Absorption Spectroscopy to Indicate Macromolecule Structural Changes
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KeywordsProtein Circular Dichroism Analysis Probe Structural Changes Sample Macromolecules Tryptophan Chromophore Residue-residue Interactions
The UV-visible absorption spectrum of a biomolecule is the combination of the electronic transitions of all of its component parts or chromophores. The spectrum thus depends on the electronic structures of its component parts, which in turn depend on their environment. Thus, e.g., the absorbance of a tryptophan chromophore that is buried in the hydrophobic core of the protein will be at least slightly different from that of an exposed tryptophan. Two particularly useful applications of UV absorption spectroscopy to probe structural changes are outlined below.
Measuring a UV-visible spectrum with an absorption spectrometer may be used to follow the condensation of a macromolecule sample into particles, though what is actually being probed is scattering of the light rather than absorption. A monotonic increase in absorbance signal outside the absorption bands of the molecules being studied is observed as condensation takes place. In the case of DNA, the addition of a highly charged DNA-binding ligands (such as spermine or [Co(NH3)6]3+) will effect this change. Concomitantly, with the increase in absorbance signal above 300 nm, a decrease in the 260-nm DNA absorption is observed.
DNA or Protein-Melting Curves: Absorption as a Function of Temperature
If there were no residue-residue interactions and no residue-environment interactions in a biomolecule, then the UV-visible absorption spectrum of a well-defined chromophore would be independent of its geometry. In the case of DNA, however, the π– π stacking interactions between base pairs lower the magnitude of the absorbance at 260 nm (the hypochromic effect) compared with that of the isolated bases and in general change the absorbance at most wavelengths. Thus, we can use the change in the UV absorbance signal at a chosen wavelength (usually 260 nm, though at ∼280 nm, A–T base pairs show very little change in absorbance, so this wavelength may be used to probe the role of G–C relative to A–T base pairs) to follow the disruption of base stacking and hence also base pair hydrogen bonding.
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