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

Abstract

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 fu...