Probing the Origins of Spectroscopic Responses to Analyte-Induced Conformational Changes in Fluorescently-Labeled Cod III Parvalbumin

An emerging strategy for the development of reagentless biosensors is the coupling of fluorescence responses to analyte-induced conformational changes within fluorescently-labeled proteins. In this work, we have examined the absorbance and the steady-state and time-resolved fluorescence responses to Ca2+-induced conformational changes within cod III parvalbumin (C3P), which was labeled at cysteine-18 with the fluorescent probes fluorescein, acrylodan or nitrobenzoxadiazole (NBD). The basis of the analyte-induced responses was further characterized by examining reporter group accessibility and rotational reorientation dynamics in the presence and absence of analyte. We show that the fluorescence responses are often based on a combination of direct and indirect effects, and that changes in fluorescence quantum yield can be reinforced or opposed by simultaneous changes in absorbance, dramatically affecting the sensitivity of the observed signal to analyte concentration. In the case of NBD, the response is fully consistent with an increase in the nonradiative rate constant owing to increased exposure of the reporter group to solvent as a result of the analyte-induced conformational change. However, in the case of acrylodan and fluorescein, the response reflects a specific interaction between the probe and either the added Ca2+ or the amino acid residues in the vicinity of Cys-18, which changes in response to protein conformation. Overall, the best performance is obtained from the NBD-labeled C3P, where a signal change of almost 50% was obtained upon complete binding of Ca2+, providing a detection limit of 100 nM, a dynamic range of 2 orders of magnitude. The other two probes produced poor responses (<10% total change in intensity on binding Ca2+), highlighting the difficulty associated with predicting the behavior of a labeled protein, and showing the complexity that exists at present in the design of sensitive biosensors based on labeled regulatory proteins.