Fluorescence of Alexa fluor dye tracks protein folding.
Fluorescence spectroscopy is an important tool for the characterization of protein folding. Often, a protein is labeled with appropriate fluorescent donor and acceptor probes and folding-induced changes in Förster Resonance Energy Transfer (FRET) are monitored. However, conformational changes of the protein potentially affect fluorescence properties of both probes, thereby profoundly complicating interpretation of FRET data. In this study, we assess the effects protein folding has on fluorescence properties of Alexa Fluor 488 (A488), which is commonly used as FRET donor. Here, A488 is covalently attached to Cys69 of apoflavodoxin from Azotobacter vinelandii. Although coupling of A488 slightly destabilizes apoflavodoxin, the three-state folding of this protein, which involves a molten globule intermediate, is unaffected. Upon folding of apoflavodoxin, fluorescence emission intensity of A488 changes significantly. To illuminate the molecular sources of this alteration, we applied steady state and time-resolved fluorescence techniques. The results obtained show that tryptophans cause folding-induced changes in quenching of Alexa dye. Compared to unfolded protein, static quenching of A488 is increased in the molten globule. Upon populating the native state both static and dynamic quenching of A488 decrease considerably. We show that fluorescence quenching of Alexa Fluor dyes is a sensitive reporter of conformational changes during protein folding.
Laboratory of Biochemistry, Wageningen University, Wageningen, The Netherlands.
Simon
S
Lindhoud
Adrie H
AH
Westphal
Antonie J W G
AJ
Visser
Jan Willem
JW
Borst
Carlo P M
CP
van Mierlo
D001059:Apoproteins / Q000737:chemistry*; D016948:Azotobacter vinelandii; D001426:Bacterial Proteins / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D005418:Flavodoxin / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008301:Maleimides / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013997:Time Factors
C504424:Alexa Fluor 488 C5-maleimide; D001059:Apoproteins; D001426:Bacterial Proteins; D005418:Flavodoxin; D005456:Fluorescent Dyes; D008301:Maleimides; C026932:apoflavodoxin; D003545:Cysteine