The authors thank Dr. Beata Jastrzebska, Ph.D. (Department of Pharmacology, Case Western Reserve University, OH) for her advice on the rhodopsin absorbance protocol. This work was supported by an NIH-NEI grant (EY030889 and 3R01EY030889-03S1) and, in part, by the University of Minnesota start-up funds to G.P.L.

1. Surface plasmon resonance (SPR) methodology
<ol>
	<li>Preparation and purification of mouse RBP4 protein
	<ol>
		<li>Design primers to generate full-length mouse RBP4 (msRBP4) cDNA (NCBI Reference Sequence: NM_001159487.1). Clone the msRBP4 cDNA into a pET28a His-tag Kanamycin-resistant expression vector and express in BL-21 DE3 cells (Table of Materials).</li>
		<li>Transform the BL-21 DE3 competent cells (according to the manufacturer&#39;s protocol) with ligated msRBP4-pET28a vector and then plate onto Luria-Bertani (LB) broth Agar plates containing Kanamycin 50 &#181;g/mL30,37. Incubate the plates overnight at 37 &#176;C in an incubator and screen for positive colonies the next day.</li>
		<li>Pick a single colony and incubate overnight at 37 &#176;C in 5 mL of LB broth containing Kanamycin 50 &#181;g/mL.</li>
		<li>For secondary culture in 500 mL of LB broth containing Kanamycin 50 &#181;g/mL, use 1% volume of primary culture (from step 1.1.3.) and incubate at 37 &#176;C for 3-4 h; check the optical density using a spectrophotometer. Ideally, proceed if the OD value is between 0.6 and 1 to induce protein expression with 0.5 mM IPTG at 24 &#176;C overnight.</li>
		<li>Use multiple IPTG concentrations of 0.1-1 mM and 16-30 &#176;C temperature range to optimize msRBP4 expression and stability.</li>
		<li>Centrifuge the culture (from step 1.1.5.) at 16,000 &#215; g for 10 min, discard the supernatant, and keep the pellet on ice.</li>
		<li>Homogenize the pellet with lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% (v/v) Triton X-100, 10% Glycerol, 0.5 mM PMSF, and Protease Inhibitors [pH 8.0]). Sonicate the lysate for 15 s ON and 30 s OFF cycle using a homogenizer with a 250 Watt output and 40-50% 20 kHz output frequency. Repeat 5x.</li>
		<li>Remove the cell debris and unlysed cells by centrifugation at 16,000 &#215; g for 20 min. Pass the supernatant of the lysate through a buffer-equilibrated nickel NTA column.</li>
		<li>Wash the column with lysis buffer (from step 1.1.8) containing 30 mM imidazole.</li>
		<li>In fractions, elute the bound msRBP4 protein using buffer containing 250 mM imidazole. Run the eluted fractions and visually monitor the protein quantity with a 12% SDS polyacrylamide gel, stained with Coomassie Brilliant Blue R-250 in methanol:water:acetic acid (45:45:10) solution and de-stain with solution without Coomassie stain methanol:water:acetic acid (45:45:10).</li>
		<li>Pool the unused fractions and dialyze the pooled fractions with a 10K dialysis cassette of 10 kDa MWCO.</li>
		<li>Evaluate the quality of the dialyzed fraction on a 12% SDS-PAGE gel and quantify the protein concentration. 
		NOTE: Apo-RBP4 protein was used in the SPR analysis. For generating holo-RBP4 (RBP4-retinol), refer to detailed protocols published elsewhere36,37.</li>
	</ol>
	</li>
	<li>Initial validation of RBPR2 and STRA6 peptides with RBP4 for structure and interaction by Tryptophan fluorescence assay and circular dichroism (CD) spectroscopy
	<ol>
		<li>Tryptophan fluorescence assay
		<ol>
			<li>Use the online protein composition analysis tool (https://web.expasy.org/protparam/) and paste the amino acid sequence in one-letter code to assess the structural composition of recombinant RBP4 protein and the individual peptides of RBPR2 and STRA6 to determine the tryptophan amino acid residues in the individual peptides and protein (Table of Materials).</li>
			<li>Click on the compute parameters button and evaluate the amino acid composition for tryptophan (Trp) residues. 
			NOTE: Tryptophan residues must be present in the individual peptides or proteins being analyzed.</li>
			<li>Dilute the individual RBPR2 and STRA6 peptides in various micromolar concentrations and incubate individually with 3 &#181;g msRBP4 in a 96-well plate at room temperature for 5 min. Excite the mix at 290 nm and scan the emission from 300 nm to 400 nm wavelength.</li>
			<li>Normalize the data with the Blank (buffer only, no peptide and no protein) and peptide control condition (no protein) and plot. 
			NOTE: The tryptophan fluorescence increment upon exposure to a low to high peptide concentration suggests a positive interaction between the two individual peptides and RBP4 protein.</li>
		</ol>
		</li>
		<li>Circular dichroism (CD) spectroscopy
		<ol>
			<li>Dilute the individual peptides and recombinant RBP4 protein in 1x PBS buffer, pH 7.0. Use various concentrations of RBPR2 and STRA6 peptides and RBP4 protein from 0.1 to 10 &#956;g/mL for a quality check, as described below.</li>
			<li>Add individual diluted peptides and protein in a 0.1 cm path length cuvette and measure the CD absorbance/ellipticity using a CD spectrophotometer (195-250 nm). Use the below formula to calculate the mean residue ellipticity (&#952;). 
			[v] = (S &#215; mRw)/(10cl) 
			S represents the CD signal in m&#952;; mRw represents the mean residue mass; c represents the concentration of the protein in mg/mL; and l represents the path length in cm.</li>
			<li>Calculate the percent change in molecule structure using BeStSel Secondary Structure Analysis to Protein Fold Prediction by CD Spectroscopy (https://bestsel.elte.hu). 
			NOTE: The secondary structure indicates the proper folding and conserved confirmations crucial for the interaction study and the outcome of the interactions. Inspect the predicted structure of the domain and proceed further with the interaction study.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. SPR analysis to determine the binding affinities and kinetic parameters of vitamin A membrane receptors (RBPR2 and STRA6) with their physiological ligand RBP4
<ol>
	<li>For the immobilization of RBP4 on CM5 Chip surface setup, we use the SPR sensor chip, which carries a carboxymethyl dextran linker extended to a size of ~100 nm covalently attached on a gold surface. In the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) converts the carboxyl groups on the chip&#39;s surface to N-Hydroxysuccinimide esters, which spontaneously react with the amine groups of amino acids and proteins, forcing the ligand to bond covalently onto the chip&#39;s surface (Table of Materials).
	<ol>
		<li>Immobilize the purified msRBP4 protein on the Sensor chip (Figure 1 and Figure 2). In the software Run menu, choose the Wizard and proceed with the immobilization option. Dilute the ligand (msRBP4 protein obtained from step 1.1) in immobilization buffer (10 mM Sodium acetate buffer, pH 5.0) to 10 &#181;g/mL (4 &#181;L of ligand + 396 &#181;L of buffer) and aliquot the coupling reagents in vials. Open the Immobilization Setup wizard window from the software control, select CM5 Chip, and option Immobilize flow cell 2 and target level of 1,200 Response Unit (RU) for achieving Rmax of 30 RU in the kinetic study and Wash solution: Ethanolamine. Enter the name RBP4 and click Next. 
		NOTE: Optional: System Preparations dialog box for the Prime before run option is not required if priming with running buffer PBST (phosphate-buffered saline solution with a 0.05% Tween 20 detergent solution) was performed previously.</li>
		<li>In the Rack position dialog box, click Eject Rack. Take the rack and place the vials with assigned volumes for each reagent: 156 &#181;L of msRBP4, 177 &#181;L of ethanolamine, 89 &#181;L of EDC, 89 &#181;L of NHS, and an empty vial. Insert the rack with sample vials and click OK and Next.</li>
		<li>In the Prepare Run Protocol dialog, click Start and Save.</li>
	</ol>
	</li>
	<li>RBPR2 and STRA6 peptides kinetic assay setup
	<ol>
		<li>Serially dilute the mouse RBPR2, mutant RBPR2, and STRA6 peptides (chemically synthesized previously31 and listed in Table of Materials) in running buffer in a range of 0.8-26.6 &#956;M.</li>
		<li>Open the control software and select Kinetics/Affinity wizard. Click File | Open template and select Kinetics/Affinity. Input the injection sequence dialog flow path 2-1 (Flow cell 2 as active and Flow cell 1 as reference), check the Chip Type CM5, and click Next. In the setup dialog, check the Run startup cycles, enter Solution: buffer and cycles: and click Next.</li>
		<li>In the injection parameters dialog, enter the values for sample parameters: contact time: 120 s, flowrate 30 &#956;L/min, Dissociation time 300 s; for regeneration parameters: enter the Regeneration solution name: Glycine-HCl (pH 2.5), contact time: 30 s, flowrate: 30 &#956;L/min and click Next.</li>
		<li>In the samples dialog, enter the name of the peptides, molecular weight, and concentrations and click next. In the Rack position dialog, assign the diluted peptides as individual ~120 &#181;L samples of various concentrations 1 to 100 &#181;M and ~230 &#181;L of Buffer, ~120 &#181;L of Buffer, and ~1,000 &#181;L of Glycine, pH 2.5, to the positions on the Rack. Click Eject Rack and insert all the vials. Click Next, and in the Prepare Run Protocol dialog, click Start, enter a file name, and Save (Figure 2).</li>
		<li>In the software, click the Start menu and select Evaluation. Choose open the file in the software, and from the dropdown selection menu, choose Fc2-1 to analyze the Reference cell Blank-subtracted Active sensorgram for the association and dissociation phases of the msRBP4 and mutant msRBPR2 and msSTRA6 peptides.</li>
		<li>Click on the dropdown Kinetics/Affinity button and choose Surface bound; to open the Select Curves dialog, check include column in the table and click Next. Select Data dialog to show blank-subtracted sensorgrams; click Kinetics and Affinity, keep the default parameters, and click Fit option for curve fitting. Inspect the Report tab for equilibrium dissociation constant Kd and Ka or Kon association rate and Kd or Koff dissociation rate. Save the Kinetics data sensorgram in .txt tab delimited format and use the values to plot graphs (Figure 3).</li>
		<li>To determine the affinity kinetics constant Kd (ligand concentration that binds to half the receptors at equilibrium), copy and paste the data columns in the .txt format file generated from the Evaluation to XY plot used in the software. Click Analyze and Nonlinear regression (curve fit), check the data column, and click Ok. In the parameter dialog, click Binding saturation and One site Specific binding fitting model with the formula of: 
		Y= (Bmax*X)/(Kd + X) 
		Where Bmax is the maximum number of binding sites (Response Unit), X is the concentration of the peptides, and Y is the binding (Response unit) (Figure 3).</li>
	</ol>
	</li>
</ol>
3. Spectrophotometry methodology to quantify the GPCR-11- cis retinal protein complex (retinylidene protein rhodopsin) in retinal lysates
<ol>
	<li>Transport the predetermined group of mice consisting of adult WT-C57BL/6J mice to the dark room.</li>
	<li>Dark adapt the mice overnight for 12-16 h, and under red light, place the mouse in a euthanasia chamber and open the CO2 gas valve on the regulator to 5 L/min for ~5 min. Ensure death by applying pressure on the neck and tail pulled backward (cervical dislocation).</li>
	<li>Enucleate the eye globes and homogenize the eyes in an ice-cold buffer containing 20 mM bis-tris propane (1,3-bis (tris(hydroxymethyl)methylamino)propane; BTP) pH 7.4, 150 mM NaCl, 1 mM EDTA and with protease inhibitor (Table of Materials).</li>
	<li>Centrifuge the homogenate for 15 min at 16000 x g at 4 &#176;C, discard the supernatant containing water soluble cytosolic proteins. Re-suspend and solubilize the pelleted membrane and membrane proteins in buffer containing 20 mM n-dodecyl-&#946;-d-maltoside (DDM), 20 mM bis-tris propane (BTP) pH 7.4, 100 mM NaCl, 1 mM EDTA and with protease inhibitor for 1 h at 4 &#176;C on a rotating platform.</li>
	<li>Centrifuge the resolubilized membrane fraction for 1 h at 16000 x g, 4 &#176;C. Mix the supernatant with 30 &#956;L of Rho 1D4 antibody beads and incubate for 1 h at 4 &#176;C on a rotating platform.</li>
	<li>Using a magnetic stand, separate the pulldown using beads, discard the supernatant, and wash the beads with buffer 20 mM bis-tris propane (BTP) pH 7.4, 500 mM NaCl, and 2 mM DDM.</li>
	<li>Elute the rhodopsin protein by incubating for 5-10 min at room temperature with buffer 20 mM bis-tris propane (BTP) pH 7.4, 100 mM NaCl, 2 mM DDM containing 0.2 mg/mL 1D4 peptide (-TETSQVAPA) (Figure 4 and Figure 5).</li>
	<li>Analyze the elute for absorbance from 200 nm to 800 nm using a rectangular, sub-micro, quartz, 2 mm aperture, 10 mm path length, 50 &#956;L cuvette in a fast scan setting with aspectrophotometer. 
	NOTE: Use only high-quality Quartz cuvettes (Figure 4).</li>
	<li>To validate and determine quality, check and expose the elute to bright light for 1 min, then repeat step 3.8.</li>
	<li>Subtract the Blank absorbance and calculate the ratio of the analyzed absorbance spectra with absorbance at 500 nm for 11-cis retinal bound opsin, 380 nm for light-exposed all-trans retinal bound opsin, and 280 nm for ligand-free Apo-Opsin protein (Figure 6). The free opsin with no ligand retinal gives a baseline absorbance at 500 nm.</li>
	<li>To quantify rhodopsin concentration, use Beer-Lambert&#39;s Law: 
	A=&#949;&#183;C&#183;l or C=A/&#949;&#183;l 
	Where A is the absorbance, &#949; the molar extinction coefficient (rhodopsin &#949;500 = 40,600 M&#8722;1 cm&#8722;1 for free Apo Opsin &#949;280 = 81,200 M&#8722;1 cm&#8722;1), C is the concentration, and l path length.</li>
	<li>For quantifying liganded-Opsin in percentage, use a ratio of A280/A500.</li>
	<li>Plot the Blank-subtracted absorbance spectra and calculate the free opsin value32.</li>
	<li>Calculate the concentration of rhodopsin using the extinction coefficient &#949;500 = 40600 M&#8722;1 cm&#8722;1. The concentration of ligand-free opsin is calculated with Beer-Lambert Law using the extinction coefficient &#949;280 = 81,200 M&#8722;1 cm&#8722;1.</li>
</ol>

Investigating Vitamin A Receptor Kinetics and Function for Retinoid Homeostasis

<ol>
	<li>Dowling, J. E., Wald, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+A+deficiency+and+night+blindness.">Vitamin A deficiency and night blindness.</a> Proc Natl Acad Sci U S A. 44 (7), 648-6618 (1958).</li><li>Dowling, J. E., Wald, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biological+function+of+vitamin+A+acid.">The biological function of vitamin A acid.</a> Proc Natl Acad Sci U S A. 46 (5), 587-608 (1960).</li><li>Kiser, P. D., Palczewski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoids+and+retinal+diseases.">Retinoids and retinal diseases.</a> Annu Rev Vis Sci. 2 (1), 197-234 (2016).</li><li>Harrison, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+involved+in+the+intestinal+absorption+of+dietary+vitamin+A+and+provitamin+A+carotenoids.">Mechanisms involved in the intestinal absorption of dietary vitamin A and provitamin A carotenoids.</a> Biochim Biophys Acta. 1821 (1), 70-77 (2012).</li><li>Harrison, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carotenoids,+&#946;-apocarotenoids,+and+retinoids:+the+long+and+the+short+of+it.">Carotenoids, &#946;-apocarotenoids, and retinoids: the long and the short of it.</a> Nutrients. 14 (7), 1411 (2022).</li><li>Li, Y., Wongsiriroj, N., Blaner, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multifaceted+nature+of+retinoid+transport+and+metabolism.">The multifaceted nature of retinoid transport and metabolism.</a> Hepatobiliary Surg Nutr. 3 (3), 126-139 (2014).</li><li>O'Byrne, S. M., Blaner, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinol+and+retinyl+esters:+biochemistry+and+physiology.">Retinol and retinyl esters: biochemistry and physiology.</a> J Lipid Res. 54 (7), 1731-1743 (2013).</li><li>Blomhoff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+and+metabolism+of+vitamin+A.">Transport and metabolism of vitamin A.</a> Nutr Rev. 52 (2 Pt 2), S13-S23 (2009).</li><li>Chelstowska, S., Widjaja-Adhi, M. A. K., Silvaroli, J. A., Golczak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+for+vitamin+A+uptake+and+Storage+in+Vertebrates.">Molecular basis for vitamin A uptake and Storage in Vertebrates.</a> Nutrients. 8 (11), 676 (2016).</li><li>Martin Ask, N., Leung, M., Radhakrishnan, R., Lobo, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+A+transporters+in+visual+function:+A+mini+review+on+membrane+receptors+for+dietary+vitamin+A+uptake,+storage,+and+transport+to+the+eye.">Vitamin A transporters in visual function: A mini review on membrane receptors for dietary vitamin A uptake, storage, and transport to the eye.</a> Nutrients. 13 (11), 3987 (2021).</li><li>Yamamoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+transthyretin+(TTR)+and+retinol-binding+protein+(RBP)+in+the+uptake+of+retinol+by+primary+rat+hepatocytes.">Interactions of transthyretin (TTR) and retinol-binding protein (RBP) in the uptake of retinol by primary rat hepatocytes.</a> Exp Cell Res. 234 (2), 373-378 (1997).</li><li>Chen, C. C., Heller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+of+retinol+and+retinoic+acid+from+serum+retinol-binding+protein+by+retinal+pigment+epithelial+cells.">Uptake of retinol and retinoic acid from serum retinol-binding protein by retinal pigment epithelial cells.</a> J Biol Chem. 252 (15), 5216-5221 (1977).</li><li>Gj&#248;en, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+takes+up+retinol-binding+protein+from+plasma.">Liver takes up retinol-binding protein from plasma.</a> J Biol Chem. 262 (23), 10926-10930 (1987).</li><li>Blomhoff, R., Norum, K. R., Berg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+uptake+of+[3H]retinol+bound+to+the+serum+retinol+binding+protein+involves+both+parenchymal+and+perisinusoidal+stellate+cells.">Hepatic uptake of [3H]retinol bound to the serum retinol binding protein involves both parenchymal and perisinusoidal stellate cells.</a> J Biol Chem. 260 (25), 13571-13575 (1985).</li><li>Kanai, M., Raz, A., Goodman, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinol-binding+protein:+The+transport+protein+for+vitamin+A+in+human+plasma.">Retinol-binding protein: The transport protein for vitamin A in human plasma.</a> J Clin Invest. 47 (9), 2025-2044 (1968).</li><li>Quadro, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+extrahepatic+retinol+binding+protein+in+the+mobilization+of+retinoid+stores.">The role of extrahepatic retinol binding protein in the mobilization of retinoid stores.</a> J Lipid Res. 45 (11), 1975-1982 (2004).</li><li>Blaner, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STRA6,+a+cell-surface+receptor+for+retinol-binding+protein:+The+plot+thickens.">STRA6, a cell-surface receptor for retinol-binding protein: The plot thickens.</a> Cell Metab. 5 (3), 164-166 (2007).</li><li>Kawaguchi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+receptor+for+retinol+binding+protein+mediates+cellular+uptake+of+vitamin+A.">Membrane receptor for retinol binding protein mediates cellular uptake of vitamin A.</a> Science. 315 (5813), 820-825 (2007).</li><li>Kawaguchi, R., Zhong, M., Kassai, M., Ter-Stepanian, M., Sun, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STRA6-catalyzed+vitamin+A+influx,+Efflux,+and+Exchange.">STRA6-catalyzed vitamin A influx, Efflux, and Exchange.</a> J. Membr. Biol. 245 (11), 731-745 (2012).</li><li>Pasutto, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+STRA6+cause+a+broad+spectrum+of+malformations+including+anophthalmia,+congenital+heart+defects,+diaphragmatic+hernia,+alveolar+capillary+dysplasia,+lung+hypoplasia,+and+mental+retardation.">Mutations in STRA6 cause a broad spectrum of malformations including anophthalmia, congenital heart defects, diaphragmatic hernia, alveolar capillary dysplasia, lung hypoplasia, and mental retardation.</a> Am J Hum Genet. 80 (3), 550-560 (2007).</li><li>Golzi, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matthew-Wood+Syndrome+is+caused+by+truncating+mutations+in+the+retinol-binding+protein+receptor+gene+STRA6.">Matthew-Wood Syndrome is caused by truncating mutations in the retinol-binding protein receptor gene STRA6.</a> Am J Hum Genet. 80 (14), 1179-1187 (2007).</li><li>Isken, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RBP4+disrupts+vitamin+A+uptake+homeostasis+in+a+STRA6-deficient+animal+model+for+Matthew-Wood+Syndrome.">RBP4 disrupts vitamin A uptake homeostasis in a STRA6-deficient animal model for Matthew-Wood Syndrome.</a> Cell Metab. 7 (3), 258-268 (2008).</li><li>Ruiz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoid+content,+visual+responses,+and+ocular+morphology+are+compromised+in+the+retinas+of+mice+lacking+the+retinol-binding+protein+receptor,+STRA6.">Retinoid content, visual responses, and ocular morphology are compromised in the retinas of mice lacking the retinol-binding protein receptor, STRA6.</a> Investig Opthalmol Vis Sci. 53 (6), 3027-3039 (2012).</li><li>Kelly, M., von Lintig, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STRA6:+Role+in+cellular+retinol+uptake+and+efflux.">STRA6: Role in cellular retinol uptake and efflux.</a> Hepatobiliary Surg Nutr. 4 (4), 229 (2015).</li><li>Berry, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+STRA6+receptor+is+essential+for+retinol-binding+protein-induced+insulin+resistance+but+not+for+maintaining+vitamin+A+homeostasis+in+tissues+other+than+the+eye.">The STRA6 receptor is essential for retinol-binding protein-induced insulin resistance but not for maintaining vitamin A homeostasis in tissues other than the eye.</a> J Biol Chem. 288 (34), 24528-24539 (2013).</li><li>Amengual, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STRA6+is+critical+for+cellular+vitamin+A+uptake+and+homeostasis.">STRA6 is critical for cellular vitamin A uptake and homeostasis.</a> Hum Mol Genet. 23 (20), 5402-5417 (2014).</li><li>Steinhoff, J. S., Lass, A., Schupp, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoid+homeostasis+and+beyond:+How+retinol+binding+protein+4+contributes+to+health+and+disease.">Retinoid homeostasis and beyond: How retinol binding protein 4 contributes to health and disease.</a> Nutrients. 14 (6), 1236 (2022).</li><li>Alapatt, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+retinol+transporter+and+receptor+for+serum+retinol-binding+protein+(RBP4).">Liver retinol transporter and receptor for serum retinol-binding protein (RBP4).</a> J Biol Chem. 288 (2), 1250-1265 (2013).</li><li>Radhakrishnan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+lacking+the+systemic+vitamin+A+receptor+RBPR2+show+decreased+ocular+retinoids+and+loss+of+visual+function.">Mice lacking the systemic vitamin A receptor RBPR2 show decreased ocular retinoids and loss of visual function.</a> Nutrients. 14 (12), 2371 (2022).</li><li>Solanki, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+binding+domain+in+the+Rbpr2+receptor+is+required+for+vitamin+A+transport,+ocular+retinoid+homeostasis,+and+photoreceptor+cell+survival+in+zebrafish.">A functional binding domain in the Rbpr2 receptor is required for vitamin A transport, ocular retinoid homeostasis, and photoreceptor cell survival in zebrafish.</a> Cells. 9 (5), 1099 (2020).</li><li>Radhakrishnan, R., Leung, M., Solanki, A. K., Lobo, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+the+extracellular+RBP4+ligand+binding+domain+on+the+RBPR2+receptor+for+vitamin+A+transport.">Mapping of the extracellular RBP4 ligand binding domain on the RBPR2 receptor for vitamin A transport.</a> Front Cell Dev Biol. 11 (11), 1105657 (2023).</li><li>Ramkumar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vitamin+A+transporter+STRA6+adjusts+the+stoichiometry+of+chromophore+and+opsins+in+visual+pigment+synthesis+and+recycling.">The vitamin A transporter STRA6 adjusts the stoichiometry of chromophore and opsins in visual pigment synthesis and recycling.</a> Hum Mol Genet. 31 (4), 548-560 (2022).</li><li>Tian, H., Sakmar, T. P., Huber, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+energetics+of+chromophore+binding+in+the+visual+photoreceptor+rhodopsin.">The energetics of chromophore binding in the visual photoreceptor rhodopsin.</a> Biophys J. 113 (1), 60-72 (2017).</li><li>Fan, J., Rohrer, B., Frederick, J. M., Baehr, W., Crouch, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rpe65-/-+and+Lrat-/-+mice:+comparable+models+of+leber+congenital+amaurosis.">Rpe65-/- and Lrat-/- mice: comparable models of leber congenital amaurosis.</a> Invest Ophthalmol Vis Sci. 49 (6), 2384-2389 (2008).</li><li>Breen, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+functional+human+vitamin+A+transporter/RBP+receptor+(STRA6)+for+structure+determination.">Production of functional human vitamin A transporter/RBP receptor (STRA6) for structure determination.</a> PLoS One. 10 (3), e0122293 (2015).</li><li>Kawaguchi, R., Zhong, M., Sun, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+analyses+of+retinol+transport+by+the+membrane+receptor+of+plasma+retinol+binding+protein.">Real-time analyses of retinol transport by the membrane receptor of plasma retinol binding protein.</a> J Vis Exp. 28 (71), e50169 (2013).</li><li>Shi, Y., Obert, E., Rahman, B., Rohrer, B., Lobo, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retinol+binding+protein+receptor+2+(Rbpr2)+is+required+for+photoreceptor+outer+segment+morphogenesis+and+visual+function+in+zebrafish.">The retinol binding protein receptor 2 (Rbpr2) is required for photoreceptor outer segment morphogenesis and visual function in zebrafish.</a> Sci Rep. 7 (1), 16207 (2017).</li></ol>

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Critical steps in the protocol 
SPR Methodology 
In silico modeling and docking analysis: The predicted structure of RBPR2 (https://alphafold.ebi.ac.uk/entry/Q9DBN1) and STRA6, and the known structure for&#160;msRBP4 PDB database (RSCB PDB ID: 2wqa), should be used for docking study29,31. Additionally, in vitro methods (cell culture) should be used to confirm the interaction of the putative bi...

Dietary obtained Vitamin A/all-trans retinol/ROL is an important component playing a role in visual function1,2. The chromophore 11-cis retinal, a metabolite of dietary vitamin A, binds to the G protein-coupled receptor (GPCR) opsin to generate the retinylidene protein, rhodopsin, in the photoreceptors. When light falls on the eye, the configuration of rhodopsin undergoes a fundamental change via the conversion of its 11-cis-retinal component to the all-trans-retinal. This configuration change triggers a phototransduction cascade within the rod photoreceptors, converting light into an electrical signal, which is transmitted to the visual cortex in the brain via the optic nerve3,4,5,6,7,8,9,10. Decreased concentrations of serum and ocular ROL can impact retinylidene protein formation, which in turn causes opsin mislocalization, apoprotein opsin accumulation, night blindness, and photoreceptor OS degeneration, leading to Retinitis Pigmentosa or Leber Congenital Amaurosis, which can cause blindness3,10.
All-trans retinol is the fundamental transport form of dietary vitamin A and it is the source from which all functional retinoids and dietary vitamin A metabolites are derived. The liver serves as the primary organ for dietary vitamin A storage. Hepatic retinol is transported via the serum as its complex with retinol-binding protein 4 (RBP4). RBP4, primarily expressed in the liver, forms a holo-complex with the retinol substrate and transthyretin (TTR), which enters the circulation11,12,13,14,15,16,17. The report of a cell surface receptor for RBP4 in the 1970s led to the hypothesis of membrane transport proteins aiding the transport of retinoids in and out of cells. The cell surface receptor for RBP4-bound retinol (RBP4-ROL) was identified as STimulated by Retinoic Acid 6 Retinol (STRA6) in the retinal pigment epithelium (RPE) of the eye. STRA6 binds to the circulatory holo-RBP4 complex and shuttles the RBP4-bound retinol across the RPE to be utilized by photoreceptors18,19. Mutations in STRA6 can lead to a myriad of diseases and phenotypes associated with reduced ocular ROL concentrations. STRA6 mutations during development can lead to anophthalmia, microphthalmia, and other non-ocular symptoms that overlap with phenotypes associated with Matthew-Wood syndrome20,21,22,23,24,25,26,27. STRA6 is expressed in different organs and tissues, such as the RPE in the eye, but not in all tissues26,27. Although the primary site of retinoid storage is the liver, STRA6 is not expressed in the liver.
Alapatt and colleagues discovered that the Retinol Binding Protein 4 Receptor 2 (RBPR2) bound RBP4 with high affinity and was responsible for the uptake of RBP4-bound retinol in the liver, similar to STRA6 in the RPE28. RBPR2 has been reported to share structural homology with STRA629,30,31. RBP4 is proposed to bind to residues S294, Y295, and L296 on RBPR2, an amino acid-binding domain partially conserved between RBPR2 and STRA6 as well29,30,31. From these studies, vitamin A membrane receptors such as STRA6 and RBPR2, which contain one or more extracellular binding residues/domains, are proposed to interact with circulatory RBP4-ROL. Membrane receptors, therefore, play an important role in receptor binding to circulatory RBP4 for ROL internalization into target tissues, such as the liver and eye.
In the first part of this study, we utilized Surface Plasmon Resonance (SPR) to investigate the interaction of two vitamin A membrane receptors (RBPR2 and STRA6) with their physiological ligand RBP431. The binding affinities and association/dissociation kinetics of protein to ligand complexes can be measured in real time by using SPR. This methodology aimed to provide critical kinetic, structural, and biochemical information on RBP4-binding motifs in RBPR2 and STRA6, which are critical for understanding pathological states of vitamin A deficiency31,32. As mentioned above, circulatory ROL is internalized into the RPE via STRA6 to generate the chromophore 11-cis retinal, which binds to opsin to generate the retinylidene protein, rhodopsin, in photoreceptors33. We used spectrophotometry methodologies to quantify the GPCR-opsin and its ligand 11-cis retinal complex in murine retinal lysates, which is critical for understanding mechanisms of reduced retinylidene protein, rhodopsin, in Retinitis Pigmentosa or Leber Congenital Amaurosis ocular pathological states34. In general, these protocols can be applied to study in vitro the physiological consequences of mutant RBP4, STRA6, or RBPR2 in influencing systemic and ocular vitamin A homeostasis or the impact of mutant rhodopsin or retinoid cycle proteins on visual function35,36.

<table><tbody><tr><td>2-D Quant Kit</td><td>Cytiva</td><td>80648356</td><td></td></tr><tr><td>Amine Coupling Kit</td><td>Cytiva</td><td>BR100050</td><td></td></tr><tr><td>Biacore evaluation software</td><td>Biacore S200</td><td>Version 1.1</td><td></td></tr><tr><td>Biacore Sensor chip CM5</td><td>Cytiva</td><td>BR100530</td><td></td></tr><tr><td>Bis tris propane</td><td>Sigma</td><td>B6755-25G</td><td>20 mM</td></tr><tr><td>BL21 DE3 competent cells</td><td>Thermo Scientific</td><td>EC0114</td><td></td></tr><tr><td>CD spectrophotometer</td><td>Jasco</td><td>J-815 Spectropolarimeter</td><td></td></tr><tr><td>Glycine HCL</td><td>Fisher Bioreagents</td><td>BP381-1</td><td></td></tr><tr><td>GraphPad Prism&amp;nbsp;</td><td></td><td></td><td>Model fitting, data analysis</td></tr><tr><td>LB broth</td><td>Fisher Bioreagents</td><td>BP1426-500</td><td></td></tr><tr><td>n-dodecyl-&amp;beta;-d-maltoside (DDM)</td><td>EMD Millipore</td><td>324355-1GM</td><td>2-20 mM</td></tr><tr><td>pET28a His-tag Kanamycin-resistant expression vector&amp;nbsp;</td><td>Addgene</td><td>69864-3</td><td></td></tr><tr><td>Plasmid purification kit</td><td>Qiagen</td><td>27106</td><td></td></tr><tr><td>Rho1D4 MagBeads</td><td>CubeBiotech</td><td>33299</td><td></td></tr><tr><td>Slide-A_Lyzer 10K dialysis cassette</td><td>Thermo Scientific</td><td>66810</td><td></td></tr><tr><td>Tween20</td><td>Fisher Bioreagents</td><td>BP337-500</td><td>0.05%</td></tr><tr><td>UV vis Spectrophotometer</td><td>Agilent&amp;nbsp;</td><td>Cary 60 UV-Vis</td><td></td></tr><tr><td>&lt;strong&gt;Peptide name&lt;/strong&gt;</td><td>&lt;strong&gt;Peptide sequence&lt;/strong&gt;</td><td>&lt;strong&gt;HPLC-purity&lt;/strong&gt;</td><td>&lt;strong&gt;Mass Spec&lt;/strong&gt;</td></tr><tr><td>Mouse Rbpr2 (42)</td><td>HVRDKLDMFEDKLESYLTHM&lt;br/&gt; NETGTLTPIILQVKELISVTKG</td><td>92.14%</td><td>Conforms</td></tr><tr><td>Mouse Stra6 (40)</td><td>SVVPTVQKVRAGINTDVSYL&lt;br/&gt; LAGFGIVLSEDRQEVVELVK</td><td>90.84%</td><td>Conforms</td></tr><tr><td>Mouse Rbpr2 mutant S294A (42)</td><td>HVRDKLDMFEDKLEAYLTHM&lt;br/&gt; NETGTLTPIILQVKELISVTKG</td><td>0.92%</td><td>Conforms</td></tr></tbody></table>

methodology for studying interactions of vitamin a membrane receptors and opsin protein with their ligands in generating the retinylidene protein

Distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and generating the retinylidene protein for visual function. RBP4-ROL is the complex of ROL with retinol-binding protein 4 (RBP4), which is present in the blood. Two membrane receptors, Retinol Binding Protein 4 Receptor 2 (RBPR2) in the liver and STimulated by Retinoic Acid 6 Retinol (STRA6) in the eye, bind circulatory RBP4 and this mechanism is critical for internalizing ROL into cells. Establishing methods to investigate receptor-ligand kinetics is essential in understanding the physiological function of vitamin A receptors for retinoid homeostasis. Using Surface Plasmon Resonance (SPR) assays, we can analyze the binding affinities and kinetic parameters of vitamin A membrane receptors with its physiological ligand RBP4. 
These methodologies can reveal new structural and biochemical information of RBP4-binding motifs in RBPR2 and STRA6, which are critical for understanding pathological states of vitamin A deficiency. In the eye, internalized ROL is metabolized to 11-cis retinal, the visual chromophore that binds to opsin in photoreceptors to form the retinylidene protein, rhodopsin. The absorbance of light causes the cis-to-trans isomerization of 11-cis retinal, inducing conformational changes in rhodopsin and the subsequent activation of the phototransduction cascade. Decreased concentrations of serum and ocular ROL can impact retinylidene protein formation, which in turn can cause rhodopsin mislocalization, apoprotein opsin accumulation, night blindness, and photoreceptor outer segment degeneration, leading to Retinitis Pigmentosa or Leber Congenital Amaurosis. 
Therefore, spectrophotometric methodologies to quantify the G protein-coupled receptor opsin-11-cis retinal complex in the retina are critical for understanding mechanisms of retinal cell degeneration in the above-mentioned pathological states. With these comprehensive methodologies, investigators will be able to better assess dietary vitamin A supply in maintaining systemic and ocular retinoid homeostasis, which is critical for generating and maintaining retinylidene protein concentrations in photoreceptors, which is critical for sustaining visual function in humans.

Quantitative methods are described to study protein-ligand interactions of vitamin A membrane receptors and photoreceptor opsin with their respective physiological ligands. The recombinant mouse RBP4 should be expressed in E. coli and the purified protein used as a conjugated ligand on a SPR Chip. The chemically synthesized RBPR2, STRA6, and mutant S294A RBPR2 &#34;SYL motif RBP4 interacting extracellular site&#34; of a ~40 amino acid peptide is used as analyte at various concentrations to measure the kinetics o...

Author Spotlight: Unraveling Vitamin A Transport Mechanisms &#8212; Linking Liver Receptors to Vision Health Through RBPR2 and RBP4 Interactions

Here, we describe two quantitative methods for studying the protein-ligand interactions of vitamin A membrane receptors and photoreceptor opsin with their respective physiological ligands.

Methodology for Studying Interactions of Vitamin A Membrane Receptors and Opsin Protein with their Ligands in Generating the Retinylidene Protein

Distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and ...

methodology-for-studying-interactions-vitamin-membrane-receptors

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JoVE Journal

Biochemistry

1.0K Views.  University of Minnesota. Ons onderzoek onderzoekt hoe vitamine A-homeostase wordt gereguleerd, waarbij we ons richten op de fysiologische interactie tussen retinale bindende eiwitreceptor twee, RRBPR2, en zijn ligand, RRBP4, voor het transport van vitamine A in het hele lichaam. We onderzoeken ook hoe 11-cis retinaal bindt aan rodopsine, wat cruciaal is voor fototransductie in het gezichtsvermogen. We gebruiken twee bekende technieken om een verband te leggen tussen de opslag en dist...

Video: Auteur in de schijnwerpers: Vitamine A-transportmechanismen ontrafelen - Leverreceptoren koppelen aan de gezondheid van het gezichtsvermogen via RBPR2- en RBP4-int

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Vitamin A Metabolite Analysis in Murine Tissue for Rhodopsin Function Studies

Quantitative Analysis of Dietary Vitamin A Metabolites in Murine Ocular and Non-Ocular Tissues Using High-Performance Liquid Chromatography

G protein-coupled receptors (GPCRs) are a superfamily of transmembrane proteins that initiate signaling cascades through activation of its G protein upon association with its ligand. In all mammalian vision, rhodopsin is the GPCR responsible for the initiation of the phototransduction cascade. Within photoreceptors, rhodopsin is bound to its chromophore 11-cis-retinal and is activated through the light-sensitive isomerization of 11-cis-retinal to all-trans-retinal, which activates the transducin G protein, resulting in the phototransduction cascade. 
While phototransduction is well understood, the processes that are involved in the supply of dietary vitamin A precursors for 11-cis-retinal generation in the eye, as well as diseases resulting in disruption of this supply, are not yet fully understood. Once vitamin A precursors are absorbed into the intestine, they are stored in the liver as retinyl esters and released into the bloodstream as all-trans-retinol bound to retinol-binding protein 4 (RBP4). This circulatory RBP4-retinol will be absorbed by systemic organs, such as the liver, lungs, kidney, and eye. Hence, a method for the quantification of the various metabolites of dietary vitamin A in the eye and systemic organs is critical to the study of proper rhodopsin GPCR function. 
In this method, we present a comprehensive extraction and analytical method for vitamin A analysis in murine tissue. Through normal-phase, high-performance liquid chromatography analysis, all relevant isomers of retinaldehydes, retinols, and retinyl esters can be detected simultaneously through a single run, which allows for the efficient use of experimental samples and increases internal reliability across different vitamin A metabolites within the same sample. With this comprehensive method, investigators will be able to better assess systemic vitamin A supply in rhodopsin GPCR function.

Author Spotlight: Investigating Physiological Functions of Vitamin A Transporters Using HPLC-Based Vitamin A Profiling

Here, a normal-phase, high-performance liquid chromatography method is described to detect and quantify critical retinoids involved in the facilitation of visual function in both ocular and systemic tissue, in the context of the systemic vitamin A supply to generate the essential photosensitive rhodopsin chromophore 11-cis-retinal.

G protein-coupled receptors (GPCRs) are a superfamily of transmembrane proteins that initiate signaling cascades through activation of its G protein upon ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

Methodology for Studying Interactions of Vitamin A Membrane Receptors and Opsin Protein with their Ligands in Generating the Retinylidene Protein

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