Glutamic Acid-rich Proteins of Rod Photoreceptors Are Natively Unfolded
2005; Elsevier BV; Volume: 281; Issue: 3 Linguagem: Inglês
10.1074/jbc.m505012200
ISSN1083-351X
AutoresRenu Batra, Karin Abarca-Heidemann, Heinz G. Körschen, Christos Tziatzios, Matthias Stoldt, Ivan L. Budyak, Dieter Willbold, Harald Schwalbe, Judith Klein‐Seetharaman, U. Benjamin Kaupp,
Tópico(s)bioluminescence and chemiluminescence research
ResumoThe 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. 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. Photoreceptors transduce the absorption of light into an electrical signal (for review see Ref. 1Pugh Jr., E.N. Lamb T.D. Stavenga D.G. DeGrip W.J. Pugh Jr., E.N. Handbook of Biological Physics. Elsevier Science Publishers B.V., Amsterdam2000: 183-255Google Scholar). The outer segment of vertebrate photoreceptors is a specialized compartment that hosts all the signaling components required for photoelectrical transduction. Rod photoreceptors harbor an unusual set of three glutamic acid-rich proteins (GARP) 2The abbreviations used are: GARPglutamic acid-rich proteinRSStokes radiusROSrod outer segmentsDTTdithiothreitolbis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolPDEphosphodiesteraseMALDI-TOFmatrix-assisted laser desorption ionization-time-of-flightNRMSDnormalized root mean square deviationBS3bis(sulfosuccinimidyl)suberateBMDB1,4-bismaleimidyl-2,3-dihydroxybutanePONDRprediction of natural disordered regionsIUPintrinsically unstructured proteinrGARPrecombinant GARPGCguanylyl cyclase. 2The abbreviations used are: GARPglutamic acid-rich proteinRSStokes radiusROSrod outer segmentsDTTdithiothreitolbis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolPDEphosphodiesteraseMALDI-TOFmatrix-assisted laser desorption ionization-time-of-flightNRMSDnormalized root mean square deviationBS3bis(sulfosuccinimidyl)suberateBMDB1,4-bismaleimidyl-2,3-dihydroxybutanePONDRprediction of natural disordered regionsIUPintrinsically unstructured proteinrGARPrecombinant GARPGCguanylyl cyclase. (see Fig. 1A) as follows: two soluble forms, GARP1 and GARP2 (2Colville C.A. Molday R.S. J. Biol. Chem. 1996; 271: 32968-32974Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 3Körschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.-W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (117) Google Scholar, 4Sugimoto Y. Yatsunami K. Tsujimoto M. Khorana H.G. Ichikawa A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3116-3119Crossref PubMed Scopus (45) Google Scholar), and a third form, which represents the cytoplasmic N-terminal domain (GARP′ part, almost identical in sequence to GARP1) of the B1 subunit of the cGMP-gated ion channel (5Körschen H.G. Illing M. Seifert R. Sesti F. Williams A. Gotzes S. Colville C. Müller F. Dosé A. Godde M. Molday L. Kaupp U.B. Molday R.S. Neuron. 1995; 15: 627-636Abstract Full Text PDF PubMed Scopus (207) Google Scholar). The B1 subunit and GARP1/GARP2 are derived from a single gene by alternative promoters and splicing (6Ardell M.D. Makhija A.K. Oliveira L. Miniou P. Viegas-Péquignot E. Pittler S.J. Genomics. 1995; 28: 32-38Crossref PubMed Scopus (20) Google Scholar, 7Ardell M.D. Aragon I. Oliveira L. Porche G.E. Burke E. Pittler S.J. FEBS Lett. 1996; 389: 213-218Crossref PubMed Scopus (28) Google Scholar, 8Ardell M.D. Bedsole D.L. Schoborg R.V. Pittler S.J. Gene (Amst.). 2000; 245: 311-318Crossref PubMed Scopus (37) Google Scholar). GARPs are characterized by their extremely high content of glutamate residues (∼150 residues in GARP1 and in the GARP′ part) and repetitive sequence motifs, in particular four short repeats designated R1–R4 (2Colville C.A. Molday R.S. J. Biol. Chem. 1996; 271: 32968-32974Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 3Körschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.-W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (117) Google Scholar, 5Körschen H.G. Illing M. Seifert R. Sesti F. Williams A. Gotzes S. Colville C. Müller F. Dosé A. Godde M. Molday L. Kaupp U.B. Molday R.S. Neuron. 1995; 15: 627-636Abstract Full Text PDF PubMed Scopus (207) Google Scholar) (Fig. 1A). The highest number of glutamate residues is found in a 110-amino acid-long segment toward the C terminus containing 61% glutamate residues, present in GARP1 and GARP′ but not in GARP2. Nonetheless, GARP2 contains approximately twice the number of glutamate residues than typical globular proteins (see supplemental table). GARPs probably serve a function specific to rods, because the GARP′ part is lacking in splice variants of the rod B1 subunit expressed in olfactory sensory neurons (9Sautter A. Zong X. Hofmann F. Biel M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4696-4701Crossref PubMed Scopus (106) Google Scholar, 10Bönigk W. Bradley J. Müller F. Sesti F. Boekhoff I. Ronnett G.V. Kaupp U.B. Frings S. J. Neurosci. 1999; 19: 5332-5347Crossref PubMed Google Scholar) and testes (11Wiesner B. Weiner J. Middendorff R. Hagen V. Kaupp U.B. Weyand I. J. Cell Biol. 1998; 142: 473-484Crossref PubMed Scopus (165) Google Scholar). Furthermore, the B3 subunit of the cGMP-gated channel of cone photoreceptors, which is encoded by a different gene, has no GARP-related sequences (12Gerstner A. Zong X. Hofmann F. Biel M. J. Neurosci. 2000; 20: 1324-1332Crossref PubMed Google Scholar), and soluble GARPs are absent in cones.GARPs have been proposed to organize an oligomeric protein complex near the cGMP-gated channel (3Körschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.-W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (117) Google Scholar) and to interact with peripherin (13Poetsch A. Molday L.L. Molday R.S. J. Biol. Chem. 2001; 276: 48009-48016Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), a protein located at the disc rim (14Molday R.S. Hicks D. Molday L. Investig. Ophthalmol. Vis. Sci. 1987; 28: 50-61PubMed Google Scholar). The tethering of the GARP′ part to peripherin is expected to produce a circular arrangement of cGMP-gated channels in juxtaposition to the disc rim (13Poetsch A. Molday L.L. Molday R.S. J. Biol. Chem. 2001; 276: 48009-48016Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 15Kaupp U.B. Seifert R. Physiol. Rev. 2002; 82: 769-824Crossref PubMed Scopus (917) Google Scholar, 16Kaupp U.B. Tränkner D. Frings S. Bradley J. Transduction Channels in Sensory Cells. Wiley-VCH, Weinheim2004: 209-235Google Scholar). The distance between the plasma membrane and the disc rim is ∼10 nm (17Roof D.J. Heuser J.E. J. Cell Biol. 1982; 95: 487-500Crossref PubMed Scopus (140) Google Scholar). However, if the GARP′ part adopts a globular shape with a calculated diameter of 6.6 nm, this would be too small to reach for peripherin across the 10-nm gap. Either the GARP′ part adopts an elongated structure or additional proteins, including soluble GARPs, may help to fill that gap.Here we have studied the structure and hydrodynamic properties of GARP1 and GARP2 by various biophysical techniques. We identify GARPs as members of a class of proteins that, in their native state, are intrinsically unfolded. The unstructured GARP′ part of B1 probably serves as an elongated tether that secures the cGMP-gated channel to the disc rim and thus provides for a unique geometric arrangement of the channel.EXPERIMENTAL PROCEDURESCalculation 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 R values of GARP1 and 2 were calculated using the program ProtParam at the EXPASY server (/www.expasy.org/tools). The mean hydrophobicity (H) is the sum of normalized hydrophobicities of individual residues divided by the total number of amino acid residues minus 4 residues (to take into account the fringe effects in the calculation of hydrophobicity). Individual hydrophobicities were determined using the Protscale program at the EXPASY server, selecting the option "Hphob/Kyte and Doolittle," a window size of 5, and a normalized scale from 0 to 1. HBoundary (Hb) was computed as described by Uversky (18Uversky V.N. Protein Sci. 2002; 11: 739-756Crossref PubMed Scopus (1492) Google Scholar): Hb = (R + 1.15)/2.785.Secondary Structure Predictions—Secondary structure predictions of repeat peptides (R1–R4) and proteins (GARP1 and GARP2) were carried out using JPRED, PredictProtein, nnPredict, APSSP, and AGADIR available on the EXPASY server.PONDR Prediction of GARP Proteins—Protein sequences were submitted to the PONDR server (www.pondr.com) using the neural network predictor VL-XT (19Li X. Romero P. Rani M. Dunker A.K. Obradovic Z. Genome Inform. Ser. Workshop Genome Inform. 1999; 10: 30-40PubMed Google Scholar, 20Romero P. Obradovic Z. Li X. Garner E.C. Brown C.J. Dunker A.K. Proteins. 2001; 42: 38-48Crossref PubMed Scopus (1330) Google Scholar). Access to PONDR® was provided by Molecular Kinetics (Indianapolis, IN).Purification of GARP1 and GARP2—Rod outer segments (ROS) were prepared from dark-adapted retina as described elsewhere (21Schnetkamp P.P.M. Daemen F.J.M. Methods Enzymol. 1982; 81: 110-116Crossref PubMed Scopus (63) Google Scholar). Unless specified, all steps were performed at 4 °C under dim red light. All buffers, except for gel filtration, contained the protease inhibitor mixtures mPIC (Sigma) and Complete (Roche Applied Science) according to the manufacturers' instructions. The rhodopsin content of ROS was determined spectrophotometrically from the absorption at 500 nm using an extinction coefficient of ϵ = 40,600 cm–1 m–1. ROS were suspended in isotonic buffer (20 mm bis-Tris/propane (BTP)), pH 7.4, 120 mm KCl, 0.2 mm MgCl2, 2 mm DTT, to a rhodopsin concentration of 4 mg/ml. The suspension was homogenized and diluted in isotonic buffer to a final rhodopsin content of 1 mg/ml. Additional MgCl2 was added to a final concentration of 2 mm. Membranes were recovered by centrifugation for 20 min at 100,000 × g and washed two more times. Under these conditions, GARPs, transducin, and PDE remained in the membrane fraction, whereas most of the other soluble proteins were removed. The membrane pellets were resuspended (1 mg/ml rhodopsin) in hypotonic buffer (5 mm Tris-HCl, pH 7.4, 0.2 mm MgCl2, 5 mm DTT) and illuminated for 5 min. The soluble fraction (containing GARP1, GARP2, and PDE) was separated from the membranes by centrifugation for 20 min at 314,000 × g. The pellet was washed twice and stored at 4 °C.The pooled supernatants of the hypotonic washings were loaded onto a 5-ml TSK heparin column (TosoHaas, Frankfurt, Germany) equilibrated with hypotonic buffer. The column was washed at 1 ml/min with hypotonic buffer until the absorbance reached the base line. GARP1 and -2 bound weakly to the column and eluted when the buffer was changed to TSK-buffer A (25 mm BTP, pH 7.4, 1 mm MgCl2, and 1 mm DTT). After washing with TSK-buffer A, PDE and other proteins were eluted in a gradient of 0–60% TSK-buffer B (TSK-buffer A containing 1 m NaCl). The proteins were collected in 1.5-ml fractions (GARP) or in 3-ml fractions (PDE) and checked by SDS-PAGE with Coomassie Blue staining and Western blotting. GARP1 and -2 eluted from the heparin column were finally separated on a Superdex-200 HiLoad 16/60 column (Amersham Biosciences) with a flow rate of 1 ml/min (20 mm BTP, pH 7.4, 130 mm NaCl, 1 mm MgCl2, 1 mm DTT). GARP1- and GARP2-containing fractions were pooled and concentrated to ∼2 mg/ml using Centriplus 30 or Centriplus 10 spin columns (Millipore), respectively. The purity of the samples was analyzed by Coomassie staining. The protein standards used for the calibration of the Superdex columns were as follows: thyroglobulin 669 kDa, ferritin 440 kDa, catalase 232 kDa, aldolase 158 kDa, ovalbumin 44 kDa, and chymotrypsinogen 25 kDa. Blue dextran (2000 kDa) was used to determine the void volume of the column.Coomassie Staining of SDS-PAGE—SDS-polyacrylamide gels were stained overnight by a solution containing 30% (v/v) ethanol, 10% (v/v) acetic acid, and 1% (w/v) Coomassie Brilliant Blue R-250. Destaining was performed by using the same solution without the dye. Under these conditions at least 50 ng of most proteins are visible.Construction and Purification of Recombinant GARP2 Expressed in Escherichia coli—Recombinant GARP2 (rGARP2) was expressed in E. coli strain BL21 (DE3) pLysE (Novagen) as His-tagged fusion protein using the pET30a vector. Cells were resuspended and sonicated (3 × 10 s) in 10 mm Na+ phosphate, pH 7.0. After addition of DNase (1,000 units/liter culture) and 2 mm MgCl2, the suspension was incubated on ice for 15 min and then centrifuged at 60,000 × g. The supernatant, containing recombinant GARP2, was adjusted to binding buffer (20 mm Na+ phosphate, pH 7.0, 35 mm imidazole, 2% glycerol, and 500 mm NaCl) and loaded onto a CoCl2-activated nitrilotriacetic acid-HiTrap column (Amersham Biosciences). The column was washed with 10 volumes of binding buffer and then with 10 volumes of binding buffer containing 100 mm imidazole. Recombinant GARP2 was eluted with binding buffer containing 500 mm imidazole and further purified by size-exclusion chromatography using a Superdex 200 column (Amersham Biosciences).Mass Spectrometry—MALDI-TOF mass spectra of purified native GARP2 samples were obtained by using a MALDI-TOF mass spectrometer (Voyager System 4197, PerSeptive Biosystems Inc., Framingham, MA). The instrument was operated in a linear mode (25-kV acceleration, 93% grid, 0.15% guide wire, and 200-MHz digitizer) and employed delayed extraction (320 ns). The m/z scale was calibrated using a mixture of known protein samples as follows: apomyoglobin (16.95 kDa), thioredoxin (11.67 kDa), and their respective dimers. Samples (16–20 μg) were mixed with a saturated solution (10 mg/ml) of sinapinic acid matrix (1:1 v/v) and then mixed with acetonitrile containing 0.1% (w/v) trifluoroacetic acid. The samples were deposited on the probe, air-dried, and inserted into the instrument. Spectra arising from 50 to 100 laser shots were averaged, and a 19-point smoothing of data were utilized for protein samples.Dynamic Light Scattering—Measurements were made with a DynaPro-MS/X (Protein Solutions Inc., Lakewood, NJ) at 20 °C using ∼47 μl of 0.5 mg of protein/ml of gel filtration buffer. All samples were either filtered (0.2 μm; Whatman) or centrifuged (5000 × g, 5 min) prior to the measurements. Diffusion coefficients were obtained from the analysis of the decay of the scattered intensity autocorrelation function. The hydrodynamic radius could be deduced from the diffusion coefficients using the Stokes-Einstein equation. All calculations were performed using the software Dynamics V6 provided by the manufacturer.Quantification of GARPs in ROS—The amount of GARP proteins relative to each other and to other proteins in the ROS (rhodopsin, PDE) was determined by SDS-PAGE and densitometric image analysis of Western blots on a Kodak Image Station. Three different ROS preparations were used. We performed the Western blot analysis using a polyclonal "anti-GARP" antibody (3Körschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.-W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (117) Google Scholar) that labels the following three bands: namely the B1 subunit of the cGMP-gated channel, GARP1, and GARP2. The amount of rhodopsin in ROS preparations was determined spectrophotometrically (22Moritz O.L. Molday R.S. Investig. Ophthalmol. Vis. Sci. 1996; 37: 352-362PubMed Google Scholar). The samples covered a concentration range of 0.1–0.7 μg of rhodopsin. The intensities of bands belonging to each GARP protein in the Western blots were determined densitometrically. The ratio of GARP proteins is a result of two independent experiments performed in duplicate using three different ROS preparations (total of 12 values).NMR Spectroscopy—NMR samples contained 0.125 mm purified rGARP2 protein in 10 mm Na+ phosphate, 120 mm NaCl, pH 5.2 or 6.8, and 95% H2O, 5% D2O. NMR spectra were recorded at 298 K on a Varian Unity INOVA spectrometer operating at a 600-MHz proton frequency equipped with a 5-mm triple resonance probe and z axis pulsed field gradient. Suppression of the water resonance was achieved through the WATERGATE technique (23Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3501) Google Scholar). For each spectrum a sum of 128 transients was accumulated to achieve a good signal-to-noise ratio. Chemical shifts were referenced against the methyl signal of external sodium 2,2-dimethyl-2-silapentane-5-sulfonate. Data were processed and plotted using the VnmrJ software (Varian Inc., Palo Alto, CA). Natural abundance 1H, 15N-HSQC experiments were measured using standard pulse sequences at 600- and 700-MHz with Bruker spectrometers, each equipped with a 5-mm triple resonance cryoprobe and z axis pulsed field gradients. Spectra were acquired at 298 K in 90% H2O, 10% D2O, pH 3. The concentration of the peptides was 1.5 mm in each case. 1H chemical shifts were referenced to sodium 3-trimethylsilyl-2,2,3,3,-d4-proprionate at 0.00 ppm, and 13C and 15N chemical shifts were calculated from the 1H frequency (24Wishart D.S. Bigam C.G. Yao J. Abildgaard F. Dyson H.J. Oldfield E. Markley J.L. Sykes B.D. J. Biomol. NMR. 1995; 6: 135-140Crossref PubMed Scopus (2053) Google Scholar). HSQC type experiments were obtained and processed using XWINNMR 3.0 (Bruker Inc.) and TOP-SPIN 1.3b (Bruker Inc.).Circular Dichroism Spectroscopy—CD spectra were recorded with a Jasco J810 spectropolarimeter. 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-β-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. Spectra were analyzed using the Dichroweb on-line server (25Whitmore L. Wallace B.A. Nucleic Acids Res. 2004; 32: 668-673Crossref PubMed Scopus (1967) Google Scholar, 26Lobley A. Whitmore L. Wallace B.A. Bioinformatics. 2002; 18: 211-212Crossref PubMed Scopus (643) Google Scholar). Reference data sets 4 and 7 (27Sreerama N. Woody R.W. Anal. Biochem. 2000; 287: 252-260Crossref PubMed Scopus (2478) Google Scholar, 28Sreerama N. Venyaminov S.Y. Woody R.W. Anal. Biochem. 2000; 287: 243-251Crossref PubMed Scopus (502) Google Scholar) were used, which are compatible with the wavelength range used in this study. To test the reliability of the secondary structure determinations, a number of alternative algorithms were used for the structure calculations as follows: SELCON3 (29Sreerama N. Woody R.W. Anal. Biochem. 1993; 209: 32-44Crossref PubMed Scopus (942) Google Scholar, 30Sreerama N. Venyaminov S.Y. Woody R.W. Protein Sci. 1999; 8: 370-380Crossref PubMed Scopus (637) Google Scholar), CONTIN (31Provencher S.W. Glockner J. Biochemistry. 1981; 20: 33-37Crossref PubMed Scopus (1868) Google Scholar, 32Van Stokkum I.H.M. Spoelder H.J.W. Bloemendal M. Van Grondelle R. Groen F.C.A. Anal. Biochem. 1990; 191: 110-118Crossref PubMed Scopus (432) Google Scholar), and CDSSTR (27Sreerama N. Woody R.W. Anal. Biochem. 2000; 287: 252-260Crossref PubMed Scopus (2478) Google Scholar, 33Compton L.A. Johnson Jr., W.C. Anal. Biochem. 1986; 155: 155-167Crossref PubMed Scopus (480) Google Scholar, 34Manavalan P. Johnson Jr., W.C. Anal. Biochem. 1987; 167: 76-85Crossref PubMed Scopus (660) Google Scholar). The analysis provided calculated secondary structures and the goodness-of-fit parameter, normalized root mean square deviation (NRMSD). The NRMSD parameter (25Whitmore L. Wallace B.A. Nucleic Acids Res. 2004; 32: 668-673Crossref PubMed Scopus (1967) Google Scholar) is defined as follows: Σ(θexp – θcal)2/(θexp)2)1/2 summed over all wavelengths, where θexp and θcal are the experimental ellipticities and the ellipticities of the back-calculated spectra, respectively. High NRMSD values (>0.1) indicate that the back-calculated and experimental spectra are not in good agreement (25Whitmore L. Wallace B.A. Nucleic Acids Res. 2004; 32: 668-673Crossref PubMed Scopus (1967) Google Scholar). However, a low NRMSD value is not always sufficient to indicate an accurate analysis. Because Dichroweb defines the NMRSD parameter in the same way for all analyses, the NMRSD parameter provides a direct means to compare the results obtained using different data bases and algorithms and in the selection of the most appropriate reference data set for the relevant protein. The NMRSD values obtained in this study were all well below 0.1.Peptide Synthesis—Peptide sequences corresponding to repeats R1–R4 in GARP sequences were synthesized according to standard protocols (72Pennington M.W. Dunn B.M. Peptide Synthesis Protocols. 35. Humana Press Inc., Totowa, NJ1994Crossref Google Scholar). R1 (residues 1–14) corresponds to MLGWVQRVLPQPPG; R2 (residues 100–117) corresponds to VLTWLRKGVEKVVPQPAH; R3 (residues 167–184) corresponds to LLRWFEQNLEKMLPQPPK; and R4 (residues 255–271) corresponds to LMAWILHRLEMALPQPV.Chemical Cross-linking of Purified GARP2 Protein—Native GARP2 was purified as described above. The purified protein (100 ng/ml) was adapted to cross-linking conditions (10 mm Hepes/KOH, pH 7.4, 150 mm NaCl, 2 mm MgCl2, 1 mm CaCl2, 1 mm tris(2-carboxyethyl)phosphine hydrochloride), and the cross-linking reaction was carried out by adding 10× stock solution of the amino-specific cross-linker bis(sulfosuccinimidyl)suberate (BS3) or the thiol-specific cross-linker 1,4-bismaleimidyl-2,3-dihydroxybutane (BMDB) at room temperature (35Weitz D. Ficek N. Kremmer E. Bauer P.J. Kaupp U.B. Neuron. 2002; 36: 881-889Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Final concentrations of the cross-linkers BS3 and BMDB in the reactions were 0.5 mm and 2.5 μm, respectively. Intermediate cross-link products were identified at various times by termination of the reaction with SDS sample buffer. Cross-link products were analyzed by SDS-PAGE and Western blot analysis (35Weitz D. Ficek N. Kremmer E. Bauer P.J. Kaupp U.B. Neuron. 2002; 36: 881-889Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar).Analytical Ultracentrifugation—Experiments were performed in a Beckman Optima XL-A ultracentrifuge, using an An-50Ti rotor at a temperature of 4 °C. Absorbance versus radius data, A(r) or A(r,t), were recorded at 280 nm. For the sedimentation equilibrium experiments, Epon 6-channel cells with a path length of 1.2 cm were used with sample and reference volumes of 130 and 135 μl, respectively. The experimental A(r) profiles were evaluated as described earlier (36Schubert D. Schuck P. Prog. Colloid Polym. Sci. 1991; 86: 12-22Crossref Google Scholar, 37Schuck P. Legrum B. Passow H. Schubert D. Eur. J. Biochem. 1995; 230: 806-812Crossref PubMed Scopus (27) Google Scholar, 38Musco G. Tziatzios C. Schuck P. Pastore A. Biochemistry. 1995; 34: 553-561Crossref PubMed Scopus (13) Google Scholar), using the computer program DISCREEQ developed by Schuck (39Schuck P. Prog. Colloid Polym. Sci. 1994; 94: 1-13Crossref Google Scholar). Sedimentation velocity runs used Epon double sector cells of 1.2 cm optical length. Sample and reference volumes were 380 and 400 μl, respectively. The experimental A(r,t) data were analyzed by the program SEDFIT applying direct boundary modeling with distribution of Lamm equation solutions (40Schuck P. Biophys. J. 2000; 78: 1606-1619Abstract Full Text Full Text PDF PubMed Scopus (3005) Google Scholar, 41Lebowitz J. Lewis M.S. Schuck P. Protein Sci. 2002; 11: 2067-2079Crossref PubMed Scopus (614) Google Scholar). The partial specific volume, v̄, 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. 42Durchschlag H. Hinz H.J. Thermodynamic Data for Biochemistry and Biotechnology. Springer-Verlag, Berlin1986: 45-128Crossref Google Scholar. This led to v̄ = 0.722 ml/g for GARP2 and v̄ = 0.702 ml/g for GARP1. The densities and viscosities of the buffers were calculated using the program SEDNTERP (43Laue T.M. Shah B.D. Ridgeway T.M. Pelletier S.L. Harding S.E. Rowe A.J. Horton J.C. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge, UK1992: 90-125Google Scholar).RESULTSRationale—GARP proteins display abnormally slow migration in SDS-PAGE (2Colville C.A. Molday R.S. J. Biol. Chem. 1996; 271: 32968-32974Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 3Körschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.-W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (117) Google Scholar, 5Körschen H.G. Illing M. Seifert R. Sesti F. Williams A. Gotzes S. Colville C. Müller F. Dosé A. Godde M. Molday L. Kaupp U.B. Molday R.S. Neuron. 1995; 15: 627-636Abstract Full Text PDF PubMed Scopus (207) Google Scholar, 13Poetsch A. Molday L.L. Molday R.S. J. Biol. Chem. 2001; 276: 48009-48016Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). The apparent molecular mass (Mr) of GARP1 and GARP2 (∼130 and ∼62 kDa, respectively) is twice as large as that predicted by the amino acid sequence (64.5 and 31.9 kDa, respectively). The electrophoretic mobility of GARP proteins could be anomalously low for a number of different reasons. 1) Bands may represent dimers of GARP1 and GARP2, respectively. 2) The high content of glutamate residues may result in poor binding of SDS and, thereby, reduced mobility. 3) GARPs may adopt an unusual shape. Previous studies have provided evidence that GARP2 associates both with other retinal proteins (3Körschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.-W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (117) Google Scholar, 13Poetsch A. Molday L.L. Molday R.S. J. Biol. Chem. 2001; 276: 48009-48016Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) and with itself (3Körschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.-W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (117) Google Scholar), raising the possibility that GARPs serve as multivalent scaffolds for macromolecular signaling complexes. Therefore, we performed sequence analysis and studied the structural properties and the oligomeric state of native GARP1 and GARP2 purified from rod photoreceptors.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 (19Li X. Romero P. Rani M. Dunker A.K. Obradovic Z. Genome Inform. Ser. Workshop Genome Inform. 1999; 10: 30-40PubMed Google Scholar, 20Romero P. Obradovic Z. Li X. Garner E.C. Brown C.J. Dunker A.K. Proteins. 2001; 42: 38-48Crossref PubMed Scopus (1330) Google Scholar, 44Romero P. Obradovic Z. Kissinger C
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