Detecting Molecular Interactions that Stabilize Native Bovine Rhodopsin

doi:10.1016/j.jmb.2006.02.008

Journal of Molecular Biology

Volume 358, Issue 1, 21 April 2006, Pages 255-269

https://doi.org/10.1016/j.jmb.2006.02.008 Get rights and content

Using single-molecule force spectroscopy we probed molecular interactions within native bovine rhodopsin and discovered structural segments of well-defined mechanical stability. Highly conserved residues among G protein-coupled receptors were located at the interior of individual structural segments, suggesting a dual role for these segments in rhodopsin. Firstly, structural segments stabilize secondary structure elements of the native protein, and secondly, they position and hold the highly conserved residues at functionally important environments. Two main classes of force curves were observed. One class corresponded to the unfolding of rhodopsin with the highly conserved Cys110–Cys187 disulfide bond remaining intact and the other class corresponded to the unfolding of the entire rhodopsin polypeptide chain. In the absence of the Cys110–Cys187 bond, the nature of certain molecular interactions within folded rhodopsin was altered. These changes highlight the structural importance of this disulfide bond and may form the basis of dysfunctions associated with its absence.

Introduction

G protein-coupled receptors (GPCRs) represent the largest class of membrane proteins and are involved in virtually every physiological process. These receptors decode a variety of sensory, chemotactic, hormonal, and neuronal signals, which they transduce across the cell membrane via heterotrimeric G proteins. All GPCRs share the same basic architecture of a seven-transmembrane α-helical (7TM) domain linked together by loops on the extracellular (EC) and the cytoplasmic (CP) surfaces. GPCRs share highly conserved residues (80–100%), such as the D(E)RY and NPXXY motifs and a stabilizing Cys–Cys disulfide bond, that play important functional roles. GPCRs can exist as either homo or hetero-oligomers, thereby increasing their functional variability.1, 2, 3, 4, 5

Rhodopsin (Rh) is the light receptor that initiates phototransduction in the rod outer segments (ROS) of the eye. Rh is a prototypical GPCR that has served as a template for studying and understanding this family of receptors and the signaling systems that they regulate.6, 7 The specific localization of Rh in the internal discs of the ROS and its high expression level (constituting >90% of all proteins in disc membranes) have facilitated studies in this system that cannot be carried out with any other GPCR system.⁸ Rh is currently the only GPCR for which a crystal structure has been solved9, 10, 11, 12 and serves as a template in the structure prediction of other GPCRs.⁷

A number of diseases are associated with mutations that cause destabilization and misfolding of GPCRs.¹³ Understanding the molecular interactions that stabilize or destabilize GPCRs is fundamental to our understanding of their function. However, little is known about the underlying molecular mechanisms. The majority of these types of mutations in Rh lead to the neurodegenerative disease Retinitis pigmentosa (RP).14, 15About 0.05% of the global population is affected by this disease leading to photoreceptor degradation and loss of vision.¹⁶ Most mutations target the TM and EC domains of Rh¹⁷ and induce misfolding by the replacement of the conserved Cys110–Cys187 disulfide (S–S) bond with an abnormal disulfide bond between Cys185 and Cys187.¹⁸ The Rh structure has provided opportunities to identify regions critical for proper folding by computational approaches.15, 19 However, experimental insights into the molecular interactions that stabilize and destabilize the protein in native membranes are currently limited due to the lack of experimental approaches that allow for direct measurements of those interactions.

Atomic force microscopy (AFM) and single-molecule force spectroscopy (SMFS) can provide high-resolution images of individual 7TM proteins in their native environment, and can detect and locate the forces that stabilize these proteins.20, 21, 22, 23, 24, 25, 26 Forces detected by SMFS are a direct measure of interactions established within the membrane protein and depend on environmental changes such as temperature,²⁷ pH, ion concentration,²⁸ and oligomeric assembly.²⁹ Recent SMFS experiments enabled the observation of the folding kinetics of single secondary structures in the sodium/proton antiporter NhaA30, 31 from Escherichia coli, and the detection of molecular interactions induced by single ligand binding and activation.²⁸ Here, we characterize molecular interactions established within Rh embedded in native ROS disc membranes. These interactions are mapped onto the GPCR structure resolving stable structural segments. The map showed the highly conserved residues among GPCRs¹⁷ to be located at the interior of the stable structural segments in Rh. In the absence of the stabilizing Cys110–Cys187 bond, the molecular interactions establishing structural segments changed their strengths and locations. Such changes may represent the molecular fingerprint of protein destabilization, misfolding, and malfunction.

Section snippets

Imaging of native ROS disc membranes

Native ROS disc membranes containing Rh were imaged in buffer solution by AFM prior to SMFS studies. The survey shows a typical ensemble of a native ROS disc membrane preparation (Figure 1): an intact disc, a single layered burst disc membrane and lipid bilayer flatly adsorbed onto the supporting mica. In most cases, burst disc membranes adsorbed to mica resulting in single layered protein membranes (arrow 1), which exhibited an average height of 8.2(±0.9) nm (n=61). Co-isolated lipid bilayers

Discussion

The unfolding pathways of Rh from native ROS disc membranes were detected by SMFS. The force spectra revealed structural segments stabilizing Rh. Analyses of the spectra using the WLC model located these structural segments on the structure of Rh and indicated the strength of their stabilizing molecular interactions. Each stable structural segment established a barrier against unfolding and was mapped onto the tertiary structure of Rh (Figure 5, Figure 6). Force–distance curves of equal length

Conclusions

Individual unfolding events of single Rh molecules were monitored. Examining the most frequent unfolding patterns revealed that the GPCR is organized into distinct stable structural segments (Figure 5, Figure 6). Molecular interactions that govern these structural segments and their respective barriers depended on interactions with other structural regions. We have pinpointed the importance of the native Cys110–Cys187 covalent linkage and the effect that it has on protein stability.

ROS disc membrane preparation

All experimental procedures were carried out under dim red light. Centrifugation steps were performed at 4 °C. ROS were purified from fresh bovine retinas as described⁵² and stored at −80 °C. To obtain disc membranes, ROS membranes were resuspended using a glass hand-held homogenizer in 13 ml of buffer A (2 mM Tris–HCl (pH 7.4)) and incubated overnight at 4 °C. The membrane suspension was centrifuged at 26,500g for 30 min. The membranes were washed twice with 13 ml of buffer A and three times with 3 ml

Acknowledgements

This research was supported by NIH grant EY08061 and grants from the Deutsche Forschungsgemeinschaft (DFG), the Deutsche Volkswagenstiftung, the Free State of Saxony, and the European Union. P.S.-H.P. is the recipient of a postdoctoral fellowship from the Natural Sciences and Engineering Council of Canada.

References (55)

A. Salahpour et al.
Functional significance of oligomerization of G-protein-coupled receptors
Trends Endocrinol. Metab.
(2000)
K.D. Ridge et al.
Phototransduction: crystal clear
Trends Biochem. Sci.
(2003)
T. Okada et al.
The retinal conformation and its environment in rhodopsin in light of a new 2.2 Å crystal structure
J. Mol. Biol.
(2004)
J. Li et al.
Structure of bovine rhodopsin in a trigonal crystal form
J. Mol. Biol.
(2004)
D. Fotiadis et al.
The G protein-coupled receptor rhodopsin in the native membrane
FEBS Letters
(2004)
Y. Liang et al.
Organization of the G protein-coupled receptors rhodopsin and opsin in native membranes
J. Biol. Chem.
(2003)
D.J. Müller et al.
Stability of bacteriorhodopsin alpha-helices and loops analyzed by single-molecule force spectroscopy
Biophys. J.
(2002)
D.A. Cisneros et al.
Probing origins of molecular interactions stabilizing the membrane proteins halorhodopsin and bacteriorhodopsin
Structure (Camb)
(2005)
H. Janovjak et al.
Molecular force modulation spectroscopy revealing the dynamic response of single bacteriorhodopsins
Biophys. J.
(2005)
A. Kedrov et al.
Controlled unfolding and refolding of a single sodium-proton antiporter using atomic force microscopy
J. Mol. Biol.
(2004)

S.S. Karnik et al.

Assembly of functional rhodopsin requires a disulfide bond between cysteine residues 110 and 187

J. Biol. Chem.

(1990)

K. Palczewski et al.

Mechanism of rhodopsin kinase activation

J. Biol. Chem.

(1991)

C. Möller et al.

Determining molecular forces that stabilize human aquaporin-1

J. Struct. Biol.

(2003)

A. Anukanth et al.

Structure and function in rhodopsin. Requirements of a specific structure for the intradiscal domain

J. Biol. Chem.

(1994)

D.M. Engelman et al.

Membrane protein folding: beyond the two stage model

FEBS Letters

(2003)

J. Klein-Seetharaman

Dual role of interactions between membranous and soluble portions of helical membrane receptors for folding and signaling

Trends Pharmacol. Sci.

(2005)

D.S. Papermaster

Preparation of retinal rod outer segments

Methods Enzymol.

(1982)

E.-L. Florin et al.

Sensing specific molecular interactions with the atomic force microscope

Biosens. Bioelectron.

(1995)

P.S.-H. Park et al.

Oligomerization of G protein-coupled receptors: past, present, and future

Biochemistry

(2004)

P.S.-H. Park et al.

Diversifying the repertoire of G protein-coupled receptors through oligomerization

Proc. Natl Acad. Sci. USA

(2005)

G. Milligan et al.

Methods to monitor the quaternary structure of G protein-coupled receptors

FEBS J.

(2005)

G.E. Breitwieser

G protein-coupled receptor oligomerization: implications for G protein activation and cell signaling

Circ. Res.

(2004)

S. Filipek et al.

The crystallographic model of rhodopsin and its use in studies of other G protein-coupled receptors

Annu. Rev. Biophys. Biomol. Struct.

(2003)

S. Filipek et al.

G protein-coupled receptor rhodopsin: a prospectus

Annu. Rev. Physiol.

(2003)

K. Palczewski et al.

Crystal structure of rhodopsin: a G protein-coupled receptor

Science

(2000)

D.C. Teller et al.

Advances in determination of a high-resolution three-dimensional structure of rhodopsin, a model of G-protein-coupled receptors (GPCRs)

Biochemistry

(2001)

T.P. Dryja et al.

Molecular genetics of retinitis pigmentosa

Hum. Mol. Genet.

(1995)

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^†: K.T.S. and P.S.-H.P. contributed equally to this work.

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Journal of Molecular Biology

Detecting Molecular Interactions that Stabilize Native Bovine Rhodopsin

Introduction

Section snippets

Imaging of native ROS disc membranes

Discussion

Conclusions

ROS disc membrane preparation

Acknowledgements

Trends Endocrinol. Metab.

Trends Biochem. Sci.

J. Mol. Biol.

J. Mol. Biol.

FEBS Letters

J. Biol. Chem.

Biophys. J.

Structure (Camb)

Biophys. J.

J. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Struct. Biol.

J. Biol. Chem.

FEBS Letters

Trends Pharmacol. Sci.

Methods Enzymol.

Biosens. Bioelectron.

Oligomerization of G protein-coupled receptors: past, present, and future

Biochemistry

Diversifying the repertoire of G protein-coupled receptors through oligomerization

Proc. Natl Acad. Sci. USA

Methods to monitor the quaternary structure of G protein-coupled receptors

FEBS J.

G protein-coupled receptor oligomerization: implications for G protein activation and cell signaling

Circ. Res.

The crystallographic model of rhodopsin and its use in studies of other G protein-coupled receptors

Annu. Rev. Biophys. Biomol. Struct.

G protein-coupled receptor rhodopsin: a prospectus

Annu. Rev. Physiol.

Crystal structure of rhodopsin: a G protein-coupled receptor

Science

Advances in determination of a high-resolution three-dimensional structure of rhodopsin, a model of G-protein-coupled receptors (GPCRs)

Biochemistry

Molecular genetics of retinitis pigmentosa

Hum. Mol. Genet.