Light induces destabilization of photoactive yellow protein.
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 PYP(M) 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 PYP(M), respectively, in 100 mM acetate buffer, and 5.26 +/- 0.24 and 4.11 +/- 0.12 M for PYP and PYP(M), respectively, in 100 mM citrate buffer. The free energy change upon denaturation (DeltaG(D)(H2O)), obtained from the denaturation curve, was 11.0 +/- 0.4 and 7.6 +/- 0.2 kcal/mol for PYP and PYP(M), respectively, in acetate buffer, and 11.5 +/- 0.3 and 7.8 +/- 0.1 kcal/mol for PYP and PYP(M), respectively, in citrate buffer. Even though the DeltaG(D)(H2O) value for PYP(M) 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 PYP(M) are indistinguishable, the configurations of the chromophores in their denatured structures are not necessarily identical. Both denatured states are interconvertible through PYP and PYP(M). Therefore, the free energy difference between PYP and PYP(M) is 3.4-3.7 kcal/mol for the protein moiety, plus the additional contribution from the difference in configuration of the chromophore.
Department of Materials Science, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan.
S
S
Ohishi
N
N
Shimizu
K
K
Mihara
Y
Y
Imamoto
M
M
Kataoka
D000085:Acetates / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects*; D002021:Buffers; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D008027:Light; D020283:Photoreceptors, Microbial; D011489:Protein Denaturation / Q000528:radiation effects; D017510:Protein Folding; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D014508:Urea
D000085:Acetates; D001426:Bacterial Proteins; D002021:Buffers; D020283:Photoreceptors, Microbial; D012996:Solutions; C050162:photoactive yellow protein, Bacteria; D014508:Urea