Mechanisms of quenching of Alexa fluorophores by natural amino acids.
Quenching of fluorophores by the same proteins that they covalently label is a phenomenon that is neither well-known nor well-characterized. It is often assumed that fluorophores are unperturbed by their target proteins. However, it has been observed that attached fluorophores can be quenched by contact with amino acids within the same protein, and this property has been exploited to report on changing conformational states or intramolecular dynamics of proteins. We show in this communication that fluorescence of Alexa dyes is, in fact, quenched by interactions with Trp, Tyr, Met, and His residues through a combination of static and dynamic quenching mechanisms. In light of this finding, the potential effect of intramolecular quenching should be considered in the interpretation of data that involves quantitative measurements of fluorescence intensity in proteins.
Chen
Huimin
H
School of Applied and Engineering Physics, 212 Clark Hall, Cornell University, Ithaca, New York 14853, USA.
Ahsan
Syed S
SS
Santiago-Berrios
Mitk'El B
MB
Abruña
Hector D
HD
Webb
Watt W
WW
D000596:Amino Acids / Q000737:chemistry*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D006834:Hydrazines / Q000737:chemistry*; D008715:Methionine / Q000737:chemistry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
C417561:Alexa 488 hydrazide; D000596:Amino Acids; D005456:Fluorescent Dyes; D006834:Hydrazines; D011506:Proteins; D014443:Tyrosine; D006639:Histidine; D014364:Tryptophan; D008715:Methionine
J Am Chem Soc. 2003 Nov 26;125(47):14564-72
14624606
Bioconjug Chem. 2003 Nov-Dec;14(6):1133-9
14624626
Proc Natl Acad Sci U S A. 2007 Jun 19;104(25):10459-64
17556539
Chemphyschem. 2009 Jul 13;10(9-10):1389-98
19475638
Biochemistry. 2002 Mar 26;41(12):4156-64
11900559
Proc Natl Acad Sci U S A. 2001 Oct 9;98(21):11867-72
11592997
Proc Natl Acad Sci U S A. 1967 Aug;58(2):719-26
5233469
J Histochem Cytochem. 1999 Sep;47(9):1179-88
10449539