Circular dichroism spectroscopy of fluorescent proteins

• Published in: FEBS letters Vol. 521; no. 1; pp. 31 - 35
• Main Authors: Visser, Nina V, Hink, Mark A, Borst, Jan Willem, van der Krogt, Gerard N.M, Visser, Antonie J.W.G
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 19-06-2002
• Subjects: Animals; Bacterial Proteins - analysis; Bacterial Proteins - genetics; Biochemie; Biochemistry; Circulair dichroism; Circular Dichroism; EPS; Excitation splitting; Green fluorescent protein; Green Fluorescent Proteins; Laboratorium voor Moleculaire biologie; Laboratory of Molecular Biology; Luminescent Proteins - analysis; Luminescent Proteins - genetics; Recombinant Fusion Proteins - analysis; Recombinant Fusion Proteins - genetics; Rotational strength; Secondary structure; Transition energies; Ultraviolet Rays; Rotational strength; Secondary structure; Circulair dichroism; Transition energies; Green fluorescent protein; Excitation splitting
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(02)02808-9

Circular dichroism (CD) spectra have been obtained from several variants of green fluorescent protein: blue fluorescent protein (BFP), enhanced cyan fluorescent protein (CFP), enhanced green fluorescent protein (GFP), enhanced yellow fluorescent protein (YFP), all from
Aequorea victoria, and the red fluorescent protein from the coral species
Discosoma (DsRed). We demonstrate that CD spectra in the spectral fingerprint region of the chromophore yield spectra that after normalization are not coincident with the normalized absorbance spectra of GFP, YFP and DsRed. On the other hand, the CD spectra of BFP and CFP coincide with the absorbance spectra. The resolution of absorption and CD spectra into Gaussian bands confirmed the location of the different electronic band positions of GFP and YFP as reported in the literature using other techniques. In the case of BFP and CFP the location of Gaussian bands provided information of the vibrational progression of the electronic absorption bands. The CD spectrum of DsRed is anomalous in the sense that the major CD band has a clear excitonic character. Far-UV CD spectra of GFP confirmed the presence of the high β-sheet content of the polypeptide chain in the three-dimensional structure.