Encyclopedia of Biophysics

Living Edition
| Editors: Gordon Roberts, Anthony Watts, European Biophysical Societies

Absorption Spectroscopy to Indicate Macromolecule Structural Changes

  • Alison RodgerEmail author
Living reference work entry
DOI: https://doi.org/10.1007/978-3-642-35943-9_777-1
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Keywords

Protein Circular Dichroism Analysis Probe Structural Changes Sample Macromolecules Tryptophan Chromophore Residue-residue Interactions 
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Synonyms

Definition

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.

Macromolecule Condensation

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 ππ 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.

The data from such an experiment are usually illustrated as a melting curve or a derivative melting curve summarized by the so-called melting temperature, Tm (e.g., Fig. 1). Tm is the temperature where the absorbance is the average of the duplex and single-stranded DNA absorbances. Thermodynamic data, transition energy and entropy and hence Gibbs free energy, relating to the stability of the duplex may also be extracted from melting curves (Marky and Breslauer 1987). An example for calf thymus DNA and the stabilizing effect of a cationic ligand bound to the DNA are illustrated in Fig. 1.
Fig. 1

Melting curves of calf thymus DNA (100 μM) in the presence of different concentrations of P-[Fe2L3] 4+ (illustrated) measured at 260 nm in a 1-cm path length cuvette (Meistermann et al. 2002)

Cross-References

References

  1. Marky LA, Breslauer KA (1987) Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves. Biopolymers 26:1601–1620CrossRefPubMedGoogle Scholar
  2. Meistermann I, Moreno V, Prieto MJ, Molderheim E, Sletten E, Khalid S, Rodger PM, Peberdy J, Isaac CJ, Rodger A, Hannon MJ (2002) Intramolecular DNA coiling mediated by metallo-supramolecular cylinders: differential binding of P and M helical enantiomers. Proc Natl Acad Sci U S A 99:5069–5074CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© European Biophysical Societies' Association (EBSA) 2018

Authors and Affiliations

  1. 1.Department of Molecular SciencesMacquarie UniversitySydneyAustralia

Section editors and affiliations

  • Alison Rodger
    • 1
  1. 1.Department of Molecular Sciences, Macquarie UniversityNWSAustralia