187 research outputs found
Regulation of protein structural changes by incorporation of a small-molecule linker
No full textProteins have the potential to serve as nanomachines with well-controlled structural movements, and artificial control of their conformational changes is highly desirable for successful applications exploiting their dynamic structural characteristics. Here, we demonstrate an experimental approach for regulating the degree of conformational change in proteins by incorporating a small-molecule linker into a well-known photosensitive protein, photoactive yellow protein (PYP), which is sensitized by blue light and undergoes a photo-induced N-terminal protrusion coupled with chromophore-isomerization-triggered conformational changes. Specifically, we introduced thiol groups into specific sites of PYP through site-directed mutagenesis and then covalently conjugated a small-molecule linker into these sites, with the expectation that the linker is likely to constrain the structural changes associated with the attached positions. To investigate the structural dynamics of PYP incorporated with the small-molecule linker (SML-PYP), we employed the combination of small-angle X-ray scattering (SAXS), transient absorption (TA) spectroscopy and experiment-restrained rigid-body molecular dynamics (MD) simulation. Our results show that SML-PYP exhibits much reduced structural changes during photo-induced signaling as compared to wild-type PYP. This demonstrates that incorporating an external molecular linker can limit photo-induced structural dynamics of the protein and may be used as a strategy for fine control of protein structural dynamics in nanomachines. © 2018 by the authors. Licensee MDPI, Basel, Switzerland11sci
Cooperative protein structural dynamics of homodimeric hemoglobin linked to water cluster at subunit interface revealed by time-resolved X-ray solution scattering
No full textHomodimeric hemoglobin (HbI) consisting of two subunits is a good model system for investigating the allosteric structural transition as it exhibits cooperativity in ligand binding. In this work, as an effort to extend our previous study on wild-type and F97Y mutant HbI, we investigate structural dynamics of a mutant HbI in solution to examine the role of well-organized interfacial water cluster, which has been known to mediate intersubunit communication in HbI. In the T72V mutant of HbI, the interfacial water cluster in the T state is perturbed due to the lack of Thr72, resulting in two less interfacial water molecules than in wild-type HbI. By performing picosecond time-resolved X-ray solution scattering experiment and kinetic analysis on the T72V mutant, we identify three structurally distinct intermediates (I1, I2, and I3) and show that the kinetics of the T72V mutant are well described by the same kinetic model used for wild-type and F97Y HbI, which involves biphasic kinetics, geminate recombination, and bimolecular CO recombination. The optimized kinetic model shows that the R-T transition and bimolecular CO recombination are faster in the T72V mutant than in the wild type. From structural analysis using species-associated difference scattering curves for the intermediates, we find that the T-like deoxy I3 intermediate in solution has a different structure from deoxy HbI in crystal. In addition, we extract detailed structural parameters of the intermediates such as E-F distance, intersubunit rotation angle, and heme-heme distance. By comparing the structures of protein intermediates in wild-type HbI and the T72V mutant, we reveal how the perturbation in the interfacial water cluster affects the kinetics and structures of reaction intermediates of HbI. © 2016 Author(s)1571sciescopu
Role of thermal excitation in ultrafast energy transfer in chlorosomes revealed by two-dimensional electronic spectroscopy
No full textChlorosomes are the largest light harvesting complexes in nature and consist of many bacteriochlorophyll
pigments forming self-assembled J-aggregates. In this work, we use two-dimensional electronic
spectroscopy (2D-ES) to investigate ultrafast dynamics of excitation energy transfer (EET) in chlorosomes
and their temperature dependence. From time evolution of the measured 2D electronic spectra of
chlorosomes, we directly map out the distribution of the EET rate among the manifold of exciton states in
a 2D energy space. In particular, it is found that the EET rate varies gradually depending on the energies of
energy-donor and energy-acceptor states. In addition, from comparative 2D-ES measurements at 77 K and
room temperature, we show that the EET rate exhibits subtle dependence on both the exciton energy and
temperature, demonstrating the effect of thermal excitation on the EET rate. This observation suggests that
active thermal excitation at room temperature prevents the excitation trapping at low-energy states and
thus promotes efficient exciton diffusion in chlorosomes at ambient temperature.1441sciescopu
Ultrafast energy transfer in chlorosome probed by femtosecond pump-probe polarization anisotropy
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Conformational substates of myoglobin intermediate resolved by picosecond X-ray solution scattering
No full textConformational substates of proteins are generally considered to play important roles in regulating protein functions, but an understanding of how they influence the structural dynamics and functions of the proteins has been elusive. Here, we investigate the structural dynamics of sperm whale myoglobin associated with the conformational substates using picosecond X-ray solution scattering. By applying kinetic analysis considering all of the plausible candidate models, we establish a kinetic model for the entire cycle of the protein transition in a wide time range from 100 ps to 10 ms. Four structurally distinct intermediates are formed during the cycle, and most importantly, the transition from the first intermediate to the second one (B → C) occurs biphasically. We attribute the biphasic kinetics to the involvement of two conformational substates of the first intermediate, which are generated by the interplay between the distal histidine and the photodissociated CO. © 2014 American Chemical Society.114151sciescopu
Pump-probe x-ray solution scattering reveals accelerated folding of cytochrome c upon suppression of misligation
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Kinetics of the E46Q mutant of photoactive yellow protein investigated by transient grating spectroscopy
No full textTo elucidate the role of internal proton transfer in the photodynamics of photoactive yellow protein (PYP), the photocycle of the Glu46Gln mutant of PYP (E46Q-PYP) is investigated by transient grating (TG) spectroscopy. Compared with wild-type PYP (wt-PYP), the first spectrally blue-shifted intermediate of E46Q-PYP is formed more slowly, which is consistent with the absence of direct protonation from Glu46 residue, if the parallel kinetic model for wt-PYP is invoked. The smaller conformational change in E46Q-PYP, as manifested by the smaller change in the diffusion coefficient, mainly arises from the relatively larger volume of the ground state E46Q-PYP than wt-PYP rather than from the smaller volume of the pB state. (c) 2017 Elsevier B.V. All rights reserved.1
Photocycle of Photoactive Yellow Protein in Cell-Mimetic Environments: Molecular Volume Changes and Kinetics
No full textUsing various spectroscopic
techniques such as UV–visible
spectroscopy, circular dichroism spectroscopy, NMR spectroscopy, small-angle
X-ray scattering, transient grating, and transient absorption techniques,
we investigated how cell-mimetic environments made by crowding influence
the photocycle of photoactive yellow protein (PYP) in terms of the
molecular volume change and kinetics. Upon addition of molecular crowding
agents, the ratio of the diffusion coefficient of the blue-shifted
intermediate (pB) to that of the ground species (pG) significantly
changes from 0.92 and approaches 1.0. This result indicates that the
molecular volume change accompanied by the photocycle of PYP in molecularly
crowded environments is much smaller than that which occurs in vitro
and that the pB intermediate under crowded environments favors a compact
conformation due to the excluded volume effect. The kinetics of the
photocycle of PYP in cell-mimetic environments is greatly decelerated
by the dehydration, owing to the interaction between the protein and
small crowding agents, but is barely affected by the excluded volume
effect. The results lead to the inference that the signaling transducer
of PYP may not necessarily utilize the conformational change of PYP
to sense the signaling state
Highly efficient gene silencing and bioimaging based on fluorescent carbon dots in vitro and in vivo
No full textSmall interfering RNA (siRNA) is an attractive therapeutic candidate for sequencespecific gene silencing to treat incurable diseases using small molecule drugs. However, its efficient intracellular delivery has remained a challenge. Here, we have developed a highly biocompatible fluorescent carbon dot (CD), and demonstrate a functional siRNA delivery system that induces efficient gene knockdown in vitro and in vivo. We found that CD nanoparticles (NPs) enhance the cellular uptake of siRNA, via endocytosis in tumor cells, with low cytotoxicity and unexpected immune responses. Real-time study of fluorescence imaging in live cells shows that CD NPs favorably localize in cytoplasm and successfully release siRNA within 12 h. Moreover, we demonstrate that CD NP-mediated siRNA delivery significantly silences green fluorescence protein (GFP) expression and inhibits tumor growth in a breast cancer cell xenograft mouse model of tumor-specific therapy. We have developed a multifunctional siRNA delivery vehicle enabling simultaneous bioimaging and efficient downregulation of gene expression, that shows excellent potential for gene therapy.close
Coherent oscillations in chlorosome elucidated by two-dimensional electronic spectroscopy
No full textChlorosomes are the most efficient photosynthetic light-harvesting complexes found in nature and consist of many bacteriochlorophyll (BChl) molecules self-assembled into supramolecular aggregates. Here we elucidate the presence and the origin of coherent oscillations in chlorosome at cryogenic temperature using 2D electronic spectroscopy. We observe coherent oscillations of multiple frequencies superimposed on the ultrafast amplitude decay of 2D spectra. Comparison of oscillatory features in the rephasing and nonrephasing 2D spectra suggests that an oscillation of 620 cm-1 frequency arises from electronic coherence. However, this coherent oscillation can be enhanced by vibronic coupling with intermolecular vibrations of BChl aggregate, and thus it might originate from vibronic coherence rather than pure electronic coherence. Although the 620 cm-1 oscillation dephases rapidly, the electronic (or vibronic) coherence may still take part in the initial step of energy transfer in chlorosome, which is comparably fast. © 2014 American Chemical Society.112131sciescopu
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