The Short-Lived Signaling State of the Photoactive Yellow Protein Photoreceptor Revealed by Combined Structural Probes

The signaling state of the photoactive yellow protein (PYP) photoreceptor is transiently developed via isomerization of its blue-light-absorbing chromophore. The associated structural rearrangements have large amplitude but, due to its transient nature and chemical exchange reactions that complicate NMR detection, its accurate three-dimensional structure in solution has been elusive. Here we report on direct structural observation of the transient signaling state by combining double electron electron resonance spectroscopy (DEER), NMR, and time-resolved pump-probe X-ray solution scattering (TR-SAXS/WAXS). Measurement of distance distributions for doubly spin-labeled photoreceptor constructs using DEER spectroscopy suggests that the signaling state is well ordered and shows that interspin-label distances change reversibly up to 19 Å upon illumination. The SAXS/WAXS difference signal for the signaling state relative to the ground state indicates the transient formation of an ordered and rearranged conformation, which has an increased radius of gyration, an increased maximum dimension, and a reduced excluded volume. Dynamical annealing calculations using the DEER derived long-range distance restraints in combination with short-range distance information from (1)H-(15)N HSQC perturbation spectroscopy give strong indication for a rearrangement that places part of the N-terminal domain in contact with the exposed chromophore binding cleft while the terminal residues extend away from the core. Time-resolved global structural information from pump-probe TR-SAXS/WAXS data supports this conformation and allows subsequent structural refinement that includes the combined energy terms from DEER, NMR, and SAXS/WAXS together. The resulting ensemble simultaneously satisfies all restraints, and the inclusion of TR-SAXS/WAXS effectively reduces the uncertainty arising from the possible spin-label orientations. The observations are essentially compatible with reduced folding of the I(2)' state (also referred to as the 'pB' state) that is widely reported, but indicates it to be relatively ordered and rearranged. Furthermore, there is direct evidence for the repositioning of the N-terminal region in the I(2)' state, which is structurally modeled by dynamical annealing and refinement calculations

Physiological effects of formulation containing tannase-converted green tea extract on skin care: physical stability, collagenase, elastase, and tyrosinase activities

Background: Green tea contains numerous polyphenols, which have health-promoting effects. The purpose of this study was to evaluate the effect of tannase-converted green tea extract (TGE) formulation on the physical stability and activities of skin-related enzymes. Methods: Physical stability was evaluated by measuring the pH, precipitation, and colors at 25 ± 2 °C/ambient humidity and at 40 ± 2 °C/70% ± 5% relative humidity for 4 months. Activities of collagenase, elastase, and tyrosinase as skin-related enzymes were assessed on TGE formulation. Results: The concentrations of epigallocatechin-3-gallate and epicatechin-3-gallate in green tea extract were greatly decreased to the extent of negligible level when treated with tannase. The formulation containing 5% tannase-converted green tea extract showed relatively stable pH, precipitation, and color features for 16 weeks. When TGE was added to the formulation, there was a significant increase in the inhibition of elastase and tyrosinase activities (p < 0.05) compared with the formulation containing 5% normal green tea extract. Conclusion: The TGE could be used in cosmetics as skin antiwrinkling or depigmenting agent

Reply to &apos;Contradictions in X-ray structures of intermediates in the photocycle of photoactive yellow protein&apos;

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Effect of salt concentration on the strength and specificity of PNA–DNA duplexes and the corresponding DNA–DNA duplexes

<p><b>Copyright information:</b></p><p>Taken from "Diagnosis of HNF-1α mutations on a PNA zip-code microarray by single base extension"</p><p>Nucleic Acids Research 2005;33(2):e19-e19.</p><p>Published online 01 Feb 2005</p><p>PMCID:PMC548378.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Three different PNA or DNA zip sequences (Zip3, 7, and 9 in ) were used to prepare the respective zip-code microarrays. This was followed by hybridization for 3 h with a 10 nM solution of 25mer 3′-Cy3-labeled DNA (5′-AAGAAGAAAAAGGA-Cy3-3′, the sequence complementary to the Zip7 sequence is bolded and underlined.) at various ionic strengths. () Fluorescence intensities of perfectly matched PNA–DNA and DNA–DNA duplexes. () The discrimination ratio for PNA–DNA and DNA–DNA complexes, which was defined as the ratio of the signal intensity for a perfectly matched duplex (PM) to the average of the respective intensities obtained with the other two mismatched duplexes (MM)

Multiplex diagnosis of the 10 HNF-1α mutations on a PNA zip-code microarray

<p><b>Copyright information:</b></p><p>Taken from "Diagnosis of HNF-1α mutations on a PNA zip-code microarray by single base extension"</p><p>Nucleic Acids Research 2005;33(2):e19-e19.</p><p>Published online 01 Feb 2005</p><p>PMCID:PMC548378.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> In schematic representation of the PNA microarray on left side, each position in the 2 × 2 grid corresponds to an individual zip-code sequence and corresponding HNF-1α mutation. The corresponding allele is also indicated in parentheses. With each of three different samples, the right side images represent the fluorescence signals obtained from four different multiplex SBE reaction products, each containing a different biotin-labeled ddNTP. ddNTP above the right side images indicate that the image was obtained from reactions containing biotin-labeled ddATP, ddGTP, ddCTP and ddUTP, respectively. () homozygote wild-type sample () homozygote mutant sample () heterozygote mutant sample

Volume-conserving trans-cis isomerization pathways in photoactive yellow protein visualized by picosecond X-ray crystallography

Trans-to-cis isomerization, the key reaction in photoactive proteins, usually cannot occur through the standard one-bond-flip mechanism. Owing to spatial constraints imposed by a protein environment, isomerization probably proceeds through a volume-conserving mechanism in which highly choreographed atomic motions are expected, the details of which have not yet been observed directly. Here we employ time-resolved X-ray crystallography to visualize the isomerization of the p-coumaric acid chromophore in photoactive yellow protein with a time resolution of 100 ps and a spatial resolution of 1.6 Å. The structure of the earliest intermediate (IT) resembles a highly strained transition state in which the torsion angle is located halfway between the trans- and cis-isomers. The reaction trajectory of IT bifurcates into two structurally distinctcis intermediates via hula-twist and bicycle-pedal pathways. The bifurcating reaction pathways can be controlled by weakening the hydrogen bond between the chromophore and an adjacent residue through E46Q mutation, which switches off the bicycle-pedal pathway.173791sciescopu

ACCORD: an assessment tool to determine the orientation of homodimeric coiled-coils

The coiled-coil (CC) domain is a very important structural unit of proteins that plays critical roles in various biological functions. The major oligomeric state of CCs is a dimer, which can be either parallel or antiparallel. The orientation of each α-helix in a CC domain is critical for the molecular function of CC-containing proteins, but cannot be determined easily by sequence-based prediction. We developed a biochemical method for assessing differences between parallel and antiparallel CC homodimers and named it ACCORD (Assessment tool for homodimeric Coiled-Coil ORientation Decision). To validate this technique, we applied it to 15 different CC proteins with known structures, and the ACCORD results identified these proteins well, especially with long CCs. Furthermore, ACCORD was able to accurately determine the orientation of a CC domain of unknown directionality that was subsequently confirmed by X-ray crystallography and small angle X-ray scattering. Thus, ACCORD can be used as a tool to determine CC directionality to supplement the results of in silico prediction. © The Author(s) 20171101sciescopu

Combined probes of X-ray scattering and optical spectroscopy reveal how global conformational change is temporally and spatially linked to local structural perturbation in photoactive yellow protein

Real-time probing of structural transitions of a photoactive protein is challenging owing to the lack of a universal time-resolved technique that can probe the changes in both global conformation and light-absorbing chromophores of the protein. In this work, we combine time-resolved X-ray solution scattering (TRXSS) and transient absorption (TA) spectroscopy to investigate how the global conformational changes involved in the photoinduced signal transduction of photoactive yellow protein (PYP) is temporally and spatially related to the local structural change around the light-absorbing chromophore. In particular, we examine the role of internal proton transfer in developing a signaling state of PYP by employing its E46Q mutant (E46Q-PYP), where the internal proton transfer is inhibited by the replacement of a proton donor. The comparison of TRXSS and TA spectroscopy data directly reveals that the global conformational change of the protein, which is probed by TRXSS, is temporally delayed by tens of microseconds from the local structural change of the chromophore, which is probed by TA spectroscopy. The molecular shape of the signaling state reconstructed from the TRXSS curves directly visualizes the three-dimensional conformations of protein intermediates and reveals that the smaller structural change in E46Q-PYP than in wild-type PYP suggested by previous studies is manifested in terms of much smaller protrusion, confirming that the signaling state of E46Q-PYP is only partially developed compared with that of wild-type PYP. This finding provides direct evidence of how the environmental change in the vicinity of the chromophore alters the conformational change of the entire protein matrix. © the Owner Societies 201619101sciescopu