Our group pioneered the application of   wavelength-selective   fluorescence   as   a   novel   approach   to monitor organization and dynamics of probes and proteins in membranes and membrane-mimetics such as micelles and reverse micelles. Wavelength-selective fluorescence relies on the slow rates of   solvent  relaxation around an excited  state  fluorophore,  which is   a    function    of   the   motional   restriction  imposed on  the    solvent  molecules    in   the  immediate   vicinity of the   fluorophore.   Utilizing   this approach, it  becomes   possible   to probe   the mobility parameters of  the environment  itself  (which is   represented   by  the  relaxing  solvent molecules) using the fluorophore merely as the   reporter   group (Chattopadhyay (2003) Chem. Phys. Lipids). Further, since the ubiquitous solvent for biological systems is water, the information obtained in such cases   will come   from   the   otherwise ‘optically   silent’   water molecules. This makes the use of wavelength-selective fluorescence approach significant in  biology since  hydration  plays  a  crucial   modulatory   role   in   a large   number of   vital cellular events.   Important applications of this approach include monitoring the environment of the functionally relevant tryptophans in  the  prototypical  ion   channel gramicidin (Mukherjee and Chattopadhyay (1994) Biochemistry; Rawat et al. (2004) Biophys. J.; Kelkar and Chattopadhyay (2005) Biophys. J.;  Rawat et al.   (2005)  Biophys. J.;  Chattopadhyay et al.   (2008) Biophys. J.), and in  the  lytic  peptide  melittin  from  bee  venom   (Ghosh et al.   (1997)   Biochemistry;   Raghuraman   and   Chattopadhyay (2003)   Langmuir;     Raghuraman   and Chattopadhyay   (2004)  Biophys. J.;   Raghuraman and Chattopadhyay   (2007) Biophys. J.;   Haldar et al. (2008) J. Phys. Chem. B).   Interesting applications include monitoring  defined depths in  the  membrane utilizing  depth-dependent    solvent relaxation as a dipstick   (Chattopadhyay   and   Mukherjee   (1999) Langmuir) and lipid-protein interactions in membranes   (Kelkar   and   Chattopadhyay   (2007)   Biochim. Biophys. Acta; Ghosh et al. (1997) Biochemistry).

     In addition, we have applied the wavelength-selective fluorescence approach to monitor organization and dynamics of functionally important tryptophan residues in tubulin   (Guha et al. (1996) Biochemistry), erythroid   spectrin  (Chattopadhyay et al. (2003)  Protein Sci.; Kelkar et al. (2005) Biopolymers)   and alpha-lactalbumin (Chaudhuri et al. (2009), submitted for publication). In a recent work, we examined the wavelength-selective fluorescence characteristics of the green fluorescent protein (GFP).  Our results show that the slow dipolar relaxation of GFP is  due  to the  rigid  protein   matrix  of  GFP  around   its fluorophore, independent of the viscosity of the surrounding medium (Haldar and Chattopadhyay (2007) J. Phys. Chem. B)

 

Membrane Organization and Dynamics using the Wavelength-Selective Fluorescence Approach