Quenching-resolved emission anisotropy studies with single and multitryptophan-containing proteins.
Measurements of the anisotropy of protein fluorescence as a function of an added collisional quencher, such as acrylamide, are used to construct Perrin plots. For single tryptophan containing proteins, such plots yield an apparent rotational correlation time for the depolarization process, which, in most cases, is approximately the value expected for Brownian rotation of the entire protein. Apparent limiting fluorescence anisotropy values, which range from 0.20 to 0.32 for the proteins studied, are also obtained from the Perrin plots. The lower values for the limiting anisotropy found for some proteins are interpreted as indicating the existence of relatively rapid, limited (within a cone of angle 0 degrees--30 degrees) motion of the tryptophan side chains that is independent of the overall rotation of the protein. Examples of the use of this fluorescence technique to study protein conformational changes are presented, including the monomer in equilibrium dimer equilibrium of beta-lactoglobulin, the monomer in equilibrium tetramer equilibrium of melittin, the N in equilibrium F transition of human serum albumin, and the induced change in the conformation of cod parvalbumin caused by the removal of Ca+2. Because multitryptophan-containing proteins have certain tryptophans that are accessible to solute quencher and others that are inaccessible, this method can be used to determine the steady state anisotropy of each class of tryptophan residues.
D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000818:Animals; D002118:Calcium; D004798:Enzymes; D005454:Fluorescence Polarization / Q000379:methods; D006801:Humans; D008433:Mathematics; D010320:Parvalbumins / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins; D014364:Tryptophan
D004798:Enzymes; D010320:Parvalbumins; D011506:Proteins; D014364:Tryptophan; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; D002118:Calcium