Dipolar relaxation within the protein matrix of the green fluorescent protein: a red edge excitation shift study.
The fluorophore in green fluorescent protein (GFP) is localized in a highly constrained environment, protected from the bulk solvent by the barrel-shaped protein matrix. We have used the wavelength-selective fluorescence approach (red edge excitation shift, REES) to monitor solvent (environment) dynamics around the fluorophore in enhanced green fluorescent protein (EGFP) under various conditions. Our results show that EGFP displays REES in buffer and glycerol, i.e., the fluorescence emission maxima exhibit a progressive shift toward the red edge, as the excitation wavelength is shifted toward the red edge of the absorption spectrum. Interestingly, EGFP displays REES when incorporated in reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT), independent of the hydration state. We interpret the observed REES to the constrained environment experienced by the EGFP fluorophore in the rigid protein matrix, rather than to the dynamics of the bulk solvent. These results are supported by the temperature dependence of REES and characteristic wavelength-dependent changes in fluorescence anisotropy.
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India.
Sourav
S
Haldar
Amitabha
A
Chattopadhyay
D002021:Buffers; D004143:Dioctyl Sulfosuccinic Acid / Q000737:chemistry; D005454:Fluorescence Polarization / Q000379:methods*; D005990:Glycerol / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008823:Micelles; D012997:Solvents / Q000737:chemistry; D013501:Surface-Active Agents / Q000737:chemistry; D013696:Temperature; D014867:Water / Q000737:chemistry
D002021:Buffers; D008823:Micelles; D012997:Solvents; D013501:Surface-Active Agents; C485184:enhanced green fluorescent protein; D014867:Water; D004143:Dioctyl Sulfosuccinic Acid; D049452:Green Fluorescent Proteins; D005990:Glycerol