Circular Dichroism (CD)

For information on access fees, policies and getting started at the CMI, see the CMI Access Page.

CD at the CMI

Circular Dichroism (CD) Spectroscopy is used to determine the optical isomerism and secondary structure of molecules. Circular dichroism (measured in molar ellipticity) is the difference in absorption of left-handed and right-handed circularly polarized light and can be observed in optically active molecules with chiral centers. Proteins have many chiral centers. CD spectra in the Far-UV region (185 – 250 nm) can be used to determine protein secondary structure. Characteristic peaks for Thermal stability (Tm) can be measured by following changes in molar ellipticity with increasing temperature.

The CMI has a Jasco J-1500 CD Spectropolarimeter with a Peltier temperature controller and single cuvette holder.   

In April 2023, the Jasco J-815 was retired, after serving the BCMP department and HMS for almost 2 decades.

 

CD Sample Data

 

Poly-L-lysine in three conformations: alpha helix, b-sheet and random coil.  Far-uv region of the CD spectra reveal peaks characteristic of these secondary structure elements, with helices having negative peaks at 222 and 208 and a positive peak around 190, b-sheet proteins have a negative peak around 218 and a positive peak around 195, and a random coil has a negative peak around 200 (as does poly-proline II helix).

 

CD Key features

Features

  • CD scans are easy, rapid, and non-destructive (~10 min/sample)
  • Label-free
  • Far-UV CD can predict secondary structure
  • Near-UV CD can reflect changes in tertiary structure
  • Thermal stability experiments can measure reversible unfolding

Limitations

  • Some commonly used solvents absorb in the region of the CD (eg. NaCl)
  • Requires concentrated, purified samples

Improvements in J-1500

Key J-1500 improvements:

  • Higher Sensitivity
    • lower minimal recommended protein concentration
    • Faster scanning speeds 
  • Improved Peltier Cell Holder - PTC-517
    • Faster temperature gradients
    • No separate power supply -Peltier turns on when the J-1500 is on
  • Multivariate Secondary Structure Estimation (SSE) Software  
    • Uses several models with data ranges varying from 176-260 to 200-260
    • Includes optical constant calculator to convert mdeg to molecular ellipticity

Data Files - About CMI Data Files

Users are responsible for storage of all raw and processed data collected at the CMI.

  • Users should have a plan to copy or transfer all raw and process data to their own local or cloud storage system.
  • While the CMI allows temporary local storage of CMI User data on the instrument computer, we make no guarantees on the security or long-term availability of any data at the CMI.
  • For most (but not all) CMI technologies, the raw data files and recommended readable exports are relatively small and can be readily transferred electronically. 
  • See specific instruments for exceptions and for details about the software, data file types and recommended data exports. 

Data Sharing:

  • Currently, a Generalist Repository is the recommended data repository for most CMI data types, as stable specialist data repositories have not been established.

Data Files - CD - Jasco

Technology Circular Dichroism (CD)
Instruments Jasco J-815, J-1500
Recommended Repository Generalist Repository
Protein Circular Dichroism Data Bank
       
Software Type Data Collection & Analysis
Current Version Spectra Manager, Version 2.15.20.1  
Data Files (Type, ~size) experiment file (spectrum)
experiment file (interval)
.jws
.jwb
25 KB/expt
Readable Exports exported CD spectrum .txt
.cvs
5 KB/expt
25 KB/expt

CD Data Collection Services

CD Service Overview

In addition to instrument training, the CMI is now offering basic protein Circular Dichroism Data Collection services, including Far-UV scan and thermal denaturation experiments.

CMI CD Instrument: Jasco J-1500

  • Protein Far-UV Scan for Secondary Structure Analysis
    • this will strictly be a data collection service and analysis using CD deconvolution algorithms (e.g. Dichroweb) will be left to the user.
  • Protein Thermal Stability (fixed wavelength variable temperature measurement)

Data Collection Fees Summary

Data Collection

  • Limited Data Collection Services are offered.
  • Service fees are based on labor and supplies costs, and will be charged for all completed services, regardless of experimental outcome. 
  • Before submitting samples for data collection, users must approve the estimated charges and be given a date and time for sample delivery.
    • External Users will also be required to submit a PO and a signed CMI User Agreement.
  • Most CMI Data Collection Services include a setup fee plus a per-sample data collection fee.
    • Some services include replicate measurements by default in the per-sample fee. For others, there is a reduced-price replicate measurement fee, if collected in the same dataset.
  • Nanobody services not available to commercial users at this time.
  • Current Harvard Life Lab commercial users are offered a 25% discount off the standard commercial rates.

All Data Collection Fees

CMI Resources

CMI Jasco J-1500 CD Getting Started Guide, guide to experimental design and standard Far-UV CD protocols.

Additional resources are available at the instrument. Instrument and software manuals are located in the “Jasco Manuals” desktop folder on the instrument computer.

Additional Resources

Jasco Training Videos provide more information on various topics related to CD measurements and the Spectra Manager program.

DichroWeba free online tool for determining protein secondary structures based on CD spectra.

  • Academic users may apply for a username and password. 

CDToolXa free, downloadable software program that enables processing, displaying, archiving, calibrating, comparisons, and analyses of circular dichroism (CD) spectroscopic data. 

Protein Circular Dichroism Data Bank, a database of CD data

  • The  YouTube channel provides video tutorials on a variety of CD-related topics, including the successful measurement of a CD spectrum and analysis using DichroWeb and CDToolX.

BeStSel (Beta Structure Selection), a free online tool based on a novel method for the secondary structure determination and fold recognition from protein circular dichroism spectra, especially beta-sheet rich proteins.

ValiDichro tests the quality of CD data based on based on common characteristics of CD protein spectra observed in the literature. 

 

*J-815 RETIRED APRIL 2023 - CMI Jasco J-815 CD Getting Started Guide. Replaced with newer J-1500 instrument.

 

  • High Quality Quartz cuvette
    • 1 mm pathlength cuvette
      • Hellma 110-1-40, style 110-QS (available from Sigma-Aldrich)
      • Starna 21-Q-1
    • 1 cm pathlength cuvette
      • Hellma 100-10-40, style 100-QS (available from Sigma-Aldrich)
      • Starna 1-Q-10 or 21-Q-10
  • your sample
  • buffer blank

For Far-UV measurements (protein secondary structure): 1 mm
For Near-UV measurements:  1 cm or 1 mm, depending on application and concentration

 

 

Assay Buffers

Buffer selection is critical for accurate CD measurements. Solvent absorbance can severely interfere with the CD signal. Many commonly used buffers and additives absorb in the far UV region used for CD measurements.

  • The ideal CD experiment is performed in a buffer:
    • in which your protein is well behaved and soluble
    • with no buffer absorbance through the range of the CD spectrum.
  • Always take a scan of your buffer blank to determine whether absorbance interferes with your region of analysis.
    • Ensure that the buffer blank is well matched to the final dilution of your protein (even trace amounts of some solvents will interfere with the CD measurements).
  • Phosphate buffers with little-to-no NaCl are recommended. 10 mM phosphate buffer is ideal.
  • Ideal aqueous solutions contain as little chloride as possible.
    • NaCl and Tris buffers are not recommended but can be tolerated at low concentrations. 
    • If salt is required, SO42- or F- are preferred counter ions, as they are transparent in the far UV. Potassium fluoride may be a good substitute for chloride-containing salts.
    • Chloride ions will result in a loss of signal below about 200±5 nm, which affects secondary structure estimation, but generally will not block detection of the alpha-helical peaks (208 nm and 222 nm) or the beta-sheet peak (218 nm).
  • DTT, ß-ME, or EDTA can be present at low concentrations (1 mM).
  • Some detergents are fairly transparent in the far UV (e.g. Chaps and octylglucoside). Triton detergents should be avoided, as they can oxidize readily and form UV-absorbing materials.
  • DMSO, formamides, and imidazole absorb strongly in the far UV region, and thus should be avoided in a CD experiment.

Samples

  • An accurate protein concentration is required for all CD experiments.
    • Over-estimating or under-estimating the protein concentration interferes with signal
    • Algorithms that estimate secondary structure depend on correct protein concentrations
  • The amount of sample required for a CD experiment depends on the size, cuvette pathlength and type of measurement being performed.
  • 1 mm pathlength cuvettes holds 300 µl and a 1 cm cuvette holds 3 ml.
  • Required protein concentration is inversely proportional to the cuvette pathlength, and thus a 1 mm path cuvette requires 10x the concentration of a 1 cm cuvette.
  • Recommended concentrations for Far-UV measurements of protein secondary structure are (Jasco J-1500):
    • 0.1 mg/mL in 1 mm path cuvette
    • 0.01 mg/mL in 10 mm patch cuvette
    • Mol/L=115/(MW*7000)*10/pathlength(mm).
    • If possible, make a concentrated stock solution of your protein (at least 2X) and dilute as needed. Protein requirements vary with secondary structure, as alpha-helical proteins have a stronger signal than beta-sheet proteins.
  • Protein aggregates can interfere with the CD signal.
    • Assess protein heterogeneity via light scattering.
    • Purify protein samples with soluble aggregates by size-exclusion chromatography.
  • Samples can be recovered from the cell. This is not recommended for thermal melts, unless you know your thermal denaturation is reversible.