.1021/bi101345x
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Conservation of Molecular Interactions Stabilizing Bovine and Mouse Rhodopsin† Shiho Kawamura,‡, Alejandro T. Colozo,§, Daniel J. M€uller,*,‡ and Paul S.-H. Park*,§ Department of Biosystems Science and Engineering, ETH Z€ urich, 4058 Basel, Switzerland, and §Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, Ohio 44106, United States. Contributed equally to this work )
‡
Received August 19, 2010; Revised Manuscript Received October 20, 2010 ABSTRACT: Rhodopsin is the light receptor that initiates phototransduction in rod photoreceptor cells. The structure and function of rhodopsin are tightly linked to molecular interactions that stabilize and determine the receptor’s functional state. Single-molecule force spectroscopy (SMFS) was used to localize and quantify molecular interactions that structurally stabilize bovine and mouse rhodopsin from native disk membranes of rod photoreceptor cells. The mechanical unfolding of bovine and mouse rhodopsin revealed nine major unfolding intermediates, each intermediate defining a structurally stable segment in the receptor. These stable structural segments had similar localization and occurrence in both bovine and mouse samples. For each structural segment, parameters describing their unfolding energy barrier were determined by dynamic SMFS. No major differences were observed between bovine and mouse rhodopsin, thereby implying that the structures of both rhodopsins are largely stabilized by similar molecular interactions.
Rhodopsin is a G protein-coupled receptor (GPCR)1 residing in rod outer segments (ROS) of photoreceptor cells, where it initiates phototransduction upon light activation. Several crystal structures are now available for a handful of GPCRs (1-5). These structures highlight the conservation of the general architecture of GPCRs, which display seven transmembrane R-helices. Some of the mechanisms underlying receptor activation and function are likely conserved across members of this family of membrane proteins (4, 6, 7). Despite low amino acid sequence similarities, comparison of GPCR crystal structures reveals only relatively small deviations in the position of transmembrane R-helices (2, 5, 8). Yet those small differences are significant enough to facilitate the specific roles and functions of those receptors. The determinants and functional effects of these small differences must begin to be understood. Inter- and intramolecular interactions contribute to the folding, structure, and stability of proteins, and they determine the protein’s functional state. Single-molecule force spectroscopy (SMFS) is a unique tool that allows for the quantification and structural localization of molecular interactions established in membrane proteins (9, 10). Native structure-function relationships of membrane proteins can be determined by SMFS because membrane proteins are studied in their functionally relevant lipid bilayer at ambient temperatures in a buffer solution. SMFS has been applied to bovine rhodopsin to quantify the stabilization of † This work was supported in part by U.S. Public Health Service Grants R00EY018085 and P30EY11373 from the National Institutes of Health, Bethesda, MD. This work was also supported by grants from the Deutsche Forschungsgemeinschaft (DFG), the European Union (FP-7), Research to Prevent Blindness, and the Ohio Lions Eye Research Foundation. *To whom correspondence should be addressed. D.J.M.: phone, þ4161-3873307; fax, þ41-61-3873994; e-mail,
[email protected]. P.S.-H.P.: phone, 216-368-2533; fax, 216-368-3171; e-mail, paul.park@ case.edu. 1 Abbreviations: aa, amino acid residues; AFM, atomic force microscopy/microscope; DFS, dynamic SMFS; F-D, force-distance; GPCR, G protein-coupled receptor; ROS, rod outer segment(s); SMFS, single-molecule force spectroscopy; WLC, worm-like chain.
pubs.acs.org/Biochemistry
Published on Web 11/01/2010
the receptor by zinc (11) and to reveal a localized stabilization that affects transducin activation by naturally occurring palmitylation in the carboxyl-terminal region of the receptor (12). Single substitutions of amino acid residues have the potential to alter molecular interactions that lead to changes in the structure and function of GPCRs (13-18). This is especially true in rhodopsin where single amino acid residue differences can result in dramatic effects. A single amino acid residue change can cause destabilization, malfunction, or misfolding of the receptor, which leads to dramatic physiological consequences. Over 100 different point mutations in rhodopsin can cause retinitis pigmentosa, a group of retinal degenerative diseases (18). Receptor homologues provide a natural system to study amino acid residue substitutions. Rhodopsin from rod photoreceptor cells in bovine and mouse retina shares 93% sequence similarity with 23 amino acid residue differences (Figure 2C and Supporting Information Figure S1). Bovine rhodopsin has been extensively studied, and there are numerous crystal structures available (19-32). The only other rhodopsin crystal structure solved is that of invertebrate squid rhodopsin (33, 34). Significant differences are observed between the structures of vertebrate bovine rhodopsin and invertebrate squid rhodopsin, including extended transmembrane helices 5 and 6 and an additional R-helix in the carboxyl-terminal region. No crystal structure has been solved for mouse rhodopsin. Thus, it is unclear whether the differences in amino acid residues between bovine and mouse rhodopsin affect the structure, stability, and function of the receptor. The purpose of this study was to determine whether the naturally occurring 23 amino acid residue differences in the sequences of bovine and mouse rhodopsin have a significant effect on the molecular interactions that stabilize each structure. SMFS was applied to quantify the molecular interactions of bovine and mouse rhodopsin embedded in native ROS disk membranes. Furthermore, insights about the kinetic stability and mechanical properties of rhodopsin structure were gained using dynamic SMFS (DFS). r 2010 American Chemical Society
Article MATERIALS AND METHODS ROS Disk Membrane Preparation. ROS were purified from either fresh bovine retina or fresh mouse retina of C57BL/ 6 mice, and disk membranes were prepared as described previously (12, 35). Fresh bovine eyes were obtained from a local slaughterhouse (Mahan Packing, Bristolville, OH), and mice were from Jackson Laboratories (Bar Harbor, ME). Mice were 3-4 weeks old and were dark adapted overnight prior to being sacrificed. Disk membranes were either resuspended in buffer A (2 mM Tris-HCl, pH 7.4) for immediate use or were resuspended in buffer B (67 mM potassium phosphate, 1 mM magnesium acetate, 0.1 mM EDTA, 18% sucrose, pH 7.0) for storage at -80 °C. Membranes stored in buffer B at -80 °C were thawed and washed twice in buffer A prior to use. Unless otherwise stated, all disk preparation procedures were carried out under dim red light at 4 °C. Purification of Rhodopsin. Rhodopsin from dark-adapted mice was purified from whole eyes. Eyes from three mice were homogenized in 3 mL of buffer C (10 mM Bis-Tris propane, 150 mM NaCl, pH 7.5) using a hand-held homogenizer. The homogenate was centrifuged at 124740g for 5 min. The pellet was resuspended in buffer D (10 mM Bis-Tris propane, 500 mM NaCl, 20 mM n-dodecyl β-D-maltoside (Anatrace, Inc., Maumee, OH), pH 7.5) and shaken at room temperature for 15 min. The suspension was then centrifuged at 124740g for 20 min. The supernatant was loaded on a 6 30 mm column packed with anti-1D4 antibody (36) coupled to CNBr-activated Sepharose 4B (Santa Cruz Biotechnology, Santa Cruz, CA) preequilibrated with buffer E (10 mM Bis-Tris propane, 500 mM NaCl, 2 mM n-dodecyl β-D-maltoside, pH 7.5). The column was washed with 10 mL of buffer E. Purified rhodopsin was obtained by eluting the column with buffer E supplemented with 800 μM 1D4 peptide (TETSQVAPA) synthesized by United Biochemical Research, Inc. (Seattle, WA). Bovine rhodopsin was purified from frozen bovine retinas purchased from InVision BioResources (Seattle, WA). Tissue from approximately two retinas was used for purification. Bovine retinal tissue was homogenized in 3 mL of buffer C using a handheld glass homogenizer, and the subsequent steps of the purification procedure are identical to that carried out for the purification of mouse rhodopsin described above. The absorbance spectrum of purified rhodopsin was obtained using a Lambda 35 UV/vis spectrophotometer (Perkin-Elmer, Waltham, MA). All purification steps were conducted under dim red light. SMFS and DFS. SMFS and DFS were performed on disk membranes isolated from mouse and bovine samples essentially as described previously (12, 35, 37). Data were collected using two different atomic force microscopes (AFM): a Multimode AFM (Veeco Metrology, Santa Barbara, CA) and a NanoWizard Ultra AFM with an 850 nm laser detection system (JPK Instruments, Berlin, Germany). Disk membranes were adsorbed on freshly cleaved mica in buffer A. SMFS and DFS were conducted using NPS Si3N4 cantilevers (nominal spring constant 0.6-0.8 N/m; Veeco Metrology). Spring constants of cantilevers were determined in buffer solution using the thermal noise method (38, 39). DFS on bovine and mouse rhodopsin was performed at six different pulling velocities: 100, 300, 700, 1500, 3000, and 6000 nm/s. DFS at 1500, 3000, and 6000 nm/s was conducted only with the Nanowizard Ultra AFM, which was equipped with a 16-bit data acquisition card (NI PCI-6221; National
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Instruments, Munich, Germany) to allow higher sampling frequencies. SMFS and DFS experiments were performed in the dark under dim red light at room temperature (≈28 °C) in SMFS assay buffer (150 mM KCl, 25 mM MgCl2, 20 mM Tris, pH 7.8). Analysis of Force-Distance (F-D) Curves. F-D curves were selected, aligned, analyzed, and superimposed using Igor Pro 6 (WaveMetrics, Inc., Lake Oswego, OR) essentially as described previously (11, 35). F-D curves exhibiting a length corresponding to a fully stretched rhodopsin molecule with an intact Cys110-Cys187 disulfide bond (≈65 nm) were first selected (35). This selection criterion ensured that only F-D curves corresponding to rhodopsin unfolded from its terminal region were analyzed. From this selection, F-D curves were aligned using their most dominant force peaks at 15, 26, 37, 97, 108, 122, 220, and 238 aa as reference. Every peak of a single F-D curve was fitted using the worm-like chain (WLC) model (40): 1 0 F ¼
C B kBT B 1 1 xC þ C B p @ x 2 4 LA 4 1L
ð1Þ
A persistence length, p, of 0.4 nm and a monomer length, x, of 0.36 nm was used (35, 37, 41). kB denotes the Boltzmann constant and T the temperature. The contour length, L, which gives the length of stretched polypeptide chain, was estimated from fitting eq 1 to each force peak of a F-D curve. From this contour length, the number of amino acid residues stretched was estimated approximating a length of 0.36 nm for each residue. Assignment of unfolding events to specific structural segments of rhodopsin was determined as described previously (35). All contour lengths revealed for either bovine or mouse rhodopsin were pooled and represented as a frequency distribution with a bin size of three amino acid residues. DFS Analysis. The most probable rupture force and the most probable loading rate at each pulling velocity were determined from the best fit of a single Gaussian function to the frequency distribution of either force or loading rate. Loading rates were calculated as the product of the pulling velocity and the slope of the F-D curve obtained from the best fit of eq 1 to a given force peak (42). DFS data were fitted using the Bell-Evans model (43) as described previously (37). In this model, the most probable unfolding force, Fp, can be expressed as kBT xu rf ln FP ¼ ð2Þ xu k B Tku In this equation xu is the distance from the free energy minimum to the transition-state barrier, ku the rate of unfolding in the absence of applied force, and rf the loading rate. Estimates for xu and ku were obtained from the fits of eq 2 to the DFS data. Estimation of the height of the transition state barrier, ΔGu‡, and rigidity, κ, for all unfolding barriers are calculated from xu and ku, and the respective errors are propagated as described previously (37). Extra Sum of Squares F-Test. A Gaussian function was used to describe the histogram of contour lengths in Figure 2A and a function defined by eq 2 was used to describe the relationship between the most probable force and loading rate in the DFS plots of Figure 4. To test whether the fitted parameters of either function differed significantly between data from bovine and mouse samples, an extra sum of squares F-test was conducted using GraphPad Prism 5.0 (San Diego, CA) (44, 45); that is, the sum of squares
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FIGURE 1: Mechanical unfolding of rhodopsin. (A) Schematic representation of the experimental setup that depicts the mechanical unfolding of rhodopsin from disk membranes of native ROS. The cantilever forms a bond at the amino-terminal region of rhodopsin to mechanically unfold the receptor out of the membrane. (B) A single F-D curve with peaks fit by the WLC model. The WLC model fits are shown with the contour length indicated above each fit. (C, D) A selection of representative F-D curves from bovine (C) and mouse (D) samples are shown. (E, F) Superimpositions of all F-D curves collected at six pulling velocities are shown for bovine (E, n = 901) and mouse (F, n = 627) rhodopsin. The dotted lines represent WLC model fitted curves with contour lengths determined from Gaussian analysis of frequency histograms (Figure 2).
obtained from the analysis where the parameters of either function were estimated separately from bovine and mouse data was compared to the sum of squares obtained from the analysis where both data sets shared single estimates of those parameters from either function. The level of significance was taken as P
