TITLE: Processing of Circular Dichroism Data Collected at B23
AUTHORS: Rohanah  Hussain, Charlotte S. Hughes, Giuliano  Siligardi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">CD spectroscopy is the essential tool to quickly ascertain in the far-UV region the global conformational changes, the secondary structure content, and protein folding and in the near-UV region the local tertiary structure changes probed by the local environment of the aromatic side chains, prosthetic groups (hemes, flavones, carotenoids), the dihedral angle of disulfide bonds, and the ligand chromophore moieties, the latter occurring as a result of protein–ligand binding interaction. Qualitative and quantitative investigations into ligand-binding interactions in both the far- and near-UV regions using CD spectroscopy provide unique and direct information whether induced conformational changes upon ligand binding occur and of what nature that are unattainable with other techniques such as fluorescence, ITC, SPR, and AUC.</div><br/><div class = "text-block">This chapter provides an overview of how to perform circular dichroism (CD) experiments, detailing methods, hints and tips for successful CD measurements. Descriptions of different experimental designs are discussed using CD to investigate ligand-binding interactions. This includes standard qualitative CD measurements conducted in both single-measurement mode and high-throughput 96-well plate mode, CD titrations, and UV protein denaturation assays with and without ligand.</div><br/><div class = "text-block">The highly collimated micro-beam available at B23 beamline for synchrotron radiation circular dichroism (SRCD) at Diamond Light Source (DLS) offers many advantages to benchtop instruments. The synchrotron light source is ten times brighter than a standard xenon arc light source of benchtop instruments. The small diameter of the synchrotron beam can be up to 160 times smaller than that of benchtop light beams; this has enabled the use of small aperture cuvette cells and flat capillary tubes reducing substantially the amount of volume sample to be investigated. Methods, hints and tips, and golden rules tomeasure good quality, artifact-free SRCD and CD data will be described in this chapter in particular for the study of protein–ligand interactions and protein photostability.</div></div>

[STEPS]
?. [Loading CDApps for Processing of Circular Dichroism Data Collected at B23]
Below are the initial steps required to load CDApps software:Open the CDApps software by:(a) On-site—Computer > Software (W:) > cd_programs > CD Apps > CD Apps.exe. Double click the CD Apps icon.(b) Off-site—double-clicking the CD Apps icon.The following options page enables the User to select the type of analysis appropriate for the data set.(a) CD Measurement (including Automation)—analysis options available include 1 to n spectra, titration, UV denaturation, thermal melt using both the Quantum Peltier (+5 °C to +95 °C) and Linkam MDS 600 (–150 °C to +350 °C) controllers and 96-well format.(b) CD Titration.The analysis using both of these methods shall be discussed. For all types of analyses, the most important bits of information for subsequent analyses including conversion of units and secondary structure estimations (SSE) are concentration, pathlength, concentration and molecular weight and Δε (the average amino acid molecular weight).
?. 
?. [Analysis]
Select the “CD Measurement (including Automation)” (highlighted in Fig. 1).
?. [Analysis]
Click on the “Sample Description” section of the sample line to be completed and enter details, e.g., name of the sample (Fig. 2).
?. [Analysis]
To add experimental data to the spreadsheet:Select the “Experiment Data” tab, selecting the appropriate button for the type of file to be uploaded (.ols or .csv/.txt).Generally, files collected at the beamline are saved as .ols files. Files collected using both the benchtop Chirascan instrument and the beamline modules can be converted into .csv format.Attach the appropriate data file to the sample line by clicking on the file to highlight (line becomes blue), then drag and drop the file across to the sample line.Experiment details (number of scans, wavelength ranges, wavelength increment) are automatically completed for the attached file.
?. [Analysis]
Complete the remaining empty experiment details (concentration, concentration units (mg/mL or μM), volume (μL),Δε calculated, molecular weight (Da), temperature (°C), pathlength (cm), slit (mm), and integration time(s)).
?. [Analysis]
New rows can be created by selecting “Add blank row” and repeating steps 4–6. If all of the details of a row are to be replicated for additional rows, this can easily be done by a single right-click and selecting the “replicate” option. Appropriate data can then be attached using step 5.
Note: For any type of analysis, the correct scan type needs to be selected and appropriate baseline allocated. Baseline is assigned to background or buffer measurements, which will be subtracted from all other measurements selected in the group during the analysis. Depending on the analysis selected, the software will subtract the baseline differently. For 1 to n Spectra multiple entries should be used during the analysis, one per sample measured which can each contain multiple scans per loaded file. The 1 to n Spectra option creates an average scan for each loaded file and subtracts the baseline from each sample. UV Denaturation analyses subtract the average spectra of the baseline from every individual scan in the file.
?. [Analysis]
From here, a range of analyses are available for different types of data sets. The type of analysis is selected using the “Scan type” drop-down menu for each sample line (see Note 17). Each analysis option will be discussed below.
?. [Analysis]
Complete the workbook with the appropriate number of sample lines and data.Ensure that the data files to be analyzed are within the same wavelength range and increments. For analyses in the far-UV region (180–260 nm) where secondary structure estimations (SSE) are desired as part of the analysis, endure that loaded files are collected in 1 nm increments.Select “1 to n Spectra” option from drop-down menu for “Scan type.”
?. [Analysis]
Highlight (blue) samples to be analyzed by clicking on the desired sample line and click “Analyse Experimental Data.”
?. [Analysis]
Select the zeroing region for the data which will be used to off-set all data sets. This is often in a region where no CDsignal is observed. Click “Graph Data.”
?. [Analysis]
Graphs will appear in the Excel Workbook, detailing the different units for CD presentation (Δε, ΔA).
Note: Only after the zeroing has been performed can SSE be calculated. A new tab will appear under the zeroing section (Fig. 3b, c).
?. [Analysis]
For SSE, select the appropriate algorithm (CONTINLL [19–21], CDSSTR, or SECLON3 [20]) and database (SP29, SP23, SP1, SP37, SP2, SP42, SMP50, SP43, SDP48, SMP56, CLSTR).
?. [Analysis]
Data will be output into an Excel Workbook containing graphical and numerical outputs. Graphs can be used as they are by copying, or numerical outputs can be used for further analysis and redesigning of graphs for data presentation.
?. Select UV denaturation option from drop-down menu for “Scan type” (Fig. 2).
?. Highlight the Sample and Baseline sample lines. Highlighted lines will appear blue.
Note: Unlike the 1 to n analysis option, only one sample can be analyzed at a time.
?. Click “Analyse Experiment Data.”
?. Set the off-set region for the data (where no CD signal is observed).
?. Select the wavelength to monitor for UV denaturation by either clicking on the region of the observed signal in CDApps or selecting the wavelength from the drop-down menu (Fig. 4).
?. Click “Graph Wavelength to Monitor” button.
?. Under the UV Denaturation Fitting tab in CDApps, select the most appropriate fit for the data points (Fig. 4).
?. Alternatively, copy the data from the “Folding Change” tab of the Excel workbook for analysis using alternative software.
?. Under the SSE (Protein) tab, select appropriate algorithm for protein secondary structure estimation (Fig. 5).
?. Select Thermal Melt option from drop-down menu for “Scan type.”
?. Highlight (click to make line blue) the Sample and Baseline sample lines.
Note: Unlike the 1 to n analysis option, only one sample can be analyzed at a time.
?. Click “Analyse Experiment Data.”
?. Set the off-set region for the data (where no CD signal is observed).
?. Select the wavelength to monitor for thermal denaturation by either clicking on the region of the observed signal in CDApps or selecting the wavelength from the drop-down menu.
?. Click “Graph Wavelength to Monitor” button.
?. Under the Boltzmann tab in CDApps, select the “Auto Fit Boltzmann Curve” button or, if the kinetics are known for the system and the analysis serves as a check to fit the details that can be manually inputted, then click “Manual Fit Boltzmann Curve” button (Fig. 6).
?. Alternatively, copy the data from the “Folding Change” tab of the Excel workbook for analysis using alternative software.
?. Under the SSE (Protein) tab, select appropriate algorithm for protein secondary structure estimation (Fig. 7).
?. Allocate baseline and sample labels to the datasets by selecting the appropriate scan type from drop-down menu (Fig. 8a):(a) 96 HTCD Baselines for baseline dataset.(b) 96 HTCD Samples for sample dataset.
?. Input experimental parameters into the workbook (see Note 17).
?. Insert the sample description for each sample in the plate by either clicking a cell to highlight and type in details directly, alternatively complete from the “Description” section of the “Sample Details” tab. Do this from the “Experiment details” section for each sample line when highlighted. A dialog box informing the User of the number of experimental details entered will appear when the overall sample line is changed, or if the “Analyse Experimental Data” button is selected. Click OK to continue analysis (see Notes 18–20).
?. Select the Sample and Baseline sample lines by clicking the desired line (the line becomes blue).
?. Click “Analyse Experiment Data” (see Note 17).
?. Set the off-set region for the data (where no CD signal is observed). Click “Graph Data” button (Fig. 8b).
?. If a particular wavelength is to be monitored across the plate for a change in signal, this can be selected from the “Wavelength to monitor (nm) section of CDApps. Data for this analysis will be available in the “Folding Change” tab of the Excel Workbook (Fig 8b)”.
?. For SSE, under the SSE (protein) tab, choose the algorithm (CONTINLL, SECLON3, CDSSTR) and the database (SP29, SP23, SP1, SP37, SP2, SP42, SMP50, SP43, SDP48, SMP56, CLSTR) most appropriate for the sample measured. Click “Estimate.” Data from this analysis is available in the Excel workbook. The easiest way to access is via the Index page of the Excel workbook, click the cell containing the Sheet number reference next to the appropriate title which generally contains reference to the algorithm and database used followed by “SSE component Data.”
?. [Analysis]
Load CDApps and click the “CD Titration” button on the option window (Fig. 9).
?. [Analysis]
Select the “Fixed” volume option at the top of the workbook.
?. [Analysis]
Under the “Experiment Settings” tab, complete the details for the cell parameters, Host and Ligand. The information provided here will be used to determine the volumes required at each step of the titration (Fig. 10a).
?. [Analysis]
To determine the Ligand stock concentration to be used and the volumes to be used at each titration point, in the spreadsheet section of the workbook enter the titration points under the “Molar Ratio” column (Fig. 10a).
?. [Analysis]
Add the number of the Ligand stock (1, 2, or 3) which will be used at each point (Fig. 10a) to the “Stock Number” column. Ensure aliquot volumes are feasible within the error of the pipettes. For ease of the User, each Ligand stock is color coded to quickly identify which stock to use.
?. [Analysis]
Complete experimental parameters under the “Spectrometer Settings” tab (Fig. 10b).
?. [Analysis]
Import data files into the workbook under the “Experiment Data” tab by selecting the appropriate button for the data to be imported (either .ols or .csv/.txt files) and locate the files. When multiple files are to be imported, highlight all appropriate files (Ctrl + Click/Shift + Click). Click Open. The files will be imported into CDApps in the “Imported Data Files” column (Fig. 10c).
?. [Analysis]
Match the appropriate files to the “Experiment Component.” This can be done one by one or in bulk. Four arrows are used to control the order and matching of files (Fig. 10c). (a) ↑ and ↓ control the position of the file in the “Imported Data Files” column. (b) ← and → control the transfer of files between the “Imported Data Files” and “Matched Data Files” columns.
?. [Analysis]
Only when in the “Imported Data Files” column can the order of the files be changed. Once they have been moved to the “Matched Data Files” column they cannot be reordered. To rearrange the order, files will have to be moved to the “Imported Data Files” column, ordered, and then transferred to the “Matched Data Files” column.
?. [Analysis]
Files can be transferred between columns either individually by selecting individual files or in bulk by highlighting multiple files (Ctrl + Click/Shift + Click) before using ← and →.
?. [Analysis]
Once the workbook has been completed, save the spreadsheet by clicking ““Save As” Spreadsheet.” This will enable the spreadsheet to be loaded at another time to continue the analysis and to ensure that a copy has been saved in case of any downstream erroneous analysis.
?. [Analysis]
The Workbook is saved as an Excel workbook, and the experimental plan can be printed from the “Titration” tab of theworkbook for the convenience of the User to perform the experiment. The plan details the volumes required at each step, and ligand stocks are color coded for quick identification by the User (Fig. 11).
?. [Analysis]
Instruction on how to perform the analysis is provided below.
?. [Analysis]
Select the “Increasing” volume option at the top of the workbook.
?. [Analysis]
Under the “Experiment Settings” tab, complete the details for the cell parameters, Host and Ligand. The informationprovided here will be used to determine the volumes required at each step of the titration (Fig. 12a). CDApps will automatically calculate the maximum volume of ligand which can be added within acceptable dilution limits.
?. [Analysis]
To determine the Ligand stock concentration to be used and the volumes to be used at each titration point, in the spreadsheet section of the workbook enter the titration points under the “Molar Ratio” column (Fig. 12a).
?. [Analysis]
Add the number of the Ligand stock (1, 2, or 3) which will be used at each point (Fig. 12a) to the “Stock Number” column. CDApps will notify you if the maximum volume of added ligand is exceeded. If this happens, adjust the concentrations of the ligand stocks to reduce the total added volume, using a maximum of three stocks using CDApps. Ensure aliquot volumes are feasible within the error of the pipettes. For ease of the User, each Ligand stock is color coded to quickly identify which stock to use.
?. [Analysis]
Complete experimental parameters under the “Spectrometer Settings” tab (Fig. 12b).
?. [Analysis]
Import data files into the workbook under the “Experiment Data” tab by selecting the appropriate button for the data to be imported (either .ols or .csv/.txt files) and locate the files. When multiple files are to be imported, highlight all appropriate files (Ctrl + Click/Shift + Click). Click Open. The files will be imported into CDApps in the “Imported Data Files” column (Fig. 12c).
?. [Analysis]
Match the appropriate files to the “Experiment Component.” This can be done one by one or in bulk. Four arrows are used to control the order and matching of files (Fig. 12c).(a) ↑ and ↓ control the position of the file in the “Imported Data Files” column.(b) ← and → control the transfer of files between the “Imported Data Files” and “Matched Data Files” columns.
?. [Analysis]
Only when in the “Imported Data Files” column can the order of the files be changed. Once they have been moved to the “Matched Data Files” column they cannot be reordered. To rearrange the order, files will have to be moved to the “Imported Data Files” column, ordered, and then transferred to the “Matched Data Files” column.
?. [Analysis]
Files can be transferred between columns either individually by selecting individual files or in bulk by highlighting multiple files (Ctrl + Click/Shift + Click) before using ← and →.
?. [Analysis]
Once the workbook has been completed, save the spreadsheet by clicking ““Save As” Spreadsheet.” This will enable the spreadsheet to be loaded at another time to continue the analysis and to ensure that a copy has been saved in case of any downstream erroneous analysis.
?. [Analysis]
The Workbook is saved as an Excel workbook, and the experimental plan can be printed from the “Titration” tab of the workbook for the convenience of the User to perform the experiment. The plan details the volumes required at each step, and ligand stocks are color coded for quick identification by the User (Fig. 13).
?. [Analysis]
Instruction on how to perform the analysis is provided below.
?. [Determination of the Binding Constants]
Load CDApps, select CD Titration button, and load the appropriate Spreadsheet. All details and
?. [Determination of the Binding Constants]
Click “Analyse Experiment Data” button.
?. [Determination of the Binding Constants]
Off set data to a region where no CD signal is detected.
?. [Determination of the Binding Constants]
If necessary, restrict the wavelength range of the data to remove noise. For measurements in the far-UV region requiring SSE, ensure restriction does not compromise the wavelength ranges required by the algorithms and datasets, e.g., 190 nm and above.
?. [Determination of the Binding Constants]
From the “Titrations –A –B” tab or the “Wavelength to monitor (nm)” section, select the wavelength from which binding constant calculations will be made. This is usually the region on the spectra which shows the biggest change in magnitude over the course of the titration. Click “Graph Wavelength to monitor” button (Fig. 14).
?. [Determination of the Binding Constants]
A “Binding” tab will then appear. From here fitting can be performed for the points to determine the Kd of the binding interaction. Fitting can either be automatically fitted through CDApps using the “Auto Fit Binding Curve” button, or manually by entering Δε values and clicking “Add Binding Model” button.
?. [Determination of the Binding Constants]
Load CDApps, select CD Titration button, and load the appropriate Spreadsheet. All details and
?. [Determination of the Binding Constants]
Click “Analyse Experiment Data” button.
?. [Determination of the Binding Constants]
Off set data to a region where no CD signal is detected.
?. [Determination of the Binding Constants]
If necessary, restrict the wavelength range of the data to remove noise. For measurements in the far-UV region requiring SSE, ensure restriction does not compromise the wavelength ranges required by the algorithms and datasets, e.g., 190 nm and above.
?. [Determination of the Binding Constants]
From the “Titrations –A –B” tab or the “Wavelength to monitor (nm)” section, select the wavelength from which binding constant calculations will be made. This is usually the region on the spectra which shows the biggest change in magnitude over the course of the titration. Click “Graph Wavelength to monitor” button (Fig. 14).
?. [Determination of the Binding Constants]
A “Binding” tab will then appear. From here fitting can be performed for the points to determine the Kd of the binding interaction. Fitting can either be automatically fitted through CDApps using the “Auto Fit Binding Curve” button, or manually by entering Δε values and clicking “Add Binding Model” button.