Circular dichroism software

The program works well for globular proteins. The main advantage of this method is that it provides very good estimates of the structure of globular proteins. A neural network is first trained using a set of known proteins so that the input of the CD at each wavelength results in the output of the correct secondary structure.

The trained network is then used to analyze unknown proteins. K2D gives a good estimate of the helical and sheet contents of both proteins and polypeptides. However, the K2D program does not estimate turns. CDNN is not suitable for the analysis of polypeptides and it currently is not being distributed. Convex Constraint Algorithm CCA : The CCA algorithm 44 deconvolutes a set of spectra into a desired number of basis spectra, which, when recombined, generate the entire data set with a minimum deviation between the original data set and the reconstructed curves.

It is very useful to determine how many different states contribute to the changes in CD as a function of ligand or temperature 26 , 45 — The method was developed to estimate protein conformation, but is poorer than least squares, SVD or neural net analysis. The protocol described here will cover: setting up CD machines to collect data; procedures for obtaining high quality, reproducible data; and methods to analyze CD spectra to estimate the secondary structures of proteins.

For more detailed information, there are several reviews which discuss CD spectroscopy in detail, including the preparations of buffers, protein samples and cuvettes and converting raw data to molar and mean residue elllipticity 48 — 53 and analysis of data to yield secondary structure information 3 , 26 , 50 , Three further protocols will cover 1 the determination of the thermodynamics of protein folding from CD data collected as a function of temperature 47 ; 2 determination of the free energy of folding and binding constants from CD data collected as a function of denaturants, osmolytes or ligands 55 and 3 determination of the kinetics of folding from CD data collected as a function of time All of the following reagents are available from SigmaAldrich.

Circular dichroism cuvettes Hellma cells inc. Wear gloves. Dissolve Adjust the pH to 7. Adjust the volume to ml. Keeps indefinitely in a glass bottle with a plastic cap.

Adjust the pH to Adjust the pH to 6. Keeps indefinitely in a bottle with a plastic cap. Combine 50 grams of sodium carbonate with Dissolve by stirring on hot plate. Filter though Whatman 1 filter paper.

Add 8. Dilute to ml. This solution lasts about 1 year in a brown bottle at room temperature. Dissolve 2 mg of bovine serum albumin in 1 ml of water and filter through a Millipore filter. CD machines must be calibrated on a regular basis to check that the ellipticity values and wavelengths are correct 52 , In addition, the ratio of ellipticity of camphorsulfonic acid at Cuvettes for CD measurements must be clean and dry.

Protein residues can be dissolved using 6M guanidine-HCl. After soaking, cells should be rinsed with water and ethanol and either dried by suction using an aspirator or blown dry with nitrogen or compressed air that has passed through a filter to remove impurities.

This method is the best method for cleaning cells with 0. CD machines have very powerful lamps that promote the ionization of oxygen to ozone. Ozone is toxic and also will quickly destroy the mirrors in the optics of the machines. Most CD machines must be flushed with nitrogen to remove oxygen before the machine is turned on and during operation.

Nitrogen sources include tanks of pre-purified nitrogen, which last about 5 hours and high-pressure liquid nitrogen tanks, which produce gas, and will last one to two weeks depending on usage.

Commercial nitrogen should be free of oxygen and most other impurities, but some manufacturers suggest using a trap to remove impurities to be on the safe side. If you are new to CD and there is no one to teach you the operation of your specific machine call the manufacturer and ask for their start up protocol.

Determine the concentration of the protein stock solutions. Options A and B require that the protein have tyrosines or tryptophans. Option C does not work with collagen-like proteins with high proline contents. Option A. Determination of protein concentration from the difference spectrum of the protein dissolved in 6 M guanidine at pH Pipette exactly the same volume 0. Add exactly the same volume e. Correct the spectrum for the baseline.

Calculate the concentration of protein. Correct the measured concentration for dilution. The mean residue concentration can be calculated by multiplying the molar concentration by the number of amino acids in the protein.

The number of milligrams per milliliter of protein is calculated by multiplying the molar concentration by the molecular weight. If the proteins are denatured by the Guanidine solution, the difference method should give one band at nm. Option B. Determination of protein concentration from the aromatic spectrum determined in 6M guanidine-HCl, pH 6.

Run a baseline spectrum of two cells with an equal volume of Guanidine-HCl in each side. Add a small aliquot of protein solution to the sample side, and an equal volume of the buffer to the reference side.

The oxidized form of these compounds absorb strongly at nm and the oxidation rates are faster in solutions containing protein than in plain buffers, so it is hard to subtract their contributions.

Collect the spectrum of the protein between and nm and calculate the protein concentration using the formulas:. The concentrations determined at and nm should agree. Option C. Determination of protein concentration using a microbiuret procedure Aliquot protein samples in buffer e.

Prepare a standard curve containing 0. Add 0. Mix with a vortex mixer. Correct the absorbance of each unknown sample for the contribution of the buffer and read the protein concentration from the curve. Correct for dilution.

This assay can be done in microtiter plates using half the volume of each reagent. The plates can be read at nm although the color intensity is lower at this wavelength than at nm. The microbiuret method using freshly prepared Benedict's reagent should be linear for protein concentrations ranging between 0 and 1.

Prepare the protein samples. For typical measurements in a 0. For measurements in 0. Nitrogen displaces oxygen. Only operate a CD machine in a well ventilated room. Do not close the door.

If a tank of liquid nitrogen begins to vent because pressure has built up, leave the room and allow the excess nitrogen to disperse before reentering. Turn on the water supply or circulating water bath chiller to the lamp housing, if the lamp is water cooled. If a water supply is used, make sure the filter is clean.

If a bath is used, make sure the water is clean. Turn on the circulating water bath for temperature control. If the temperature is controlled using a temperature-regulated cell compartment that requires a heat sink set the temperature of the bath to 20 or 25 degrees. If the temperature is controlled using water-jacketed cells set the temperature of the bath to the desired temperature see step 9. Turn on the lamp, if it has a separate switch, before you turn on the power to the rest of the CD machine or computers.

Set the desired temperature. For a previously uncharacterized protein, one should collect spectra at multiple temperatures and correlate the spectra with some measurement of the activity or the protein, e. Once a stability range is established, data may be collected at more closely-spaced intervals to determine whether there are spectral changes indicative of folding intermediates using the CCA algorithm 44 or singular value decomposition 47 , 61 , Collecting CD spectra for the determination of secondary structure - TIMING 2 -4 hours for collection of 5 samples and 5 baselines at a single temperature, depending on wavelength range and interval and number of repeat spectra.

Set the desired equilibration time. Usually globular proteins reach equilibrium within 2 minutes, but some proteins, e. Set the half-band width between 1 and 1. These values give spectra with good signal to noise levels and adequate spectral resolution, since UV bands are broad Figures 2a and 2b. Lyoszyme was obtained from Sigma L and dissolved in sodium phosphate, 10 mM. Set the wavelength range: from to nm for 0.

Set the wavelength interval, 0. Data collected at 0. Set the time for data collection at each point i. For samples with a concentration of approximately 0. If one is collecting replicate spectra, at intervals from 0. If the protein concentration is low or the buffer has high absorbance, increase the averaging time.

The relative signal to noise will increase as the square root of signal averaging time. Set the instrument time constant.

For routine collection of CD spectra this should be milliseconds. For rapid collection of data, e. Set the instrument to record the ellipticity and the photomultiplier tube PMT voltage. When light hits the photomultiplier of the CD machine a current is induced.

Most CD machines maintain constant current by raising the voltage as the amount of light decreases. As it scans to lower wavelengths, the absorbance will increase and the PMT voltage will rise. The signal to noise will greatly diminish once the dynode voltage goes above volts and the data are often become very noisy and unreliable.

For protein concentrations ranging from 0. With 0. When the dynode voltage 2c goes above V e. Determine the spectrum of the blank. Fill the cell with water and determine its spectrum. Suitable buffers should have no ellipticity, but their increased absorbance compared to water decreases the signal to noise. The spectrum of the cell containing water should be relatively flat, but may be displaced from that of pure air due to the birefringence of the cell Figure 2a. Collect a spectrum of the buffer to make sure that it does not have any ellipticity due to dichroic components or a high absorbance leading to a very poor signal to noise and possibly false peaks.

Note that phospholipids are asymmetric and have CD bands. If samples are suspended in phospholipids it is essential that the spectrum of the blank contains the same concentration of protein-free lipids.

The spectrum of the buffer and water should overlay each other, within the experimental error, but the spectrum of the buffer usually has a lower signal to noise than the spectrum of water at low wavelengths see Figure 2a. Clean the cell, fill with protein solution and collect the CD spectrum. It is a good idea to collect data 2 or 3 times for new samples to make sure that the sample is at equilibrium and the signal is not changing as a function of time. Many CD machines can collect multiple spectra automatically.

If the protein is at equilibrium, replicate scans of the protein solutions should overlay each other and not drift as a function of time Figures 2a and 2b. If the data sets overlay each other one, average the data sets.

For the most accurate estimates of protein secondary structure, data should be collected to nm or lower wavelengths, in 0.

Since data below nm may have low signal to noise, three to five scans should be collected and averaged. Smooth the spectra of the sample and blank. Most CD machines have built-in smoothing algorithms and some will automatically pick the best smoothing parameters - refer to the manual. The smoothing algorithms that are used depend on the manufacturer. If the machine uses Savitsky-Golay smoothing 63 , and data is collected at 0. If data is collected at shorter wavelength intervals increase the number of points.

Some smoothing protocols give estimates of the goodness of smoothing by calculating whether the difference between the raw and smoothed data has the statistical characteristics of noise. Check that the data have not been over-smoothed by subtracting the smoothed curve from the raw data. The points should be evenly distributed around zero. Some CD machines will automatically calculate the residuals from the smoothing and these can be viewed on the spectrometer in its data viewing mode.

Subtract the smoothed baseline from the smoothed spectrum of the sample. The ellipticity for most proteins should be close to zero between and nm. The mean residue weight of a peptide is the molecular weight divided by the number of backbone amides number of amino acids -1 if the protein is not acetylated. If the protein or peptide is monomeric and does not aggregate under the experimental conditions used to collect the CD data, the spectra collected at different protein concentrations and path lengths should give the same mean residue ellipticities Figure 2d.

Binary data cannot be edited by a text editor or imported into a spread sheet or converted to formats that can be used for analysis programs. If data has been collected at different wavelength ranges, e. If the data do not agree with each other, make sure the proper baseline has been subtracted for each sample and that the path lengths of the cells are correct.

If they still do not agree, redo the measurements! Analyze the data using appropriate methods Options A—D. It is best to use as many methods as possible for the most accurate results. Option A Evaluating the secondary structure of globular proteins using data collected between and nm. The CDPro package or the on-line analysis programs at DicroWeb are recommended for the most accurate estimates of secondary structure.

The DicroProt suite of programs uses older versions of the programs available at DicroWeb and in CDPro, but the results are similar to those of the more modern versions. Evaluating the secondary structure of globular proteins using data truncated below nm or above nm. For data sets collected over truncated wavelength ranges e.

G, and Wallace, B. Whitmore, L. Biopolymers 89 : Nucleic Acids Res 32 : W - DichroWeb is produced in the lab of Professor B. Grants to B. It takes format-independent input data from any type of CD instrument, enables a wide range of standard and advanced processing methods, and, in a single user-friendly graphics-based package, takes raw data through the entire processing procedure and, importantly, uses data-mining techniques to retain in the final output all the information associated with the processing.

It permits the facile comparison of data obtained from different instruments without the need for reformatting and displays it in graphical formats suitable for publication.

It also includes the ability to automatically archive the processed data.