The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

The overall goal of this video article is to provide answers to the following two questions. One, can studies of protein interactions be made more reliably by measuring the fraction of a protein sample that is actually capable of binding to another protein? Two, how can kinetic analysis give us a better understanding of structure, function relationships?

Both of these questions will be addressed in an experiment involving Corp X 100, an instrument that detects protein interactions in real time without using labels. This experiment will examine the interaction of bovine cystatin B with the cystine protease papain, the crystal structure of the two proteins in complex points to several residues involved in binding to the protease to determine how each of the interacting residues affects the cystatin B papin interaction. Several cystatin B variants are produced via site directed mutagenesis.

In such experiments, there is always the possibility that recombinant proteins will not fold correctly, aggregate or deteriorate leading to incorrect and irreproducible results. VIACORE X 100 has the capability of determining the fraction of protein that is available for binding by calibration free concentration analysis abbreviated CFCA. This approach allows for determining concentration of protein with retained binding activity even when calibration standards are not available.

CFCA will be used here to determine the concentration of the cystatin B variants. These data will then be used as input in the determination of the binding kinetics of the cystatin B variants to Pepin, which is also assessed using BOR X 100. Hi, I'm CLA gram from GE Healthcare in Sweden.

Today my colleague Eva Pool will show you how to do structure function relationship analysis using BOR X 100 Recombinant proteins may exhibit improper folding or loose bin activity over time, and this behavior cannot be detected by methods that rely on the total concentration measurement. This may in effect lead to incorrect kinetic data following the concentration measurements by calibration free concentration analysis. B.A core X 100 is used to analyze the kinetics and affinity of the interaction of cystatin B variants with pepane.

These parameters will give an insight into the binding mechanism. We start with a brief explanation of the experimental design, so let's get started. The interaction between the cystine, protease, papin, and the inhibitor cystatin B is dominated by hydrophobic contacts as revealed by the crystal structure of the two proteins in complex.

From the inhibitor side, the contact areas are the N and C terminal Ns and two hairpin loops. In this study, four mutants are examined, each of which contain cysteine three to Syrian mutation introduced in order to prevent dimerization cystatin B.Based on the context revealed in the crystal structure, leucine 73 was mutated to glycine histamine 75 to glycine and tyrosine 97 was changed to alanine. The two glycine mutations were introduced to examine the importance of the histidine 75 and leucine 73 side chains for the binding to Pepin, the tyrosine residue was mutated to alanine to investigate the interaction between the C terminus of cystatin B and Pepin.

The three double mutants as well as INE three to Syrian single mutant are tested for their interaction with a cataly inactive variant of Papain S methyl popin. This cataly inactive form of the Proteus has a methyl group attached to CYS 25 in the active cleft. For simplicity, we will call this variant propan throughout the protocol.

CFCA relies on measurement of binding rate under conditions where the rate is limited by diffusion of analyte molecules to the surface. This is favored by high immobilization levels of the ligand. Before starting the immobilization procedure, placed the HEPs buffer used in the experiments on the left hand tray and insert both tubings.

Also place an empty waste bottle on the right hand tray. Then insert a new sensor gypsum five into the instrument initialize via core X 100 software, and at the start screen, click the dock chip tool item. Click start doc in the software.

Next, use the immobilization wizard to set up the pape and immobilization by aiming coupling to censorship CM five from the start screen, click on the other options button, then select wizards. This displays a window with the available wizards. Select immobilization and then click the new button At the bottom right, the first screen of the immobilization wizard appears aiming coupling to sensor gypsum five is the default.

Check the box under flow cell two to immobilize in flow cell two. Flow cell one is left unmodified in order to use it as a reference surface from the three available options below. Check the specify contact time box on the right target level changes to contact time, specify a contact time of 900 seconds.

Next, enter the immobilization parameters under capturing molecule slash ligand solution. Enter papain. Then click next.

The next screen displays the layout of the sample rack. Position one will hold et ethanolamine used to deactivate the remaining active after the aiming coupling of the ligand papain to the chip surface. Insert the ethanolamine solution at position one in position two.

Place the ligand papain papain is stored in an excess of methyl sulfate and alluded to 50 micrograms per milliliter in 10 millimolar sodium acetate buffer. pH 4.5 reconstitute the EDC and NHS coupling reagents from the amine coupling kit with double distilled water pipette 85 microliters of EDC into a vial. Cap it and place it in position three.

Use the same volume for NHS in position four. Notice the file five should be capped and empty in order for the instrument to mix ED, C and NHS in this file. Also remember to fill the wash file in position H2O with fresh deionized water.

When the rack is filled according to the outline on the screen, click load samples and load the rack into the instrument. When the rack is loaded, click next. Before the run is started, a checklist is shown.

Make sure that all bullet points are fulfilled when they are. The run is started by pressing the start button. The run is started during the immobilization procedure.

Data are collected in real time. First, the coupling reagents are injected. Next pape is coupled to the chip surface and the procedure ends in deactivation of the remaining active esters.

After the run, a dialogue window summarizes the result of the run. In this window, the immobilization level is displayed. The entire coupling procedure should result in about 3000 RU immobilized papain.

Once papain has been immobilized to the sensor chip, begin CFCA analysis by diluting the cystatin B mutant samples to a total concentration of about 10 nanomolar in at least 300 microliters. Record the dilution factor as we will use it later in the assay setup. Begin by bringing up the wizard dialogue as described in the previous section in the concentration section of the assay folder.

Choose calibration free, then choose new. The first screen of the wizard shows the injection sequence. The settings can remain unchanged since sensor chip CM five and a direct immobilization assay approach are used here.

Click next in the next screen. Leave the check boxes prime before run and run startup cycles checked. Also, leave the default number of startup cycles unchanged at three.

Enter heaps buffer in the name of the solution field. Then click next. In the next screen, enter the name of the regeneration solution, which is 20 millimolar sodium hydroxide.

Leave the other parameters unchanged. Click next to view the sample table. Set up the samples according to the sample table.

Each row in the sample table results in two cycles. One with a flow rate of 10 microliters per minute and one with 100 microliters per minute. Start the run with a blank.

Then enter the name dilution factor diffusion coefficient at 20 degrees Celsius and molecular weight. For each mutant the diffusion coefficient can be calculated using the web tool on the product website. A link to this tool is provided in the support navigator of the software.

Since it is recommended to run each sample and duplicate, copy the entire table from the blank row to the last sample and paste it at the bottom of the table. Click next in order to proceed. The next screen displays the sample rack.

Place the samples and reagents in the rack according to the layout outlined by the software. Load the rack into the instrument by pressing the load samples button. Then click next.

After loading the samples, a reminder screen with a checklist is displayed. Make sure to check everything and then press start. Data are collected in real time during the run.

When the run is complete, close the window displaying the collected data. Observe that a new result item has appeared in the main window. In order to calculate the concentrations from the data, select the new result item and click the button.

Evaluate via core X 100. Evaluation software is started displaying all the collected data from the toolbar, select concentration analysis, and then calibration free from the menu. In the first window, choose the data to include in the analysis.

Scroll through the list and step through the view list in order to view data for all samples. If you want to exclude any data, remove the check mark from the list. Click next to start the calculation.

The CFCA interaction model is automatically fitted to the experimental data when the fitting is completed, the results are reported for each sample in the result window. Step through the fittings for each sample by selecting the corresponding row in the table. The column called measured concentration shows the concentration in the sample that was analyzed.

The concentration in the original sample is calculated using the dilution factor entered earlier and shown in the column calculated concentration when done, click finish to save the result. Now that we know the concentrations of the proteins from the CFCA analysis, we have an excellent basis for our subsequent kinetic measurements. Kinetic analysis will now help us to understand the important of key amino acid residues for the binding mechanism.

Let's get started with a setup of the kinetic analysis. Kinetic analysis begins by docking a new sensor chip CM five into the instrument in the start screen. Choose to create an assay workflow for kinetic's affinity.

In the top left corner, a dialogue window appears where information about the ligand is entered. Enter the name papain in the ligand name box and choose another protein from the list of types of species. Then select immobilize ligand covalently using sensor chip CM five from the two alternative ligand attachment approaches presented, the recommended assay workflow is now outlined on the right hand side of the window.

It starts with an immobilization step followed by an assay step. Press continue in order to proceed. Save the assay workflow as requested by entering a name and pressing save.

The software now outlines the assay workflow with buttons associated with every step. The first step under sensor surface preparation is find a mobilization pH. Since this pH is known, press enter known value in the dialogue box that appears.

Enter the buffer name acetate and pH 4.5 and choose save. The assay workflow is now updated with a green check mark to indicate that this step has been completed. Now it is time to immobilize press the run button in the immobilized step.

This brings up the same wizard as was used for propane and immobilization for the CFCA analysis immobilization of papin for kinetic analysis is set up in the same way here, except that a more dilute solution is injected for less time. This is in order to immobilize less papin to avoid situations where the binding rates of the cystatin be mutants are limited by diffusion so that kinetic data can be measured in isolation. Since the name of the ligand and immobilization pH were entered in previous steps in the assay workflow, these are both preset here.

The only required update is 50 seconds for the contact time. Immobilization then proceeds as shown previously. The expected binding after immobilization level is 50 R ru.

A green check mark next to the run button indicates that the immobilization is complete. Next, enter the regeneration solution in the workflow in the step. Find regeneration conditions by clicking enter known values in the dialogue box that appears.

Enter the regeneration solution and the contact time. Then click save. Now that the ligand is immobilized and the regeneration conditions entered, all is set to generate kinetics data in the run kinetics Affinity assay box, press the run button the first step in the kinetics.

Affinity wizard outlines the injection sequence of every cycle for single cycle kinetics, which is used here, there is no need to change anything. Click next to proceed. In the next step, keep the default settings indicating priming of the system before run.

Choose to run one startup cycle in order to equilibrate the chip surface. Next, define the injection parameters for the sample. Since the regeneration solution was specified earlier and the setting is predefined, there is no need to change any of the parameters in the screen.

Click next. Finally, specify the samples. Enter two rows for each mutant variant, one blank and the other with sample.

A blank cycle is indicated by setting the highest concentration parameter to zero.Two. Cystatin B variants can be fit in each run. For each mutant enter molecular weight, highest concentration, and dilution factor, press next.

In the following screen, the sample rack is outlined. Prepare a dilution series of the protein samples and place the vials with caps into the rack As outlined on the screen, a vial with buffer is placed in position 13 and a vial with the regeneration solution is placed at position 14. Make sure to fill the water position with fresh water.

Place the rack in the machine and load the samples by pressing the load samples button and click next. As before, perform a final check before proceeding with running the experiment by pressing start interaction data appear in real time. When the run has finished, immediately proceed with running the remaining cystatin B variants according to as just demonstrated.

If the remaining cystatin B variants are run in succession, omit the prime step and startup cycle by unchecking the corresponding check boxes in step. When each run is done, the data can be accessed from the link. All results in the assay workflow.

A dialogue window with all results generated within this workflow appears. In order to calculate the kinetic rate constants, select the result to evaluate and press evaluate be a core X 100 evaluation software is started again displaying all the collective data. Select kinetics affinity from the toolbar menu.

In the first screen, the cysteine three to Syrian mutant is selected for evaluation. The data from this mutant is displayed together with the blank run performed for this mutant proceed by clicking.Next. The next screen shows the data with the blank run subtracted from the run with sample.

Choose to evaluate kinetics by clicking the kinetics button. Now choose to fit the data to a one-to-one interaction model. By clicking the button fit, the fit is displayed on the screen as it is calculated.

Once it is ready, quality controls are automatically applied and highlighted at the bottom. Look through the quality control items and then look at the sensor gram data to make sure that the sensor grams show sufficient curvature. Also, take a look at the residual plot showing the differences between the collected data and the fitted model.

Pay attention to systematic and non-random deviations. This fit is acceptable. Now take a look at the kinetic data in the report tab.

The association rate constant, the dissociation rate constant and the equilibrium dissociation constant. When done, press finish repeat the same procedure for all the other mutants. Here are representative results of CFCA analysis for the different cystatin B variants examined.

The specific binding concentration data are extracted from the sensor grams obtained at flow rates of 10 and 100 microliters per minute. When comparing the concentration determined by a two 80 measurement with that determined by CFCA, it is clear that while in most cases the protein is fully active for the leucine 73 glycine variant, the fraction of active protein is very low. The kinetics of cystatin B variants binding to pepin are determined using single cycle kinetics.

The sensor grams show blank and reference subtracted data with kinetic for a one-to-one interaction model overlaid in black. The specific binding concentration measured by CFCA for the leucine 73 variant is only about 10%of the total protein concentration as determined by a two 80 measurement. When using the correct concentration in calculating the kinetic parameters, it becomes clear that the association rate for Lucien 73 is similar to that of the other variants.

While the association rates of the mutants are comparable to that of the wild type protein, the dissociation rate constant of the mutants can be higher by close to two orders of magnitude with a corresponding increase in the equilibrium dissociation constant. The reduced affinities depends mainly on increased dissociation rates, indicating that both the second binding loop and the C terminal end of cystatin B contribute to the binding affinity by keeping the inhibitor attached to the enzyme once the complex has been formed, the use of calibration free concentration analysis was essential for this conclusion because one of the mutants turned out to have a very low binding activity, My colleague Pol has now shown you how to use bi core X 100 in a structured function study. When performing these experiments, it's important to remember that although both concentration and kinetic analysis are based on the same binding procedure, the experimental conditions are quite different.