Mapping of the local environmental changes in proteins by cysteine scanning

Protein conformational changes, which regulate the activity of proteins, are induced by the alternation of intramolecular interactions. Therefore, the detection of the local environmental changes around the key amino acid residues is essential to understand the activation mechanisms of functional proteins. Here we developed the methods to scan the local environmental changes using the vibrational band of cysteine S-H group. We validated the sensitivity of this method using bathorhodopsin, a photoproduct of rhodopsin trapped at liquid nitrogen temperature, which undergoes little conformational changes from the dark state as shown by the X-ray crystallography. The cysteine residues were individually introduced into 15 positions of Helix III, which contains several key amino acid residues for the light-induced conformational changes of rhodopsin. The shifts of S-H stretching modes of these cysteine residues and native cysteine residues upon the formation of bathorhodopsin were measured by Fourier transform infrared spectroscopy. While most of cysteine residues demonstrated no shift of S-H stretching mode, cysteine residues introduced at positions 117, 118, and 122, which are in the vicinity of the chromophore, demonstrated the significant changes. The current results are consistent with the crystal structure of bathorhodopsin, implying that the cysteine scanning is sensitive enough to detect the tiny conformational changes

Mapping the ultrafast vibrational dynamics of all- trans and 13- cis retinal isomerization in Anabaena Sensory Rhodopsin

International audienceDiscrepancies in the isomerization dynamics and quantum yields of the trans and cis retinal protonated Schiff base is a well-known issue in the context of retinal photochemistry. Anabaena Sensory Rhodopsin (ASR) is a microbial retinal protein that comprises a retinal chromophore in two ground state (GS) conformations: all-trans, 15-anti (AT) and 13-cis, 15-syn (13C). In this work, we apply impulsive vibrational spectroscopic techniques (DFWM, pump-DFWM and pump-IVS) to ASR to shed more light on how the structural changes take place in the excited state within the same protein environment. Our findings point to distinct features in the ground state structural conformations as well as to drastically different evolutions in the excited state manifold. The ground state vibrational spectra show stronger Raman activity of the C14-H out-of-plane wag (at about 805 cm-1) for 13C than for AT isomer, which hints at a pre-distortion of the 13C in the ground state. Evolution of the Raman frequency after interaction with actinic pulse shows a blue shift for the C=C stretching and CH3 rocking mode for both isomers. For AT, however, the blue shift is not instantaneous as observed for the 13C isomer, rather it takes more than 200 fs to reach the maximum frequency shift. This frequency blue shift is rationalized by a decrease of the effective conjugation length during the isomerization reaction, which further confirms a slower formation of the twisted state for the AT isomer and corroborates the presence of a barrier in the excited state trajectory previously predicted by quantum chemical calculations

Spectral Tuning Mechanism of Primate Blue-sensitive Visual Pigment Elucidated by FTIR Spectroscopy

Protein-bound water molecules are essential for the structure and function of many membrane proteins, including G-protein-coupled receptors (GPCRs). Our prior work focused on studying the primate green- (MG) and red- (MR) sensitive visual pigments using low-temperature Fourier transform infrared (FTIR) spectroscopy, which revealed protein-bound waters in both visual pigments. Although the internal waters are located in the vicinity of both the retinal Schiff base and retinal β-ionone ring, only the latter showed differences between MG and MR, which suggests their role in color tuning. Here, we report FTIR spectra of primate blue-sensitive pigment (MB) in the entire mid-IR region, which reveal the presence of internal waters that possess unique water vibrational signals that are reminiscent of a water cluster. These vibrational signals of the waters are influenced by mutations at position Glu113 and Trp265 in Rh, which suggest that these waters are situated between these two residues. Because Tyr265 is the key residue for achieving the spectral blue-shift in λmax of MB, we propose that these waters are responsible for the increase in polarity toward the retinal Schiff base, which leads to the localization of the positive charge in the Schiff base and consequently causes the blue-shift of λmax

Structural changes in bacteriorhodopsin in response to alternate illumination observed by high-speed atomic force microscopy

金沢大学理工研究域数物科学系Blue light, green light: High-speed atomic force microscopy visualized light-induced structural changes of the D96N bacteriorhodopsin (bR) mutant under alternate two-color illumination. With green light, each bR molecule is displaced outward from the trimer center. This activated structure is driven back to the ground state by the subsequent blue-light illumination (see picture). Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

High-speed atomic force microscopy shows dynamic molecular processes in photoactivated bacteriorhodopsin

金沢大学理工研究域数物科学系Dynamic changes in protein conformation in response to external stimuli are important in biological processes, but it has proved difficult to directly visualize such structural changes under physiological conditions1-10. Here, we show that high-speed atomic force microscopy7 can be used to visualize dynamic changes in stimulated proteins. High-resolution movies of a light-driven proton pump, bacteriorhodopsin, reveal that, upon illumination, a cytoplasmic portion of each bacteriorhodopsin monomer is brought into contact with adjacent trimers. The bacteriorhodopsin-bacteriorhodopsin11,12 interaction in the transiently formed assembly engenders both positive and negative cooperative effects in the decay kinetics as the initial bacteriorhodopsin recovers and, as a consequence, the turnover rate of the photocycle is maintained constant, on average, irrespective of the light intensity. These results confirm that high-resolution visualization is a powerful approach for studying elaborate biomolecular processes under realistic conditions. © 2010 Macmillan Publishers Limited. All rights reserved

Vibrational spectroscopy analysis of ligand efficacy in human M₂ muscarinic acetylcholine receptor (M₂R)

振動分光法を駆使した薬剤効能測定法の開発 --アセチルコリン受容体を標的とした神経疾患の治療薬開発への期待--. 京都大学プレスリリース. 2021-12-01.The intrinsic efficacy of ligand binding to G protein-coupled receptors (GPCRs) reflects the ability of the ligand to differentially activate its receptor to cause a physiological effect. Here we use attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to examine the ligand-dependent conformational changes in the human M₂ muscarinic acetylcholine receptor (M₂R)). We show that different ligands affect conformational alteration appearing at the C=O stretch of amide-I band in M2R. Notably, ATR-FTIR signals strongly correlated with G-protein activation levels in cells. Together, we propose that amide-I band serves as an infrared probe to distinguish the ligand efficacy in M₂R) and paves the path to rationally design ligands with varied efficacy towards the target GPCR

Salivary Mucocele in a Laboratory Beagle

The histologic characteristics of a salivary mucocele in a beagle used in a toxicity study are described in this report. A pale yellowish cyst under the mandibular skin containing frothy mucus was observed at necropsy. Microscopically, numerous villous projections arose from the internal surface of the cyst and were lined by stratified epithelial-like macrophages, which were immunopositive for macrophage scavenger receptor A. A ruptured sublingual interlobar duct connected to the lumen was observed near the cyst. Luminal amorphous material showed a positive reaction with Alcian blue and periodic acid-Schiff staining as did mucin in the sublingual gland. Ultrastructurally, the epithelial-like macrophages had numerous vacuoles containing electron-lucent material, which was presumed to be lysosomal in origin, and had pseudopods on their cell surfaces interdigitating with those on the adjacent cells. This case report helps to understand the diversity of the background findings in beagles used in toxicity studies

Role of trimer-trimer interaction of bacteriorhodopsin studied by optical spectroscopy and high-speed atomic force microscopy

Bacteriorhodopsin (bR) trimers form a two-dimensional hexagonal lattice in the purple membrane of Halobacterium salinarum. However, the physiological significance of forming the lattice has long been elusive. Here, we study this issue by comparing properties of assembled and non-assembled bR trimers using directed mutagenesis, high-speed atomic force microscopy (HS-AFM), optical spectroscopy, and a proton pumping assay. First, we show that the bonds formed between W12 and F135 amino acid residues are responsible for trimer-trimer association that leads to lattice assembly; the lattice is completely disrupted in both W12I and F135I mutants. HS-AFM imaging reveals that both crystallized D96N and non-crystallized D96N/W12I mutants undergo a large conformational change (i.e., outward E-F loop displacement) upon light-activation. However, lattice disruption significantly reduces the rate of conformational change under continuous light illumination. Nevertheless, the quantum yield of M-state formation, measured by low-temperature UV-visible spectroscopy, and proton pumping efficiency are unaffected by lattice disruption. From these results, we conclude that trimer-trimer association plays essential roles in providing bound retinal with an appropriate environment to maintain its full photo-reactivity and in maintaining the natural photo-reaction pathway. © 2013 Elsevier Inc. All rights reserved