Abstract
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.
Highlights
The manner by which proteins attain their functional conformation has been a major puzzle in Biochemistry since the seminal experiments of Anfinsen [1]
Based on extensive stopped-flow fluorescence spectroscopy data, we showed that apoflavodoxin kinetic folding is described by [25,26]: Iof f ' unf olded apof lavodoxin ' Ion ' native apof lavodoxin ðSðSchchememee11ÞÞ
In which U, I and N represent unfolded protein, off-pathway folding intermediate and native apoflavodoxin, respectively, Kij is the equilibrium constant of the i-j equilibrium, mij is the constant that describes the denaturant concentration-dependence of Kij, superscript 0 designates the parameter at zero denaturant concentration, [D] is the denaturant concentration, fi is the fractional population of state i at a particular denaturant concentration, Yobs is the observed spectroscopic signal, ai is the spectroscopic property of state i at zero denaturant concentration, and bi is the constant describing the denaturant concentrationdependence of the spectroscopic signal of state i
Summary
The manner by which proteins attain their functional conformation has been a major puzzle in Biochemistry since the seminal experiments of Anfinsen [1]. In which U, I and N represent unfolded protein, off-pathway folding intermediate and native apoflavodoxin, respectively, Kij is the equilibrium constant of the i-j equilibrium, mij is the constant that describes the denaturant concentration-dependence of Kij, superscript 0 designates the parameter at zero denaturant concentration, [D] is the denaturant concentration, fi is the fractional population of state i at a particular denaturant concentration, Yobs is the observed spectroscopic signal, ai is the spectroscopic property of state i at zero denaturant concentration, and bi is the constant describing the denaturant concentrationdependence of the spectroscopic signal of state i.
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