Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent protein (EGFP): Acid-base specificity

Ahmed A. Heikal, Samuel T. Hess, Watt W. Webb

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130 Scopus citations


Green fluorescent protein (GFP), isolated from Aequorea victoria jellyfish, has been used extensively as a noninvasive intracellular pH indicator and site-specific fluorescent marker in biochemistry, cell biology, and molecular genetics. Numerous mutations, aimed at optimizing spectroscopic and thermodynamic properties of GFP, have been created for different applications. Fluorescence correlation spectroscopy (FCS) reveals that the enhanced green fluorescent protein mutant (EGFP; S65T/F64L) undergoes external proton exchange with the buffer on ∼45-300 μs time scale with pKa = 5.8 ± 0.1 [Proc. Natl. Acad. Sci. USA 95 (1998) 13573]. This contribution represents a comprehensive characterization of pH and excitation mode (wavelength, one and two photon (2P)) effects on the spectroscopy, excited-state dynamics, and rotational mobility of EGFP aiming at elucidating the significant electronic states of this molecular system. EGFP exhibits a large 2P action cross-section and, therefore, is well suited for intracellular imaging using 2P fluorescence microscopy.

Original languageEnglish (US)
Pages (from-to)37-55
Number of pages19
JournalChemical Physics
Issue number1
StatePublished - Dec 1 2001
Externally publishedYes

Bibliographical note

Funding Information:
The authors are very grateful to Dr. Petra Schwille for her help with the 2P excitation cross-section measurements at the early stage of this project. The research at Cornell was carried out in the Developmental Resource for Biophysical Imaging Opto-electronics with funding provided by NIH (P41 RR04224) and NSF (BIR 8800278). STH benefited from NSF Graduate Research Fellowship and NIGMS-NIH Molecular Biophysics Training Grant (GM08267).


  • Enhanced green fluorescent protein
  • Excited-state dynamics
  • Green fluorescent protein
  • Two-photon excitation cross-section


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