CD - Circular Dichroism Spectroscopy

CD - Circular Dichroism Spectroscopy

Please acknowledge the SIP core facility ( RRID: SCR_018986) in publications, on posters, or in talks if you use any instruments in the SIP core facility. Please include SIP's RRID (RRID: SCR_018986) and the grant numbers for instruments funded through instrumentation grants in your acknowledgements. This is a requirement from the funding agencies and is crucial for future funding. Find example text on theÌýAcknowledgement PageÌýor the individual instrument pages.

CD in in Biochemistry, Biophysics and Structural Biology

Circular Dichroism (CD) spectroscopy is a technique used to measure the differential absorption of left- and right-handed circularly polarized light by chiral molecules, providing insights into their structural properties. Chiral molecules, which are non-superimposable on their mirror images, interact differently with polarized light depending on their spatial arrangement. This unique interaction forms the basis of CD spectroscopy.

CD spectroscopy has a wide range of applications across various scientific disciplines, including Chemistry, Biochemistry, Biology, Physics, Chemical and Biological Engineering, and Material Sciences. Within the realm of Biochemistry, Biophysics, and Structural Biology, CD spectroscopy is typically divided into two distinct applications based on the ultraviolet (UV) light region used: far UV and near UV.

Far UV CD spectroscopy, which operates within the 180 to 240 nm wavelength range, is particularly useful for studying the optical activity of the protein backbone. This method is employed to analyse the secondary structure of proteins, providing information about elements like alpha helices, beta sheets, and random coils. It also serves as a powerful tool to assess protein stability under various conditions, as changes in the secondary structure can be detected through alterations in the CD spectrum.

On the other hand, near UV CD spectroscopy focuses on the 250 to 350 nm wavelength range. This technique produces a "fingerprint" of the environment surrounding aromatic amino acids, such as phenylalanine, tyrosine, and tryptophan, within proteins. These aromatic residues are sensitive to their local environment, and any conformational changes, such as those induced by ligand binding, complex formation, or changes in buffer conditions, can lead to detectable shifts in the CD spectrum. This makes near UV CD spectroscopy a valuable tool for investigating protein structure and structural dynamics.

The utility of CD spectroscopy extends beyond proteins. It is also employed to study other biomolecules, including peptides, lipids, RNA, and DNA. The ability of CD spectroscopy to provide detailed information about the conformational and structural properties of these molecules makes it an indispensable tool in the investigation of various biological and chemical systems.

Methods and Instruments

Left, upper panel shows Lysozyme spectra, native, unfolded and refolded, left lower panel shows Lysozyme refolding measured by stopped-flow CD, right upper panel shows CD spectra of an RNA g-quadruplex taken between 15 and 95 0C in 10 0C steps.

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Key highlights of CD in Biology, Biochemistry, and Biophysics

  • Investigations into structure, stability, and kinetics
  • Secondary structure analysis of proteins, peptides, RNA, and DNA
  • Thermodynamic stability assessments
  • Studies on protein folding and refolding
  • Tertiary structure and conformational changes in proteins, particularly using near UV CD to observe contributions from phenylalanine, tyrosine, and tryptophan residues
  • Ligand-induced changes in both structure and stability
  • Stopped-flow experiments in single or sequential mixing mode
  • And more...
Chirascan Plus CD spectrometer
Cuvette holder and detectors

Instrument and Accesories

CD spectrometerÌý

We have a modular Applied Photophysics Chirascan Plus CD and Fluorescence Spectrometer.

Cuvette holders

  • 4 single cell holders for rectangular cells for different path lengths (0.5, 1, 4 and 10 mm) and applications
  • One 4 position turret for 10 mm path length rectangular cells

CD Cells:

  • Rectangular CD cuvettes with 0.5, 1, 4 and 10 mm path lengths

Temperature Control:

  • Peltier controlled sample holders and in-cell temperature sensors

Accessories:

  • Fluorescence and fluorescence polarization detector with scanning emission monochromator
  • Stopped-flow module for absorbance, CD, fluorescence and polarization
  • Automatic titration unit

Essential Information for Using SIP's CD

Acknowledgements Protocols Training Scheduling Cost Location
Acknowledgements

Why?

Acknowledgements are essential for ensuring the continued success of the Shared Instruments Pool (SIP). They enable us to secure the necessary funding to sustain and expand the SIP, ensuring that our instruments are in optimal working condition and that the methods we offer are at the forefront of biochemical and biophysical research.

Please include SIP's RRID number (RRID: SCR_018986) in your acknowledgements. This allows funding organisations and potential grant reviewers to easily locate publications supported by SIP, helping to evaluate the impact of SIP on our research community.

If facility staff have provided substantial assistance, please consider acknowledging them. If they contributed significantly to the intellectual aspects or conducted important experiments, co-authorship may also be appropriate.

Example text:

We thank the Shared Instruments Pool (RRID: SCR_018986), Department of Biochemistry, University of Colorado Boulder for the use of the Applied Photophysics Chirascan Plus CD spectrometer. The CD was funded by NIH S10RR028036. We also thank [Name and title of the facility member providing significant help] for their invaluable assistance with data collection and evaluation.

Please contact Dr Erbse to obtain detailed protocols and arrange an initial project consultation and personalised training sessions. Protocols are available as PDF files on the instrument computer, with printed copies stored alongside the instruments.

Interested users can contact Dr Erbse to discuss planned experiments and arrange training sessions. These sessions will involve the use of actual user samples alongside standards, enabling users to collect preliminary data during the training and receive help from core staff right away if troubleshooting or optimisation is needed. Users are welcome to request additional training or support sessions at any time. We are always happy to provide a refresher if it has been a while.

After your training is completed, you will be invited to join the CD Google calendar.Ìý

Sign Up Rules:Ìý
Up to Friday the week before the planned experiment users can sign up for a maximum of 2 days. In the week of the experiment users can sign up for additional time if available.

  • Initial consultation is free. SIP staff are happy to assist with a short pilot experiment if it can be accommodated within SIP's resources.
  • Regular user groups are expected to buy into SIP with a monthly flat fee according to their SIP usage level. For detailed information, please contact Dr Annette Erbse.
  • Users are required to provide all consumables specific to their experiments.Ìý
  • Costs for necessary repairs, services, or replacement parts due to normal wear and tear will be shared among all user groups, based on the time used over the past two years. Please note that assuming the instrument is handled properly, such repairs or replacements are infrequent, and costs may arise after a user’s period of use has ended.
  • Users are responsible for covering the costs of repairs or replacement parts needed due to damage caused by carelessness or neglect.

The CD is located on the third floor of JSCBB in the D-Wing, room D381, on East Campus. Proxcard access is required at all times.