BCH210H1 Lecture Notes - Lecture 12: Nuclear Magnetic Resonance Spectroscopy, Fluorescence Spectroscopy, Nuclear Magnetic Resonance

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BCH210 Lecture 11 studying protein structures
Tryptophan Fluorescence
- The presence of the indole ring allows tryptophan to fluoresce in UV light at a
wavelength of 270-290nm
o The indole ring absorbs light in this wavelength range
- The emission of tryptophan occurs between 310-355nm and is sensitive to the polarity of
its local environment in a protein
- In a polar environment, the fluorescence is red-shifted to longer wavelengths
o This happens when Trp is exposed to water
- Trp found on the inside of the protein, therefore in a hydrophobic environment absorbs
shorter wavelengths it is blue-shifted
- The intensity of fluorescence depends on the environment in which Trp is found
o Longer wavelength and lower intensity water-exposed environments
o Shorter wavelength and higher intensity hydrophobic environments
- You can follow the unfolding / folding of a protein using Trp fluorescence
- Tryptophan fluorescence differs at different pH conformational change
Protein Chromophores
- All proteins absorb light at 220nm
- Chromophore groups usually contain conjugated double bonds
- These chemical groups absorb UV and/or visible light at characteristic wavelengths
- Aromatic rings and amide carbonyls are important chromophores found in proteins
- A protein’s structure influences the accessibility of these groups to light
o Can be used to characterize a protein’s structure
Protein structure techniques
- Fluorescence spectroscopy
- Infra-red (IR) spectroscopy
o Use light in the infra-red region
- Circular dichroism (CD) spectroscopy
- X-ray crystallography
- Nuclear magnetic resonance (NMR) spectroscopy
- Electron microscopy (EM)
Infra-red (IR) spectroscopy
- Proteins contain vibrating, stretching and bending groups
- These motions lead to an absorption of infrared radiation as a function of wavelength
- The most prominent bonds contribute to the IR spectrum
o C-H
o N-H
o C=O
- Can be used to measure secondary structure and hydrogen bond
- Proteins absorb light in the 780-300nm range due to the peptide bonds
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