Time-resolved detection of light-induced conformational changes of heliorhodopsin

Heliorhodopsins (HeRs) are a new category of rhodopsins. They exist as a dimer and exhibit a characteristic inverted topology. HeRs bind all-trans-retinal as a chromophore in the dark, and its isomerization to the 13-cis form by light illumination leads to a photocyclic reaction involving several photo-intermediates: K, L, M, and O. In this study, the kinetics of conformational changes of HeR from Thermoplasmatales archaeon SG8-52-1 (TaHeR) were studied by the transient grating (TG) and circular dichroism (CD) methods. The TG method reveals that the diffusion coefficient (D) does not change until the O formation suggesting no significant conformation change at the surface of the protein during the early steps of the reaction. Subsequently, D decreases upon the O formation. Although two time constants (202 mu s and 2.6 ms) are observed for the conversion from the M to O by the absorption detection, D decreases only at the first step (202 mu s). Light-induced unfolding of helical structure is detected by the CD method. To examine the contribution of a characteristic helix in the intracellular loop 1 (ICL1 helix), Tyr93 on the ICL1 helix was replaced by Gly (Y93G), and the reaction of this mutant was also investigated. It was found that this replacement partially suppresses the D-change, although the CD-change is almost the same as that of the wild type. These results are interpreted in terms of different sensitivities of TG and CD methods, that is, D is sensitive to the structure of the solvent-exposed surface and selectively observes the conformational change in the ICL1 region. It is suggested that the structure of hydrophilic residues in the ICL1 helix is changed during this process

Time-resolved study on signaling pathway of photoactivated adenylate cyclase and its nonlinear optical response

Photoactivated adenylate cyclases (PACs) are multidomain BLUF proteins that regulate the cellular levels of cyclic adenosine 3', 5'-monophosphate (cAMP) in a light-dependent manner. The signaling route and dynamics of PAC from Oscillatoria acuminata (OaPAC), which consists of a light sensor BLUF domain, an adenylate cyclase domain, and a connector helix (α3-helix), were studied by detecting conformational changes in the protein moiety. Although circular dichroism and small-angle X-ray scattering measurements did not show significant changes upon light illumination, the transient grating method successfully detected light-induced changes in the diffusion coefficient (diffusion-sensitive conformational change (DSCC)) of full-length OaPAC (FL-PAC) and the BLUF domain with the α3-helix. DSCC of FL-PAC was observed only when both protomers in a dimer were photoconverted. This light intensity dependence suggests that OaPAC is a cyclase with a nonlinear light intensity response. The enzymatic activity indeed nonlinearly depends on light intensity, that is, OaPAC is activated under strong light conditions. It was also found that both DSCC and enzymatic activity were suppressed by a mutation in the W90 residue, indicating the importance of the highly conserved Trp in many BLUF domains for the function. Based on these findings, a reaction scheme was proposed together with the reaction dynamics

Relationship between Physical Activity and Health-related Quality of Life in Hospitalized and Terminally Ill Cancer Patients

Article信州医学雑誌 68(3): 149-157(2020)journal articl

Time-resolved detection of SDS-induced conformational changes in α-synuclein by a micro-stopped-flow system

An intrinsically disordered protein, α-synuclein (αSyn), binds to negatively charged phospholipid membranes and adopts an α-helical structure. This conformational change is also induced by interaction with sodium dodecyl sulfate (SDS), which is an anionic surfactant used in previous studies to mimic membrane binding. However, while the structure of the αSyn and SDS complex has been studied widely by various static measurements, the process of structural change from the denatured state to the folded state remains unclear. In this study, the interaction dynamics between αSyn and SDS micelles was investigated using time-resolved measurements with a micro-stopped-flow system, which has been recently developed. In particular, the time-resolved diffusion based on the transient grating technique in combination with a micro-stopped-flow system revealed the gradual change in diffusion triggered by the presence of SDS micelles. This change is induced not only by binding to SDS micelles, but also by an intramolecular conformational change. It was interesting to find that the diffusion coefficient decreased in an intermediate state and then increased to the final state in the binding reaction. We also carried out stopped-flow-kinetic measurements of circular dichroism and intramolecular fluorescence resonance energy transfer, and the D change was assigned to the formation of a compact structure derived from the helix bending on the micelle

セイショクコウ センサー タンパクシツ フォトトロピン ノ ブンシロンテキ ハンノウ ダイナミクス ト ドメインカン ソウゴ サヨウ ニ カンスル ケンキュウ

京都大学0048新制・課程博士博士(理学)甲第14414号理博第3411号新制||理||1499(附属図書館)UT51-2009-D126京都大学大学院理学研究科化学専攻(主査)教授 寺嶋 正秀, 教授 竹腰 清乃理, 教授 谷村 吉隆学位規則第4条第1項該当Doctor of ScienceKyoto UniversityDA

Time-Resolved Diffusion Detection with Microstopped Flow System

The transient grating (TG) method is a powerful technique for monitoring the time dependence of the diffusion coefficient during photochemical reactions. However, the applications of this technique have been limited to photochemical reactions. Here, a microstopped flow (μ-SF) system is developed to expand the technique’s applicability. The constructed μ-SF system can be used for a solution with a total volume as small as 3 μL, and mixing times for absorption and diffusion measurements were determined to be 400 μs and 100 ms, respectively. To demonstrate this system with the TG method, an acid-induced denaturation of a photosensor protein, phototropin LOV2 domain with a linker, was studied from the viewpoint of the reactivity. This system can be used not only for time-resolved diffusion measurement but also for conventional absorption or fluorescence detection methods. In particular, this system has a great advantage for a target solution in that only a very small amount is needed

Diffusion detects conformation changes during reactions of photosensor proteins

Since conformation changes of proteins and biomolecular interactions (including protein-DNA, or protein-protein interactions) are essential processes for biological functions, detections of these processes are important in chemistry and biochemistry to understand the reactions. For the detection of these processes, a variety of techniques have been developed. UV/vis absorption spectroscopy or emission spectroscopy are very powerful to trace the time development of reactions. However, these techniques have a limitation to detect the conformation changes of proteins and biomolecular interactions. Recently, our group discovered that the translational diffusion coefficient can be a useful and sensitive probe to detect the conformation change as well as the intermolecular interaction changes. Although many techniques, e.g., dynamic light scattering, Taylor dispersion, capillary method, NMR spectroscopy, have been developed to monitor molecular diffusion, molecular diffusion has never been considered as a time dependent property during reactions. We have been developing a method based on the pulsed-laser induced transient grating (TG) technique to detect the time-dependent diffusion. Here, we repot the time-resolved detection of protein conformation changes of a blue light sensor protein of phototropin by using the diffusion coefficient

Diffusion detects conformation changes during reactions of photosensor proteins

Since conformation changes of proteins and biomolecular interactions (including protein-DNA, or protein-protein interactions) are essential processes for biological functions, detections of these processes are important in chemistry and biochemistry to understand the reactions. For the detection of these processes, a variety of techniques have been developed. UV/vis absorption spectroscopy or emission spectroscopy are very powerful to trace the time development of reactions. However, these techniques have a limitation to detect the conformation changes of proteins and biomolecular interactions. Recently, our group discovered that the translational diffusion coefficient can be a useful and sensitive probe to detect the conformation change as well as the intermolecular interaction changes. Although many techniques, e.g., dynamic light scattering, Taylor dispersion, capillary method, NMR spectroscopy, have been developed to monitor molecular diffusion, molecular diffusion has never been considered as a time dependent property during reactions. We have been developing a method based on the pulsed-laser induced transient grating (TG) technique to detect the time-dependent diffusion. Here, we repot the time-resolved detection of protein conformation changes of a blue light sensor protein of phototropin by using the diffusion coefficient

Light-Induced Conformational Change and Transient Dissociation Reaction of the BLUF Photoreceptor Synechocystis PixD (Slr1694).

The light-induced reaction of the BLUF (blue light photoreceptor using flavin adenine dinucleotide) photoreceptor PixD from Synechocystis sp. PCC6803 (Slr1694) was investigated using the time-resolved transient grating method. A conformational change coupled with a volume contraction of 13 mL mol(-1) was observed with a time constant of 45 ms following photoexcitation. At a weak excitation light intensity, there were no further changes in volume and diffusion coefficient (D). The determined D-value (3.7×10(-11) m(2) s(-1)) suggests that PixD exists as a decamer in solution, and this oligomeric state was confirmed by size-exclusion chromatography and blue native polyacrylamide gel electrophoresis. Surprisingly, by increasing the excitation laser power, we observed a large increase in D with a time constant of 350 ms following the volume contraction reaction. The D-value of this photoproduct species (7.5×10(-11) m(2) s(-1)) is close to that of the PixD dimer. Combined with transient grating and size-exclusion chromatography measurements under light-illuminated conditions, the light-induced increase in D was attributed to a transient dissociation reaction of the PixD decamer to a dimer. For the M93A-mutated PixD, no volume or D-change was observed. Furthermore, we showed that the M93A mutant did not form the decamer but only the dimer in the dark state. These results indicate that the formation of the decamer and the conformational change around the Met residue are important factors that control the regulation of the downstream signal transduction by the PixD photoreceptor

Time-resolved fluctuation during the photochemical reaction of a photoreceptor protein: Phototropin1LOV2-linker

Although the relationship between structural fluctuations and reactions is important for elucidating reaction mechanisms, experimental data describing such fluctuations of reaction intermediates are sparse. In order to investigate structural fluctuations during a protein reaction, the compressibilities of intermediate species after photoexcitation of a phot1LOV2-linker, which is a typical LOV domain protein with the C-terminal linker including the J-α helix and used recently for optogenetics, were measured in the time-domain by the transient grating and transient lens methods with a high pressure optical cell. The yield of covalent bond formation between the chromophore and a Cys residue (S state formation) relative to that at 0.1 MPa decreased very slightly with increasing pressure. The fraction of the reactive species that yields the T state (linker-unfolded state) decreased almost proportionally with pressure (0.1-200 MPa) to about 65%. Interestingly, the volume change associated with the reaction was much more pressure sensitive. By combining these data, the compressibility changes for the short lived intermediate (S state) and the final product (T state) formation were determined. The compressibility of the S state was found to increase compared with the dark (D) state, and the compressibility decreased during the transition from the S state to the T state. The compressibility change is discussed in terms of cavities inside the protein. By comparing the crystal structures of the phot1LOV2-linker at dark and light states, we concluded that the cavity volumes between the LOV domain and the linker domain increase in the S state, which explains the enhanced compressibility