title: Mechanisms of quenching of Alexa fluorophores by natural amino acids.
abstract: 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.
authors.affiliation: School of Applied and Engineering Physics, 212 Clark Hall, Cornell University, Ithaca, New York 14853, USA.
authors.forename: Huimin
authors.initials: H
authors.lastname: Chen
authors.forename: Syed S
authors.initials: SS
authors.lastname: Ahsan
authors.forename: Mitk'El B
authors.initials: MB
authors.lastname: Santiago-Berrios
authors.forename: Hector D
authors.initials: HD
authors.lastname: Abruña
authors.forename: Watt W
authors.initials: WW
authors.lastname: Webb
mesh_terms: 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
chemical_list: C417561:Alexa 488 hydrazide; D000596:Amino Acids; D005456:Fluorescent Dyes; D006834:Hydrazines; D011506:Proteins; D014443:Tyrosine; D006639:Histidine; D014364:Tryptophan; D008715:Methionine
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