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Surface plasmon resonance is an optical technique utilized for detecting molecular interactions. Binding of a mobile molecule (analyte) to a molecule immobilized on a thin metal film (ligand) changes the refractive index of the film. The angle of extinction of light, reflected after polarized light impinges upon the film, is altered, monitored as a change in detector position for the dip in reflected intensity (the surface plasmon resonance phenomenon). Because the method strictly detects mass, there is no need to label the interacting components, thus eliminating possible changes of their molecular properties. We have utilized surface plasmon resonance to study the interaction of proteins of hair cells.
Surface plasmon resonance (SPR) binding analysis methodology is used to study molecular interactions (1, 2). SPR is an optical technique for detecting the interaction of two different molecules in which one is mobile and one is fixed on a thin gold film (1). In the work described here, affinity-purified fusion polypeptides are immobilized by an amine-coupling reaction on a sensor chip (Biacore, Piscataway, NJ, USA) inserted into the flow chamber of a Biacore 3000 instrument (Biacore, Uppsala, Sweden). Addition of a second polypeptide, the flow-through analyte, to the chamber, results in binding to the immobilized polypeptide ligand, producing a small change in refractive index at the gold surface (3), which can be quantified with precision (4). Binding affinities can be obtained from the ratio of rate constants, yielding a straightforward characterization of protein-protein interaction. SPR directly detects mass (concentration) with no need for special radioactive or fluorescent labeling of polypeptides (5) before measurement, presenting a great advantage in minimizing time and complexity of the studies.
We perform surface plasmon resonance experiments with a Biacore 3000 instrument. The immobilization involves activation of carboxymethyl groups on a dextran-coated chip by reaction with N-hydroxysuccinimide, followed by covalent bonding of the ligands to the chip surface via amide linkages and blockage of excess activated carboxyls with ethanolamine (6). Reference surfaces are prepared in the same manner, except that all carboxyls are blocked and no ligand is added. During analysis, each cell with an immobilized fusion polypeptide is paired with an adjacent cell on the chip, the latter serving as a reference. The final concentration of bound ligand, expressed in response units (RU), is calculated by subtracting the reference RU from the ligand RU. HBS-N, HBS-P, HBS-EP, or PBST buffer may be used as both running and analyte-binding buffer. Purified fusion polypeptide or protein (analyte), typically at 100 nM, is allowed to flow over the immobilized-ligand surface and the binding response of analyte to ligand is recorded. The chip surface is regenerated by removal of analyte with a regeneration buffer. The maximum RU with each analyte indicates the level of interaction, and reflects comparative binding affinity.
This work was supported by NIH R01 DC000156, NIH R01 DC004076, and the American Hearing Research Foundation. We thank Dr. Stanley Terlecky, Department of Pharmacology, Wayne State University, for use of the Biacore 3000 instrument.
1The Biacore CM5 chip is widely employed for SPR interaction studies. Carboxymethylated dextran molecules are attached to a gold-coated surface. The CM5 chip can detect nucleic acids, carbohydrates, and small molecules, in addition to proteins. The chip surface is prepared for binding studies by coupling ligand molecules to the carboxymethyl group via NH2, -SH, -CHO, -OH or -COOH linkages. As alternatives to the CM5 chip, CM4, CM3 and C1 chips can be used, which have a lower matrix density of functional groups. Another chip, SA, can be used for immobilization of biotinylated peptides, proteins, nucleic acids, and carbohydrates. HPA and L1 chips are employed for lipid or liposome immobilization. NTA chips are utilized for capturing histidine-tagged molecules (7).
2If no non-specific PCR bands are present, the product can be purified by phenol-chloroform extraction and alcohol precipitation. Adjust the sample volume to 200 μL by adding deionized water. Add an equal volume of phenol-chloroform (50% phenol saturated with 0.1 M Tris-HCl, pH 7.6, 48% chloroform, 2% isoamyl alcohol) to the sample and vortex to mix. Centrifuge at 14,000 g for 5 min. Carefully pipette out the upper phase into a fresh tube and add an equal volume of chloroform, vortex, and centrifuge as before. To the supernatant, add 1/10 volume of 3 M sodium acetate, pH 4.5, and 2.5 volumes of ethanol. Incubate at –20 °C for 1 h. Centrifuge at 14,000 g for 10 min at room temperature. Remove the supernatant. Wash pellet in excess 70% ethanol, centrifuge as before, decant the supernatant, and dry pellet in air.
3The pRSET vector contains sequences for six contiguous histidine residues upstream of the multiple cloning site, and thus the his-tagged fusion protein expressed in bacteria can be purified using a nickel affinity column (Qiagen). Immediately adjacent and upstream to the multiple cloning site is an eight-amino-acid epitope against which antibodies are commercially available (Invitrogen), facilitating antibody-based detection. The pGEX expression vector can also be used (see Note 10).
4The Qubit fluorescence spectrometer (Invitrogen) is a convenient instrument for measurement of nucleic acid and protein concentration by fluorescence. In a 0.5 mL tube, pipette 190 μL of the Quant-iT working solution (1/200 dye in buffer; buffer is double-stranded DNA Broad-Range reagent). Add DNA sample (1–10 μL) and bring to a total volume of 200 μL with water. Vortex 2–3 s, incubate for 2 min, and read the fluorescence.
5Add, to an autoclaved 0.5-mL microcentrifuge tube, 4 μL of 5X ligase reaction buffer, 15–60 fmol vector DNA, 45–180 fmol insert DNA (0. 1–1. 0 μg total DNA), and 1 unit of T4 DNA ligase in 1 μL (Invitrogen). Bring the total volume to 20 μL, mix gently, and centrifuge briefly to collect the contents at the bottom of the tube. Incubate at 4 °C overnight.
6Further confirmation of the correct insert sequence is accomplished by nucleotide sequencing of the insert before carrying out the protein expression.
7In verifying the expression of protein, there should be a heavily-stained protein band at the estimated molecular size of the fusion product for the IPTG-induced sample. There may be a low-to-moderately stained band in the non-induced samples. There should be no comparable band in the empty vector sample. Once expression is verified, proceed to the purification step.
8After establishing protein overexpression for the clonal sequence, the bacterial cells are grown in cultures for scaled-up protein purification. For purification of the fusion protein, generally 50–250 mL of culture is sufficient for small-to-medium-scale protein yield. From a 50 mL culture, one can purify up to 250 μg of protein.
9For Ni-NTA spin-column (Qiagen) purification, as much as 100 μg of pure protein can be obtained from 1.2 mL of the lysate solution, depending on the level of expression. In some cases, minor bands are visible in the purified sample, and a re-purification step with a fresh spin column is required.
10For purification using glutathione-agarose beads, PCR primers for domains of interest, containing desired restriction sites, are used in PCR reactions to clone the domains into the pGEX-6P-1 vector. This vector contains a GST coding region followed by a multiple cloning site. The fusion polypeptide product will contain an N-terminal, 26-kDa GST tag, which allows affinity purification. The pGEX-6P-1 vectors are utilized to transform E. coli BL21 (DE3) as described in Section 3.2.3, and the protein is purified by the glutathioneagarose beads.
11In general, both ligand and analyte should be as pure as possible, particularly for kinetic analysis of interactions. However, if a well-purified ligand is used, it is possible to determine the concentration of the analyte even if the latter is present in a mixture (9).
12It is critical that there be no glycerol in the protein samples and buffers.
13De-gas solutions by fitting a rubber stopper, with inlet tube attached to an aspirator and trap, to the top of a vessel containing the solution and reduce the pressure until air bubbles are expelled.
14The pH should be adjusted so that the protein has a net positive charge. The pI and molecular mass of the protein can be estimated using the COMPUTE pI/MW tool found at http://au.expasy.org/tools/pi_tool.html.
15The ligand should be at least 95% pure. Generally, a ligand polypeptide of smaller molecular mass should be paired with a polypeptide/protein of higher molecular mass as analyte, because the SPR response will be higher, due to the more advantageous (higher) density that can be achieved for the smaller molecular-mass molecule bound to the surface.
16A single injection of 50 mM NaOH at a flow rate of 20 μL/min usually removes all the ligand from the surface (inspect the baseline to see if it returns to the same level as before the run). If the baseline remains elevated, a second wash with the same or another solution is performed to remove the ligand completely.
17The amine coupling reaction is the reaction generally used for proteins because of readily-available amine groups in proteins. Fig. 20.2 shows a sensorgram of a typical immobilization experiment using an amine coupling reaction. However, amine coupling is not suitable if the ligand is too acidic (pH<3. 5), if the amine groups are present in the active site, or if there are too many amine groups. In such situations, if thiol groups are present in the ligand, thiol coupling is preferred as an alternative (7). If both are not suitable, capture techniques such as streptavidin-biotin or His-tag nickel affinity methods can be selected. If a well-characterized antibody is available against the ligand, the antibody is immobilized and the ligand captured for binding studies. Non-amine-coupling protocols are not covered here.
18It is important to perform DESORB before each run in order to clean the micro fluidic tubes, chamber, and injection port. Such recommended instrument maintenance should be carried out on a regular basis. In the Biacore 3000 Control program, click the TOOLS tab and then WORKING TOOLS. Place one vial each of BIAdesorb Solution 1 and BIAdesorb Solution 2 in the rack designated by the program. Click DESORB and then START. Use a maintenance chip (chip lacking gold or other surface coatings) during the DESORB; do not use the CM5 chip.
19As noted in step 2, Section 3.1, the sensor chip should be primed before each run. The total duration for the priming is 6.3 min.
20A slow flow rate (5 μL/min) is best, since the chance for the ligand to adsorb to the surface increases as the flow rate decreases.
21All the buffers, solutions, and sensor chips are brought to room temperature before the run. De-gas all the solutions. The buffers and regeneration solutions must be filtered through standard 0. 2-μm or 0. 4-μm filters.
22When analyzing the samples, make sure that there are no air bubbles trapped in the sample solution. Even with prior de-gassing, bubbles can form during the sample dilution, and sometimes the bubbles may not be clearly visible. Eliminate these bubbles by spinning the tube at top speed (18,000 g) in a benchtop centrifuge for 10–15 s.
23To minimize the mass transport effect (8), a low level of ligand immobilization is preferred.
24Regeneration is achieved with the optimal pH and ionic concentration that keep the ligand active. A series of regeneration tests should be performed with regeneration buffers, from mild to more stringent conditions of pH and ionic strength. In most cases where the binding affinity is low or moderate, begin with a pH close to neutral (pH 4.5–8.5) and an ionic strength in the range of 0.5–1.0 M. 1.0 M NaCl should be sufficient to dissociate most of the bound protein (Figs. 20.5, 20.6).
25When undocking the chip from the instrument, it is often advantageous to select not to remove the buffer from the flow cells by un-checking EMPTY FLOW CELL. The chip can then be wrapped in soft tissue paper or a piece of paper towel and stored in a tight container at 4 °C. The sensor chips with immobilized ligands can be stored for a few days to more than a month.
26SPR binding measurements (Fig. 20.7) are generally performed in duplicate or triplicate. Experimentally-obtained kinetic data can be evaluated with the SPR kinetic evaluation software, BIAevaluation. In general, analysis of kinetic data involves the following steps: (a) make overlay plots of several interaction curves, (b) select analysis region, (c) select interaction model and curve fitting, (d) store results of the fitting procedure into an analysis results file.
27In selecting the data range for the analysis of kinetic plots with BIAevaluation software, a short period before the injection start and before the stop (the beginning and end of the association phase of the kinetic plot) should be excluded in the selection to avoid the dispersion effect (the complex response in the refractive index at the beginning and end of injection, not clearly defined mechanistically).
28Perform separate fits for different regions of the association and dissociation phases to ascertain if the calculated constants are consistent.
29Although not detailed here, association and dissociation constants can also be obtained directly by determining the concentrations of analyte and ligand under steady-state conditions (9).