Abstract
The outer segment of vertebrate photoreceptors is a specialized compartment that hosts all the signaling components required for visual transduction. Specific to rod photoreceptors is an unusual set of three glutamic acid-rich proteins (GARPs) as follows: two soluble forms, GARP1 and GARP2, and the N-terminal cytoplasmic domain (GARP' part) of the B1 subunit of the cyclic GMP-gated channel. GARPs have been shown to interact with proteins at the rim of the disc membrane. Here we characterized native GARP1 and GARP2 purified from bovine rod photoreceptors. Amino acid sequence analysis of GARPs revealed structural features typical of "natively unfolded" proteins. By using biophysical techniques, including size-exclusion chromatography, dynamic light scattering, NMR spectroscopy, and circular dichroism, we showed that GARPs indeed exhibit a large degree of intrinsic disorder. Analytical ultracentrifugation and chemical cross-linking showed that GARPs exist in a monomer/multimer equilibrium. The results suggested that the function of GARP proteins is linked to their structural disorder. They may provide flexible spacers or linkers tethering the cyclic GMP-gated channel in the plasma membrane to peripherin at the disc rim to produce a stack of rings of these protein complexes along the long axis of the outer segment. GARP proteins could then provide the environment needed for protein interactions in the rim region of discs.
Highlights
Photoreceptors transduce the absorption of light into an electrical signal
The apparent molecular mass (Mr) of GARP1 and GARP2 (ϳ130 and ϳ62 kDa, respectively) is twice as large as that predicted by the amino acid sequence (64.5 and 31.9 kDa, respectively)
Amino Acid Sequence Analysis of GARP1 and GARP2—We applied a series of predictors of natural disordered regions (PONDR) to GARP1 and GARP2 and their orthologs to identify the regions that are lacking a fixed tertiary structure [19, 20, 44]
Summary
Calculation of Mean Net Charge (R) and Mean Hydrophobicity (H)—The mean net charge (R) of a protein is determined as the absolute value of the difference between the number of positively and negatively charged residues divided by the total number of amino acid residues. The ratio of GARP proteins is a result of two independent experiments performed in duplicate using three different ROS preparations (total of 12 values). Spectra of purified GARP proteins in CD buffer (5 mM Tris-HCl, pH 7.5, 100 mM Na2SO4; 5% (v/v) glycerol, 0.25 mM n-dodecyl--D-maltoside) were recorded as the average of four individual spectral scans in the far-UV region from 190 to 300 nm using a cuvette with a path length of 0.2 cm and the following parameters: instrument sensitivity, 1 millidegrees; response time, 2 s; scan speed, 50 nm/min. The partial specific volume, v, of GARP2 and GARP1 in aqueous buffers was calculated from the amino acid composition according to the method of Durchschlag, applying the data set of Cohn and Edsall, as tabulated in Ref. 42. The densities and viscosities of the buffers were calculated using the program SEDNTERP [43]
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