Raman Optical Activity Probing Structural Deformations of the 4-Hydroxycinnamyl Chromophore in Photoactive Yellow Protein

Abstract

Many biological cofactors, such as light-absorbing chromophores in photoreceptors, contain a π-electron system and are planar molecules. These cofactors are, however, usually nonplanar within a protein environment, and such structural distortions have been shown to be functionally important. Because the nonplanar structure makes the molecule chiral, Raman optical activity (ROA) provides a wealth of stereochemical information about the structural and conformational details of cofactors. The present study applied a near-infrared excited ROA to photoactive yellow protein, a blue light receptor. We successfully obtained the ROA spectra of the 4-hydroxycinnamyl chromophore embedded in a protein environment. Furthermore, calculations of the ROA spectra utilizing density functional theory provide detailed structural information, such as data on out-of-plane distortions of the chromophore. The structural information obtained from the ROA spectra includes the positions of hydrogen atoms, which are usually not detected in the crystal structures of biological samples.

Experimental Detection of the Intrinsic Difference in Raman Optical Activity of a Photoreceptor Protein under Preresonance and Resonance Conditions

“…This greatly increases the amplitude of the detected Raman bands and depends strongly on the energy difference between the position of the electronic transition and the excitation wavelength. [8,9] TheR OA signals measured in these studies are dominated by vibrational modes originating from the two distinct chromophores of these two photoreceptors.W es uggested that the ROAs pectra of the chromophore are enhanced by ap reresonance Raman effect. The difference in Raman scattered intensity between right (I R ) and left (I L )circularly polarized incident light holds information on molecular chirality [3][4][5][6] and is called Raman optical activity (ROA).…”

“…We observe aclose similarity between the shape of the RR spectrum and the resonance ROA spectrum of PYP.F urthermore,w ee xperimentally verify the theoretical prediction concerning the ratio of the amplitudes of the ROAa nd Raman spectra. [7][8][9] Since Raman scattering is ar elatively weak effect and the CID value is small (D < 10 À3 ), resonance enhancement is of great importance in ROAs tudies.H owever,e xperimental [10][11][12][13] and theoretical [14,15] ROAs tudies under resonance conditions have so far been very limited. In the case that molecules embedded in proteins exhibit an electronic transition, resonance Raman (RR) spectroscopy allows the enhancement of the vibrational modes of these chromophores with an excitation wavelength in resonance with this electronic transition.…”

“…The difference in Raman scattered intensity between right (I R ) and left (I L )circularly polarized incident light holds information on molecular chirality [3][4][5][6] and is called Raman optical activity (ROA). [17] Additionally,the shape of the ROAspectra, including the sign of the detected bands,isquite distinct from Raman spectra for these proteins.In ROAs tudies on PYP, [8,9] the availability of its highresolution crystal structures [18,19] allows quantum chemical calculations based on density functional theory (DFT) to simulate the ROAspectra. [7][8][9] Since Raman scattering is ar elatively weak effect and the CID value is small (D < 10 À3 ), resonance enhancement is of great importance in ROAs tudies.H owever,e xperimental [10][11][12][13] and theoretical [14,15] ROAs tudies under resonance conditions have so far been very limited.…”

“…[7][8][9] Since Raman scattering is ar elatively weak effect and the CID value is small (D < 10 À3 ), resonance enhancement is of great importance in ROAs tudies.H owever,e xperimental [10][11][12][13] and theoretical [14,15] ROAs tudies under resonance conditions have so far been very limited. [7][8][9] Since Raman scattering is ar elatively weak effect and the CID value is small (D < 10 À3 ), resonance enhancement is of great importance in ROAs tudies.H owever,e xperimental [10][11][12][13] and theoretical [14,15] ROAs tudies under resonance conditions have so far been very limited.…”

“…In ROAs tudies on PYP, [8,9] the availability of its highresolution crystal structures [18,19] allows quantum chemical calculations based on density functional theory (DFT) to simulate the ROAspectra. Such combined experimental and computational studies established the high sensitivity of the ROAs pectra to out-of-plane deformations of the chromophore.T hese studies demonstrated that the near-infrared excitation allows accurate measurement of structurally infor-mative preresonance ROAs pectra of ac hromophore embedded in aprotein.…”