A fast and accurate procedure to collect and analyze unfolding fluorescence signal: the case of dystroglycan domains

Biophys Chem. 2004 Feb 1;107(2):197-211. doi: 10.1016/j.bpc.2003.09.005.

Abstract

Monitoring the fluorescence signal upon unfolding often represents a very effective method to rapidly retrieve the first preliminary structural information on a protein domain. The relationship between intrinsic fluorescence signals and unfolding of proteins are discussed, including several practical considerations for properly setting fluorescence experiments and the phenomenological equations required to analyze the spectra. In particular, a fast and accurate method which allows to minimize the deleterious effect of photobleaching is provided. A number of unfolding reactions relative to immunoglobulins (IgG and IgM) and to the different domains of the adhesion molecule dystroglycan are presented. Special attention is dedicated to a alpha-dystroglycan immunoglobulin-like domain showing a "reverse" behavior of the fluorescence signal as a function of the denaturing agent concentration.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / chemistry
  • Cytoskeletal Proteins / chemistry*
  • Dystroglycans
  • Guanidines / chemistry
  • Immunoglobulins / chemistry
  • Mathematics
  • Membrane Glycoproteins / chemistry*
  • Photobleaching
  • Protein Denaturation
  • Protein Folding*
  • Recombinant Proteins / chemistry
  • Spectrometry, Fluorescence / methods

Substances

  • Amino Acids
  • Cytoskeletal Proteins
  • Guanidines
  • Immunoglobulins
  • Membrane Glycoproteins
  • Recombinant Proteins
  • Dystroglycans