Photophysics of the Red Chromophore of HcRed: Evidence for Cis−Trans Isomerization and Protonation-State Changes

• Published in: The journal of physical chemistry. B 2010-04, Vol.114 (13), p.4678-4685
• Main Authors: Mudalige, Kumara, Habuchi, Satoshi, Goodwin, Peter M, Pai, Ranjith K, De Schryver, Frans, Cotlet, Mircea
• Format: Article
• Language: English
• Subjects: ABSORPTION; Animals; B: Biophysical Chemistry; BASIC BIOLOGICAL SCIENCES; DEACTIVATION; FLUORESCENCE; GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; GROUND STATES; Hydrogen-Ion Concentration; Isomerism; ISOMERIZATION; LIFETIME; Luminescent Proteins - chemistry; NANOSCIENCE AND NANOTECHNOLOGY; PROTEINS; Protons; RELAXATION; Sea Anemones - metabolism; SEAS; SOLVENTS; Spectrometry, Fluorescence; SPECTROSCOPY; Temperature; THERMAL EQUILIBRIUM
• ISSN: 1520-6106; 1520-5207
• DOI: 10.1021/jp9102146

HcRed is a dimeric intrinsically fluorescent protein with origins in the sea anemone Heteractis crispa. This protein exhibits deep red absorption and emission properties. Using a combination of ensemble and single molecule methods and by varying environmental parameters such as temperature and pH, we found spectroscopic evidence for the presence of two ground state conformers, trans and cis chromophores that are in thermal equilibrium and that follow different excited-state pathways upon exposure to light. The photocycle of HcRed appears to be a combination of both kindling proteins and bright emitting GFP/GFP-like proteins: the trans chromophore undergoes light driven isomerization followed by radiative relaxation with a fluorescence lifetime of 0.5 ns. The cis chromophore exhibits a photocycle similar to bright GFPs and GFP-like proteins such as enhanced GFP, enhanced YFP or DsRed, with radiative relaxation with a fluorescence lifetime of 1.5 ns, singlet−triplet deactivation on a microsecond time scale and solvent controlled protonation/deprotonation in tens of microseconds. Using single molecule spectroscopy, we identify trans and cis conformers at the level of individual moieties and show that it is possible that the two conformers can coexist in a single protein due to the dimeric nature of HcRed.