Supported by National Institutes of Health grant R01EY018144.

1. Production, Refolding, and HPLC Purification of Holo-RBP
<ol>
	<li>Transform BL-21cells with the pET3a vector harboring the cDNA for human RBP with 6x His tag on the N-terminus. Grow the transformed BL-21 cells in a shaker at 37 &deg;C in 40 ml LB media with carbenicillin until O.D. at 600 nm reaches 0.5. Induce RBP protein expression by adding IPTG to 1mM. Grow the bacteria at 37 &deg;C another 5 hr.</li>
	<li>RBP produced in E.coli is mostly present in insoluble inclusion bodies. To enrich inclusion bodies, pellet cells at 10,000 x g for 20 min. Then sonicate the cells on ice in 5 ml PBS with protease inhibitors, followed by 2 cycles of freezing and thawing. Pellet down the inclusion bodies at 20,000 x g for 30 min at 4 &deg;C. Remove supernatant and keep the pellet on ice.</li>
	<li>Solubilize the inclusion body pellet by sonication in 10 ml 7.5 M guanidine hydrochloride. Add a half volume (5 ml) of 25 mM Tris buffer, pH 9.0 and DTT to a final concentration of 10 mM. Vigorously mix solution on a vortex mixer overnight at room temperature to fully reduce and denature proteins.</li>
	<li>Centrifuge the protein solution to remove insoluble material at 18,000 x g for 20 min at 4 &deg;C. After the spin, transfer the supernatant to a new tube and place the tube on ice.</li>
	<li>Prepare four volumes (60 ml) of refolding buffer (25 mM Tris, pH 9.0, 0.3 mM cystine, 3.0 mM cysteine, 1 mM EDTA). Degas the refolding buffer. Also prepare 600 &mu;l 10 mM retinol in ethanol under dim red light. Keep it in the dark on ice. Both the refolding buffer and retinol solution need to be freshly prepared.</li>
	<li>Cool both the protein solution and the refolding buffer on ice for at least 30 min. Higher temperature likely reduces the quality of refolded RBP. Add 1 ml refolding buffer dropwise and 10 &mu;l retinol solution in turn while protein solution is vigorously mixed in a beaker on ice. Repeat this step until all refolding buffer and retinol are added. Keep stirring the reaction on ice in the dark for 5 hr.</li>
	<li>Spin down the refolding reaction at 24,000 x g for 30 min at 4 &deg;C. It is essential to remove the aggregates right after the refolding reaction.</li>
	<li>Concentrate the supernatant solution using Amicon Ultra 15 concentrator (MWCO 10 K) to about 10 ml. Do not concentrate the refolded solution more than this level. Concentrating too much will lead to protein aggregation and lowers the final yield and quality of correctly folded RBP. Dilute the concentrated sample with cold PBS to 100 ml. The purpose of this step is to remove the components in the refolding reaction that interfere with nickel purification.</li>
	<li>Spin the solution one more time at 24,000 x g for 20 min at 4 &deg;C. It is important to remove any precipitate. Transfer the supernatant to two 50 ml tubes, each containing 1 ml Ni-NTA slurry that has been washed with PBS. Rotate the tubes at 4 &deg;C for one hour. Incubating diluted solution for 1 hr can cause the precipitation of misfolded proteins which need to be removed.</li>
	<li>Spin down the tubes at 500 x g for 3 min. Remove supernatant carefully. Anything floating should be removed. Add cold PBS to 30 ml for each tube. Spin again and remove the supernatant. Repeat this process until you see nothing but beads in the tube.</li>
	<li>Transfer the Ni-NTA resin to an empty column. Wash resin with 20 column volumes of 10 mM imidazole, 500 mM NaCl in PBS. Elute His-RBP in 5 column volumes of 100 mM imidazole in PBS. Do not store this solution for an extended period of time, and freezing reduces the final yield and quality. For the best result, purify RBP by HPLC immediately.</li>
	<li>Dialyze His-RBP purified from the Ni-NTA resin against 25 mM Tris, pH 8.4, and 120 mM NaCl. A concentrator can also be used for buffer exchange. Clear samples for HPLC by centrifugation at 16,000 x g for 10 min at 4 &deg;C right before each run.</li>
	<li>Purify holo-RBP on HPLC using an ion exchange column AX-300 by a NaCl step gradient (220 mM 12 min, 360 mM 15 min, and 1,000 mM 15 min) using 25 mM Tris, pH 8.4 as mobile phase at 1 ml/min. As NaCl concentration rises, holo-His-RBP is released while apo-RBP and misfolded RBP stay bound to the column (Figure 1). If RBP refolding is not done optimally, misfolded RBP (e.g. misfolded RBP-I in Figure 1) can predominate the whole preparation.</li>
	<li>Recover the correctly folded holo-RBP from the peak fractions at 360 mM NaCl. Carefully monitor the elution profile of correctly folded and misfolded His-RBP. At 1,000 mM NaCl, most of misfolded His-RBP should be released.</li>
	<li>Fractions containing holo-RBP are pooled, concentrated, and dialyzed against PBS overnight at 4 &deg;C. Check the final yield and quality (330 nm/280 nm) by spectrophotometer. Correctly folded product should have a 330 nm/280 nm ratio of slightly higher than 1 (Figure 1).</li>
	<li>To produce apo-RBP, add an equal volume of heptane to the purified holo-RBP and mix gently by rotating overnight at 4 &deg;C. Centrifuge at 16,000 g for 10 min at 4 &deg;C and carefully transfer the bottom aqueous phase to a new tube (avoiding the precipitate at the interface). Repeat the process three times with a 3 hr incubation for each. Check absorption on a Nanodrop spectrophotometer to make sure the 330 nm peak has decreased to the background level.</li>
</ol>
2. Real-time Monitoring of STRA6-catalyzed Retinol Release and Retinol Loading
<ol>
	<li>Block a black 96-well Microfluor-2 plate (Thermo Scientific) with 200 &mu;l of Blocker Casein (Pierce) overnight at 4 &deg;C to prevent nonspecific sticking of holo-RBP to the plastic. Blocker Casein gives the best blocking effect of the all the blocking solutions we tested.</li>
	<li>Membranes freshly prepared from cells expressing STRA6 or control cells are washed using PBS and passed through a Hamilton syringe (Gastight #1710) 6 times to produce a uniform suspension in PBS. We typically use membranes derived from 1/100 to 1/20 of cells grown on a 100 mm dish per reaction.</li>
	<li>Wash the coated wells of the Microfluor-2 plate once with 200 &mu;l of PBS. Cool the plate on ice before addition of membrane suspension (50 &mu;l per well).</li>
	<li>Real time monitoring of retinol fluorescence is measured using fluorescence optics of the POLARstar Omega (BMG Labtech) with the excitation filter 320ex and the emission filter 460-10. The program is set to the Endpoint reading mode. The Plate Mode can also be used to measure a time course if the time intervals are not varied. The signal from each time point is the average of 10 measurements (orbital averaging with a diameter of 2 mm) and the gain is set at 1,800. The plate is shaken for 10 sec at 500 rpm using double-orbital shaking before each measurement.</li>
	<li>To start the retinol release assay, the wells are read once using the Endpoint reading mode before holo-RBP (typically 1 &mu;M final concentration) is added at 0 min. As soon as holo-RBP is added, the reaction is monitored continuously every 5-10 min for 1-3 hr. To start the retinol loading assay, all-trans retinol (typically 1 &mu;M final concentration) is added to the wells under dim red light. The plate is read once before apo-RBP is added at 0 min. As soon as apo-RBP is added, the reaction is monitored continuously every 5-10 min for 1-3 hr.</li>
	<li>After all measurements are done, download the raw data (example shown in Figure 2) from the Omega Data Analysis software (BMG Labtech) to Microsoft Excel for data analysis. Each time point generates a file that contains the readings of all experimental conditions. For example, if there are 20 time points, consolidate 20 files into one Excel file for data analysis.</li>
	<li>For data analysis, the fluorescence signals before the addition of retinol or holo-RBP at 0 min are considered background signals and subtracted from the final fluorescence signals at all time points. Although the background signals are low compared to retinol fluorescence in holo-RBP, subtracting the background eliminates the nonspecific influence of light scattering by the membranes and makes it possible to focus on the retinol fluorescence of holo-RBP or retinol added at 0 min.</li>
</ol>
3. Real-time Monitoring of STRA6-catalyzed Transport of Retinol From Holo-RBP to CRBP-I
<ol>
	<li>Retinol-EGFP is a new fluorescence resonance transfer (FRET) pair that has recently been established 41. STRA6-catalyzed retinol transport from holo-RBP to EGFP-CRBP-I is monitored in real time by measuring retinol-EGFP FRET. EGFP-CRBP-I fusion protein with 6XHis tag (EGFP-CRBP-I) is produced in mammalian cells and is purified on Ni-NTA resin before the experiment. EGFP-CRBP-I can be stored in PBS with protease inhibitors for a short period of time at 4 &deg;C. Alternatively the fusion protein is frozen in -80 &deg;C in small aliquots.</li>
	<li>Before the reactions, prepare a blocked Microfluor-2 plate and membranes as described above. Wash the coated wells of the Microfluor-2 plate once with 200 &mu;l PBS before addition of membrane suspension (50 &mu;l per well).</li>
	<li>Real-time retinol-EGFP FRET is measured using POLARstar Omega with the excitation filter 320ex and the emission filters 460-10 and 510-10 using simultaneous duel emission fluorescence optics. The program is set to the Endpoint reading mode. The Plate Mode can also be used to measure a time course if the time intervals are not varied. The signal from each time point is the average of 10 measurements (orbital averaging with a diameter of 2 mm) and the gain is set at 1,800 per channel. The plate is shaken for 10 sec at 500 rpm using double-orbital shaking before each measurement.</li>
	<li>To start the reactions, EGFP-CRBP-I (typically 1 &mu;M final concentration) is added to the wells. The plate is read once using the Endpoint reading mode before holo-RBP is added at 0 min. As soon as holo-RBP is added, the reaction is monitored continuously by measurement every 5-10 min for 1-2 hr.</li>
	<li>After all measurements are done, download the raw data (example shown in Figure 3) from the Omega Data Analysis software to Microsoft Excel for data analysis as described above.</li>
	<li>Retinol-EGFP FRET is calculated by the dynamic change in the ratio of the acceptor/donor emission peaks47. The equation41 to calculate this ratio is [(510t-510b)/(460t-460b)], where 510t, 510b, 460t, and 460b represent emissions at 510 nm after initiation of the reaction (t=time point), at 510 nm before retinol or holo-RBP is added (b=background), at 460 nm after initiation of the reaction (t=time point), and at 460 nm before retinol or holo-RBP is added (b=background), respectively.</li>
</ol>

No conflicts of interest declared.

We share here an optimized RBP production protocol because RBP production and purification procedures are critical to generating correctly folded RBP. Given the possibility of misfolded RBP species and the presence of trace amounts of bacterial proteins even in HPLC purified bacteria-produced RBP, it is helpful to use native RBP from serum to confirm a conclusion related to RBP. Urine RBP, which is commercially available, is a complex mixture of many species of RBP including apo-RBP and holo-RBP 48,49.
<p...

Vitamin A is an organic molecule that is essential for human survival and the proper functioning of almost all human organs. Vitamin A derivatives (retinoids) participate in diverse biochemical and cellular events including the sensing of light for vision 1,2 and the regulation of gene expression and protein translation during embryonic development and in adult tissues 3-6. Although retinol has the ability to diffuse systemically, evolution came up with plasma retinol binding protein, a specific carrier protein for vitamin A transport in the blood to achieve high efficiency and specificity and to avoid toxicity associated with random diffusion 7-10. A high-affinity receptor that binds to RBP and takes up vitamin A was hypothesized in the 1970s 11-13. Despite evidence accumulated in three decades on the existence of the RBP receptor 14-31, the receptor hypothesis was debated for many years due to the existence of an incorrect definition of holo-RBP. The correct definition of holo-RBP is that it is the high affinity 1:1 complex between retinol and RBP. Repeated extraction of holo-RBP by organic solvent is necessary to produce apo-RBP. This definition is used by almost all labs studying RBP 7,9,32-35 or the RBP receptor 14-31,36-42. The incorrect definition of holo-RBP that was used to disprove the existence of the RBP receptor is the acute mixture of free retinol with apo-RBP. Since the function of the RBP receptor in vitamin A uptake from holo-RBP is to release retinol from holo-RBP, the RBP receptor would play no role in retinol uptake if retinol is free to begin with (as proposed by the incorrect definition of holo-RBP).
The recent identification of RBP receptor as a multitransmembrane domain protein called STRA636 and its function in vitamin A uptake from holo-RBP 36-43 strongly argues against the hypothesis that RBP does not need a receptor to deliver vitamin A. Detailed analyses revealed that STRA6 has 9 transmembrane domains with the N-terminus located extracellularly and C-terminus located intracellularly 40. Located between transmembrane 6 and 7 is an essential RBP binding domain 39. STRA6 is coupled to both LRAT and CRBP-I in vitamin A uptake from holo-RBP, but neither LRAT nor CRBP-I is absolutely required for enhanced STRA6 activity 41. STRA6's ability to catalyze retinol release from holo-RBP is the key to its vitamin A uptake activity 41. By relying on STRA6 to release its retinol, vitamin A delivery by RBP can transport vitamin A to target cells in peripheral tissues with high specificity and efficiency.
The critical importance of holo-RBP definition and preparation is illustrated not only by the historical debate on the existence of the RBP receptor, but also by three related recent papers based on holo-RBP definitions different from the original and correct definition 44-46. The first paper used the holo-RBP definition that was used to disapprove the existence of the RBP receptor to study the RBP receptor 44. The second and third papers came up with a third definition of holo-RBP that made it even less likely for retinol to be studied to form a proper complex with RBP 45,46. These studies prepared 3H-retinol/RBP by mixing holo-RBP (not even apo-RBP) with 3H-retinol. Since this assay did not have 3H-retinol/RBP formed and did not remove excessive free 3H-retinol 45,46, it is not an assay for 3H-retinol uptake from 3H-retinol/RBP, but is a free 3H-retinol diffusion assay. It has been shown previously that STRA6 does not enhance cellular uptake of free retinol by LRAT 38 or CRBP-I 41. Virtually all retinol is bound to RBP in the blood and there is no detectable free retinol. A main function of the RBP receptor is to catalyze retinol release from holo-RBP during retinol uptake from holo-RBP 41. If the retinol is artificially released or is in the free form to begin with 45,46, the RBP receptor is not needed. The dramatically different results obtained from the free retinol diffusion assay as compared to assays based on correctly prepared holo-RBP illustrate that correct preparation of RBP is crucial for its functional assays.
RBP can be purified from human serum 41, but the procedure is complex and the yield is low. An alternative approach is to produce RBP in E. coli. Because E. coli does not have the ability to correctly fold mammalian secreted proteins with more than one pair of disulfide bonds like RBP, it is essential to refold RBP and purify the correctly folded protein. Misfolded proteins not only behave differently from corrected folded RBP in various assays, but also cause protein aggregation during storage. For the same reason, apo-RBP is only produced from high-quality holo-RBP. We describe here an optimized protocol to produce high quality RBP 100% loaded with retinol through bacterial expression, refolding, and HPLC purification. HPLC purification not only removes incorrectly folded RBP but also significant bacterial contamination that can cause serious artifacts if RBP is used in signal transduction assays. We also describe two sensitive real-time monitoring techniques to study retinol transport by STRA6. Both techniques depend on high quality RBP. Due to space limitations, the classic techniques of radioactive retinoid-based and HPLC-based vitamin A uptake assays are not described here.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>guanidine hydrochloride</td>
 <td>EMD</td>
 <td>5010</td>
 <td></td>
 </tr>
 <tr>
 <td>cystine</td>
 <td>Sigma</td>
 <td>C8755</td>
 <td></td>
 </tr>
 <tr>
 <td>cysteine</td>
 <td>Sigma</td>
 <td>C7352</td>
 <td></td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Fisher</td>
 <td>BP118-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris</td>
 <td>Fisher</td>
 <td>7786-1</td>
 <td></td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>EMD</td>
 <td>3860</td>
 <td></td>
 </tr>
 <tr>
 <td>retinol</td>
 <td>Sigma</td>
 <td>R7632</td>
 <td></td>
 </tr>
 <tr>
 <td>carbenicillin</td>
 <td>Fisher</td>
 <td>BP2648-5</td>
 <td></td>
 </tr>
 <tr>
 <td>IPTG</td>
 <td>EMD</td>
 <td>5810</td>
 <td></td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>EMD</td>
 <td>6508</td>
 <td></td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Fisher</td>
 <td>BP358-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Ni-NTA</td>
 <td>Qiagen</td>
 <td>1018244</td>
 <td></td>
 </tr>
 <tr>
 <td>imidazole</td>
 <td>EMD</td>
 <td>5720</td>
 <td></td>
 </tr>
 <tr>
 <td>heptane</td>
 <td>EMD</td>
 <td>HX0295-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Blocker Casein</td>
 <td>Pierce</td>
 <td>37528</td>
 <td></td>
 </tr>
 <tr>
 <td>Amicon Ultra 15 concentrator (MWCO 10 K)</td>
 <td>Millipore</td>
 <td>UFC901024</td>
 <td></td>
 </tr>
 <tr>
 <td>Microfluor-2 plate </td>
 <td>Fisher</td>
 <td>14-245-177</td>
 <td></td>
 </tr>
 <tr>
 <td>Hamilton syringe Gastight #1710</td>
 <td>Fisher</td>
 <td>14-824-655</td>
 <td></td>
 </tr>
 </tbody>
 </table>

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.

We present here representative results for holo-RBP production and purification by HPLC (Figure 1), real-time analysis of STRA6-catalyzed retinol release from holo-RBP and retinol loading into apo-RBP (Figure 2) and real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to EGFP-CRBP-I (Figure 3).
Without refolding, RBP produced in bacteria is almost completely misfolded due to the presence of many incorrect disulfide bonds. Ther...

Watch this Scientific Journal Video about Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein at JoVE.com

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

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 ...

real-time-analyses-retinol-transport-membrane-receptor-plasma-retinol

Research

JoVE Journal

Biology

13.6K Views.  University of California, Los Angeles. 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.

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

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways 1,2. Of these many 'client' proteins for hsp90, the assembly of steroid receptor&bull;hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR&bull;hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. 
The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid 3. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR&bull;hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR&bull;hsp90 heterocomplex before steroid binding activity is restored 4 . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [3H]steroid. 
Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 5. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source 6. 
In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K+, which results in the reconstitution of the GR&bull;hsp90 heterocomplex and reactivation of steroid binding activity 7. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding 8-11.

Biochemical Reconstitution of Steroid Receptor&amp;#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

An in vitro method for preparing functional glucocorticoid receptor (GR)&#x2022;hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...