Application of FTIR Spectroscopy to the Structural Study on the Function of Bacteriorhodopsin

Abstract

Difference FTIR spectroscopy of the photointermediates of bacteriorhodopsin is informative for changes in H-bonding, the protonation states, and the bond orientation of functional residues such as C=O, N-H, and O-H of the chromophore, protein residues, peptide bonds, and internal water molecules. The vibrational bands of the chromophore are found at frequencies similar to those observed by resonance Raman spectra. Moreover, FTIR gives clear results on the N-H in-plane bending vibration and C 14 -C 15 stretching vibrational modes, both of which are useful for the analysis of the structure of the chromophore. The protonation states and H-bonding changes of intramembrane protonated aspartic acid residues can be revealed only by FTIR spectroscopy, in combination with proper isotope labeling and site-directed mutagenesis. The results with Asp-96 and Asp-85 in the L, M, and N photointermediates were especially useful for understanding the proton transfer mechanism. Besides amino acid groups in the protein, peptide C=O vibrations were assigned to a specific bond by the use of site-directed isotope labeling. Water molecules which undergo structural changes upon photoreaction were attributed specifically to those interacting with particular protein residues by mutational studies. On the basis of these FTIR studies, the role of the L intermediate is emphasized as the key intermediate that creates the conditions for the proton transfer reaction from the Schiff base to Asp-85 and subsequent proton uptake reactions in the cytoplasmic domain.

Changes in the hydrogen-bonding strength of internal water molecules and cysteine residues in the conductive state of channelrhodopsin-1

“…39 The IR intensity of the C-C stretching vibrations of the retinal is negligible unless the SB is protonated. 23,58 The absence of positive bands in this region (beside one weak band at 1176 cm −1 ) confirms that an intermediate with deprotonated SB is predominantly accumulated. A detailed analysis of the retinal bands has recently been published.…”

“…The first recorded spectrum (6.5 ms after the laser pulse) lacks positive bands in the C-C stretching region of the retinal (1100-1300 cm −1 ), a characteristic feature for intermediates with deprotonated SB. 23,58 Thus, the spectrum recorded at 6.5 ms after pulsed excitation represents an almost pure P 2 380 spectrum ( Fig. 2(b), top).…”

“…Because of the natural occurrence of O-H groups in proteins, in the side chains of threonine, serine, and tyrosine, the assignment of O-H vibrations to internal waters relies on the ∼11 cm −1 spectral downshift when using H 2 18 O as a solvent, as shown by pioneering studies on bacteriorhodopsin (BR). [23][24][25] Briefly, dangling and weakly H-bonded waters in proteins show bands between 3700 and 3600 cm −1 with a bandwidth of 6-20 cm −1 . This experimental approach was later extended to experiments in D 2 16 O and D 2 18 O, in the less congested X-D stretching region, which benefits from slightly larger isotopic downshifts (∼14 cm −1 ) and narrower bands.…”

Evidence for Proton Transfer from Glu-46 to the Chromophore during the Photocycle of Photoactive Yellow Protein

“…In contrast, the p-coumaryl chromophore is negatively charged in PYP but is neutral in PYP M . Despite the striking differences in the chromophores, protein moieties, and linkages, the chromophores of both retinal proteins and PYP turn neutral in their near-UV intermediates, which are thought to be formed as the result of large conformational changes (17,28). In addition, cis/trans isomerization of the chromophore is involved in both systems (15).…”

“…As shown in retinal protein systems, the changes in the hydrogen-bonding network centering on the chromophore closely correlates with the protein conformational changes (17). Therefore, it is of importance to study the internal proton movement around the chromophore and nearby amino acid residues to understand the light signal transduction mechanism of PYP.…”