Screening and detecting ligand binding to biological macromolecules by circular dichroism

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Screening and detecting ligand binding to
biological macromolecules by circular dichroism

• Matthew Engel
• BME 501
• December 12, 2006

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SRCD Basic Principles

• Chiroptical ultraviolet based spectroscopy
• Spectra is composite of all secondary structural
  motifs in sample.
• Sample can be glycoprotein, protein, nucleic acid
  or mixture.
• Patented applications includes screening of HIV-1
  and HIV-2 gp120, gp41, reverse transcriptase and
  protease alone or in the presence of a potential
  ligand.

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Spectral Properties
RED a-helix (myoglobin) YELLOW polyproline
helix (collagen VI) BLUE ß-sheet (concavalin)
SRCD vs. conventional CD -high signal to
noise -low wavelength data
Wallace, B. A., and Janes, R. W. (2001)
Synchrotron radiation circular dichroism
spectroscopy of proteins secondary structure,
fold recognition and structural genomics, Curr
Opin Chem Biol 5, 567-571. Wallace, B. A. (2000)
Synchrotron radiation circular-dichroism
spectroscopy as a tool for investigating protein
structures, Journal of synchrotron radiation 7,
289-295.
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Design
PEM
Automatic Sample Changer
50 kHz
in vacuo
R
L
I0
PMT
Circular Polarizer
Sample Chamber
PC
Sutherland, J. C. (2002) Simultaneous Measurement
of Circular Dichroism and Fluorescence
Polarization Anisotropy, in Proceedings of SPIE
(Cohn, G. E., Ed.), pp 126-137.
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Calcium Fluoride Sample Cell
1.5 cm Path length 4 micron Width 4 mm
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SRCD
B19 erythroviral capsid protein /- Calcium
Plus Calcium
Minus Calcium
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Algorithmic Analysis
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Automatic Sample Changer
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10 Sample Cells
M
M
D 3 cm
Horizontal Motor Mount
D 20 cm
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