ABSTRACT
The energetics, protein dynamics, and diffusion coefficients of three mutants of photoactive yellow protein, R52Q, P68A, and W119G, were studied by the transient grating and pulsed laser-induced photoacoustic method. We observed a new dynamics with a lifetime of approximately 1 micro s in the transient grating signal, which is silent by the light absorption technique. This fact indicates that, after the structure change around the chromophore is completed (pR(1)), the protein part located far from the chromophore is still moving to finally create another pR (pR(2)) species, which can transform to the next intermediate, pB. Although the kinetics of pR(2)-->pB-->pG are very different depending on the mutants, the enthalpies of the first long-lived (in micro seconds, 100-micro s range) intermediate species (pR(2)) are similar and very high for all mutants. The diffusion coefficients of the parent (pG) and pB species of the mutants are also similar to that of the wild-type photoactive yellow protein. From the temperature dependence of the volume change, the difference in the thermal expansion coefficients taken as indicator of the flexibility of the structure between pG and pR(2) is measured. They are also similar to that of the wild-type photoactive yellow protein. These results suggest that the protein structures of pR(2) and pB in these mutants are globally different from that of pG, and this structural change is not altered so much by the single amino acid residue mutation. This is consistent with the partially unfolded nature of these intermediate species. On the other hand, the volume changes during pR(1)-->pR(2) are sensitive to the mutations, which may suggest that the volume change reflects a rather local character of the structure, such as the chromophore-protein interaction.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Photoreceptors, Microbial/chemistry , Photoreceptors, Microbial/genetics , Acoustics , Biophysical Phenomena , Biophysics , Diffusion , Hot Temperature , Kinetics , Light , Models, Molecular , Mutagenesis, Site-Directed , Mutation , Protein Folding , Spectrophotometry , Temperature , Time FactorsABSTRACT
Themodynamic and transport properties of intermediate states of the photocyclic reaction of photoactive yellow protein (PYP) were studied by a combination of the pulsed laser-induced transient grating (TG), transient lens (TrL), and photoacoustic (PA) spectroscopies from tens of nanoseconds to hundreds of milliseconds. The diffusion coefficients (D) of PYP in the ground state (pG) and of the second intermediate state (pB) were determined by the TG analysis, and it was found that D of pG is about 1.2 times larger than D of pB. At the same time, D at various denatured conditions were measured using guanidine hydrochloride as the denaturant. D of completely unfolded protein is about 0.4 times that of the native form. The enthalpy of pB is estimated to be 60 kJ/mol by the TrL method with an assumption that the volume change of pB is not sensitive to the temperature. Since the enthalpy of the first intermediate state (pR) is as high as 160 kJ/mol, it implies that most of the photon energy is stored as the strain of the protein in pR, and this may be the driving force for the successive reaction to pB. From the temperature dependence of the volume change, the difference in the thermal expansion coefficients between pG and pR was calculated. All of the characteristic features of PYP, the negative volume change, the larger thermal expansion coefficient, and the slower diffusion process, indicate that the intermediate pR and pB are reasonably interpreted in terms of the unfolded (loosened) protein structure.
