Light Induces Destabilization of Photoactive Yellow Protein

• Published in: Biochemistry (Easton) Vol. 40; no. 9; pp. 2854 - 2859
• Main Authors: Ohishi, Shin'ichi, Shimizu, Nobutaka, Mihara, Ken'ichi, Imamoto, Yasushi, Kataoka, Mikio
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 06.03.2001
• Subjects: Acetates - chemistry; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Bacterial Proteins - radiation effects; Buffers; Circular Dichroism; Hydrogen-Ion Concentration; Light; Photoreceptors, Microbial; Protein Denaturation - radiation effects; Protein Folding; Solutions; Spectrophotometry, Ultraviolet; Thermodynamics; Urea
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi001846i

To understand the effect of visible light on the stability of photoactive yellow protein (PYP), urea denaturation experiments were performed with PYP in the dark and with PYPM under continuous illumination. The urea concentrations at the midpoint of denaturation were 5.26 ± 0.29 and 3.77 ± 0.19 M for PYP and PYPM, respectively, in 100 mM acetate buffer, and 5.26 ± 0.24 and 4.11 ± 0.12 M for PYP and PYPM, respectively, in 100 mM citrate buffer. The free energy change upon denaturation (ΔG D H 2 O), obtained from the denaturation curve, was 11.0 ± 0.4 and 7.6 ± 0.2 kcal/mol for PYP and PYPM, respectively, in acetate buffer, and 11.5 ± 0.3 and 7.8 ± 0.1 kcal/mol for PYP and PYPM, respectively, in citrate buffer. Even though the ΔG D H 2 O value for PYPM is almost identical in the two buffer systems, the urea concentration at the midpoint of denaturation is lower in acetate buffer than in citrate buffer. Although their CD spectra indicate that the protein conformations of the denatured states of PYP and PYPM are indistinguishable, the configurations of the chromophores in their denatured structures are not necessarily identical. Both denatured states are interconvertible through PYP and PYPM. Therefore, the free energy difference between PYP and PYPM is 3.4−3.7 kcal/mol for the protein moiety, plus the additional contribution from the difference in configuration of the chromophore.