Vibrational spectra of red fluorescent protein DsRed have been studied for the first time by polarization-sensitive multiplex coherent anti-Stokes Raman scattering at two excitation wavelengths, 545 and 583 nm, in resonance with the absorption bands of the immature “green” and mature “red” protein chromophores. Overall vibrational patterns of both DsRed chromophores were found to be similar to each other and to differ from that of S65T-GFP at pH8. The combined analysis of our CARS data and published structural information suggest that both “green” and “red” DsRed species possess an extended chromophore structure. Consequently, our data suggest that π-bonding system extension during isomerization around the cis peptide bond between Phe 65 and Gln 66 is a necessary but not sufficient step in DsRed chromophore maturation.
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