Structural Change of Site-Directed Mutants of PYP: New Dynamics during pR State

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.

Ultrafast infrared spectroscopy reveals a key step for successful entry into the photocycle for photoactive yellow protein

“…Probably, the inability of the chromophore to break the hydrogen bond on a sufficiently fast time scale leads to its decay to the original ground state by means of a mixture of vibrational cooling and reisomerization to the trans configuration, rather than to its entry into the photocycle by a relaxation of the cis isomer. The important role of the strength of the chromophore's hydrogen bond with the backbone N atom of Cys-69 in determining the success of the isomerization is compatible with the higher quantum yield of isomerization in the P68A mutant, where this hydrogen bond is weaker (42).…”

Arginine52 Controls the Photoisomerization Process in Photoactive Yellow Protein

“…However, mutants in which the arginine has been replaced by an electronically neutral amino acid can still enter the photocycle, albeit with a lower rate and quantum yield. 4 These findings indicate that Arg52 is important but not essential for photoactivation.…”

Tryptophan fluorescence as a reporter for structural changes in photoactive yellow protein elicited by photo-activation

“…Another mutation of W119, W119G, also does not alter the nature of the photocycle dynamics and the chromophore environment of PYP. 21,[60][61][62][63] It appears therefore that we can safely use the W119F mutation to create 'single tryptophan' mutants. Nevertheless, W119 is a highly conserved residue, 42 possibly because of its function in UV sensing, 59 rather than being essential for the chromophore photochemistry and photocycle dynamics (see further below).…”

“…19 Through further relaxation of the chromophore, the pR 1 and pR 2 intermediates are then formed. 20,21 Next, an intra-molecular proton transfer takes place from E46 to the phenolate moiety of the chromophore, 22 thereby forming the intermediate pB′. 23 From pB′, pB is formed, which is the presumed signalling state of PYP.…”

“…PYP mutants in which R52 is replaced by a non-charged amino acid have unperturbed spectra and photocycle kinetics. 21,40 Indeed, in neutron crystallography of PYP, R52 is observed to be deprotonated. 41 We are interested in resolving the structural changes preceding intra-molecular proton transfer in the different functional regions of PYP.…”