Characterization of photocycle intermediates in crystalline photoactive yellow protein

The photocycle in photoactive yellow protein (PYP) crystals was studied by single-crystal absorption spectroscopy with experimental setups for low-temperature and time-resolved measurements. Thin and flat PYP crystals, suitable for light absorption studies, were obtained using special crystallization conditions. Illumination of PYP crystals at 100 K led to the formation of a photostationary state, which includes at least one hypsochromic and one bathochromic photoproduct that resemble PYP(H) and PYP(B), respectively. The effect of temperature, light color and light pulse duration on the occupancy of these low-temperature photoproducts was determined and appeared similar to that observed in solution. At room temperature a blueshifted photocycle intermediate was identified that corresponds to the blueshifted state of PYP (pB). Kinetic studies show that the decay of this blueshifted intermediate is biphasic at -12 degrees C and 15-fold faster than that observed in solution at room temperature. These altered pB decay kinetics confirm a model that holds that the photocycle in crystals takes place in a shortcut version. In this version the key structural events of the photocycle, such as photoisomerization and reversible protonation of the chromophore, take place, but large conformational changes in the surrounding protein are limited by constraints imposed by the crystal lattice.     

Controlling Radical Formation in the Photoactive Yellow Protein Chromophore

To understand how photoactive proteins function, it is necessary to understand the photoresponse of the chromophore. Photoactive yellow protein (PYP) is a prototypical signaling protein. Blue light triggers trans–cis isomerization of the chromophore covalently bound within PYP as the first step in a photocycle that results in the host bacterium moving away from potentially harmful light. At higher energies, photoabsorption has the potential to create radicals and free electrons; however, this process is largely unexplored. Here, we use photoelectron spectroscopy and quantum chemistry calculations to show that the molecular structure and conformation of the isolated PYP chromophore can be exploited to control the competition between trans–cis isomerization and radical formation. We also find evidence to suggest that one of the roles of the protein is to impede radical formation in PYP by preventing torsional motion in the electronic ground state of the chromophore.     

Structural changes during the photocycle of photoactive yellow protein monitored by ultraviolet resonance raman spectra of tyrosine and tryptophan

Photoactive yellow protein (PYP) is a bacterial blue light photoreceptor, and photoexcitation of dark-state PYP (PYP(dark)) triggers a photocycle that involves several intermediate states. We report the ultraviolet resonance Raman spectra of PYP with 225-250 nm excitations and investigate protein structural changes accompanying the formation of the putative signaling state denoted PYP(M). The PYP(M)-PYP(dark) difference spectra show several features of tyrosine and tryptophan, indicating environmental changes for these amino acid residues. The tyrosine difference signals show small upshifts with intensity changes in Y8a and Y9a bands. Although there are five tyrosine residues in PYP, Tyr42 and Tyr118 are suggested to be responsible for the difference signals on the basis of a global fitting analysis of the difference spectra at different excitation wavelengths and the crystal structure of PYP(dark). A further experiment on the Thr50-->Val mutant supports environmental changes in Tyr42. The observed upshift of the Y8a band suggests a weaker or broken hydrogen bond between Tyr42 and the chromophore in PYP(M). In addition, a reorientation of the OH group in Tyr42 is suggested from the upshift of the Y9a band. For tryptophan, the Raman bands of W3, W16, and W18 modes diminish in intensity upon formation of PYP(M). The loss of intensities is attributable to an exposure of tryptophan in PYP(M). PYP contains only one tryptophan (Trp119) that is located more than 10 A from the active site. Thus the observed changes are indicative of global conformational changes in protein during the transition from PYP(dark) to PYP(M). These results are in line with the currently proposed photocycle mechanism of PYP.     

Novel biomimetic surfactant: synthesis and micellar characteristics

Novel biomimetic surfactants based on cholesterol as the hydrophobic segment and poly[2-(methacryloyloxy)ethyl phosphorylcholine] (pMPC) as the hydrophilic segment were synthesized in the present study by atom transfer radical polymerization (ATRP) of 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) using a cholesterol-based macroinitiator. The association behavior of cholesterol-block-poly[2-(methacryloyloxy)ethyl phosphorylcholine] (Chol-pMPCs) in aqueous solution was studied by (1)H NMR spectroscopy, fluorescence probe technique, and atomic force microscopy (AFM). The (1)H NMR spectrum of the polymer in CD(3)OD showed both the cholesterol group and the phosphorylcholine group while the cholesterol group did not appeared in the (1)H NMR spectrum of the polymer in D(2)O, which implied the formation of a micelle structure. Fluorescence excitation spectra of a pyrene probe solubilized in the aggregates of Chol-pMPCs suggested the presence of a critical micelle concentration (cmc) in water. The critical micelle concentrations of the polymers CMPC10, CMPC20 and CMPC40 were determined to be 7.27 x 10(-3), 13.47 x 10(-3), and 20.77 x 10(-3) mg . mL(-1), respectively. AFM images of the aggregates on mica suggested that the pMPC block formed the biocompatible micelle coronas and the cholesterol block formed the hydrophobic micelle cores. These new biomimetic diblock copolymers were evaluated as "stealthy" nanocapsules for the delivery of hydrophobic drugs. The anti-cancer drug adriamycin (ADR) was chosen as a hydrophobic drug to be incorporated into the inner core of the micelles and the morphology of the drug-loaded micelles were observed by AFM.     

Separation of brombuterol enantiomers in capillary electrophoresis with cyclodextrin‐type chiral selectors and investigation of structure of selector‐selectand complexes using nuclear magnetic resonance spectroscopy

The major goal of this study was to determine the affinity pattern of brombuterol (BB) enantiomers toward various cyclodextrins (CD) and to evaluate the potential of NMR spectroscopy for understanding fine mechanisms of interactions between CDs and BB enantiomers. Separation of BB enantiomers was performed in a fused‐silica capillary using a phosphate buffer, pH 2.5, at the room temperature in the normal polarity mode. It was shown once again that CE in combination with NMR spectroscopy represents a very sensitive tool for studies of affinity patterns and structure of CD complexes with chiral guests. Although opposite affinity patterns of BB enantiomers were observed toward native β‐ and γ‐CDs, no significant differences between the structures of the complexes of these two CDs with BB were detected by NMR spectroscopy. In contrary to this, the opposite affinity pattern of BB enantiomers toward β‐CD and its two sulfated derivatives, heptakis (2,3‐O‐diacetyl‐6‐sulfo)‐β‐CD (HDAS‐β‐CD) and heptakis (2‐O‐methyl‐3,6‐di‐O‐sulfo)‐β‐CD (HMDS‐β‐CD) was associated with major differences in the structure of the complexes. In addition, it was shown again that HMDS‐β‐CD provides separation of enantiomers without formation of inclusion‐type complex with the chiral analyte.     

Formation and characterization of inclusion complexes of poly(butylene succinate) with alpha- and gamma-cyclodextrins

The inclusion complexes (ICs) of alpha- and gamma-cyclodextrins (CDs) with high-molecular-weight poly(butylene succinate) (PBS) were prepared and characterized by differential scanning calorimetry, Fourier-transform infrared spectroscopy (FT-IR), wide-angle X-ray diffraction, solid-state 13C nuclear magnetic resonance (NMR) spectroscopy, and solution 1H NMR spectroscopy. The resultant ICs were found to have channel structures. FT-IR data suggested that the ICs were stabilized by hydrogen bonds between the host CD molecules and the guest PBS chains. Through the formation of ICs, the PBS chain possibly adopts the kink conformation in the included state, as indicated by NMR analysis.     

Studies on the biomimetic membrane interaction between liposome and realgar nanoparticles

The liposome of small unilamellar vesicles (SUV) made from phosphatidylcholine-cholesterol mixtures was used as a simple model for biomimetic membranes. The studies on the interaction between the liposome and realgar nanoparticles (NPs) demonstrate that the phospholipid is one of the key targeted molecules of realgar NPs, used by surface plasmon resonance (SPR) technology, fluorescence polarization, Raman spectroscopy, nuclear magnetic resonance (NMR) and atom force microscope (AFM). It was observed that th...     

Three-dimensional organization of helices: design principles for nucleobase-functionalized beta-peptides

The construction and molecular recognition of various three-dimensional biomimetic structures is based on the predictable de novo design of artificial molecules. In this regard beta-peptides are especially interesting, since stable secondary structures are obtained already with short sequences; one of them is the 14-helix in which every third residue has the same orientation. The covalent functionalization of every third 14-helix side chain with nucleobases was used for a reversible organization of two helices based on nucleobase pairing. A series of beta-peptides with various nucleobase sequences was synthesized and the stability of double strand formation was investigated. As few as four nucleobases are sufficient for considerable duplex stability. The stability of base pairing was examined by temperature-dependent UV spectroscopy and the formation of the 14-helix was confirmed by circular dichroism (CD) spectroscopy. The preferred strand orientation of complementary-nucleobase-modified beta-peptide helices was investigated as well as the influence of helix content on the duplex stability. The preorganization of a 14-helix in regard to double-strand recognition was tuned by the sequential order of polar beta-amino acids or by the amount of 2-aminocyclohexanecarboxylic acid units incorporated, which are known to facilitate 14-helix formation, respectively.     

两性离子自组装仿生表面的制备、 表征及抗黏附性能

设计与合成了磺酸甜菜碱型的两性离子化合物: N,N-二甲基氨甲酸乙酯基丙基三乙氧基硅烷磺酸内盐(SiNNS), 利用红外光谱(FTIR)和氢核磁共振波谱(¹H NMR)对其分子组成与结构进行了表征. 通过自组装技术将SiNNS分子构筑在玻璃基材表面, 形成了模拟细胞外层膜的仿生表面. 利用原子力显微镜(AFM)、 X光电子能谱(XPS)和接触角测量仪对表面的形貌特征、 化学组成和润湿性进行了表征. 以空白玻璃为对照样品, 研究了这一表面的防雾性能和抗细菌黏附性能. 结果表明, 所制备的两性离子自组装仿生表面具有超亲水性和水下超疏油特性, 其水滴接触角为9.2°, 水下油滴接触角接近180°; 与对照样品相比, 两性离子自组装表面具有优异的防雾性与抗细菌黏附性.     

Vibrational assignment of the 4-hydroxycinnamyl chromophore in photoactive yellow protein

Photoactive yellow protein (PYP) is a bacterial photoreceptor containing a 4-hydroxycinnamyl chromophore. We report the Raman spectra for the dark state of PYP whose chromophore is isotopically labeled with 13C at the carbonyl carbon atom or at the ring carbon atoms. Spectra have been also measured with PYP in D2O where the exchangeable protons are deuterated. Most of the observed Raman bands are assigned on the basis of the observed isotope shifts and normal mode calculations using a density functional theory. We discuss the implication for the analysis of the infrared spectra of PYP. The comprehensive assignment provides a satisfactory framework for future investigations of the photocycle mechanism in PYP by vibrational spectroscopy.     

Structural evolution of the chromophore in the primary stages of trans/cis isomerization in photoactive yellow protein

We have studied the structural changes induced by optical excitation of the chromophore in wild-type photoactive yellow protein (PYP) in liquid solution with a combined approach of polarization-sensitive ultrafast infrared spectroscopy and density functional theory calculations. We identify the nuC8-C9 marker modes for solution phase PYP in the P and I0 states, from which we derive that the first intermediate state I0 that appears with a 3 ps time constant can be characterized to have a cis geometry. This is the first unequivocal demonstration that the formation of I0 correlates with the conversion from the trans to the cis state. For the P and I0 states we compare the experimentally measured vibrational band patterns and anisotropies with calculations and find that for both trans and cis configurations the planarity of the chromophore has a strong influence. The C7=C8-(C9=O)-S moiety of the chromophore in the dark P state has a trans geometry with the C=O group slightly tilted out-of-plane, in accordance with the earlier reported structure obtained in an X-ray diffraction study of PYP crystals. In the case of I0, experiment and theory are only in agreement when the C7=C8-(C9=O)-S moiety has a planar configuration. We find that the carboxylic side group of Glu46 that is hydrogen-bonded to the chromophore phenolate oxygen does not alter its orientation on going from the electronic ground P state, via the electronic excited P state to the intermediate I0 state, providing conclusive experimental evidence that the primary stages of PYP photoisomerization involve flipping of the enone thioester linkage without significant relocation of the phenolate moiety.     

Inclusion Compounds Formation of Poly(azomethine ether)s and β‐Cyclodextrin

In this study, two poly(azomethine ether)s were synthesized and they can form inclusion compounds (ICs) with β‐cyclodextrin (β‐CD). Fourier transform infrared (FTIR) spectroscopy, ¹H nuclear magnetic resonance spectroscopy (¹H‐NMR), thermogravimetric analysis (TGA), X‐ray diffraction (XRD) have been utilized to observe the formation of polymer‐CD‐ICs. The differentiation in their FTIR spectra may indicate the formation of the inclusion compounds between poly(azomethine ether)s and β‐CD. Compared the ¹H‐NMR of polymer‐CD‐ICs with β‐CD, proton signals belonging to both β‐CD and poly(azomethine ether)s can be found in the spectrum. The chemical shift of the protons H‐3, H‐5 has changed after the formation of inclusion compounds, which is perhaps due to the interaction of these protons with polymers. TGA scans showed the much higher decomposition temperatures observed for two polymer‐CD‐ICs may imply that polymer chains included inside the β‐CD's cavity can greatly improve β‐CD's stabilities. The X‐ray diffraction patterns were confirmed to be the new crystal structures.     

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 ? 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.     

Anharmonic vibrational calculations modeling the raman spectra of intermediates in the photoactive yellow protein (PYP) photocycle

The role of anharmonic effects in the vibrational spectroscopy of the dark state and two major chromophore intermediates of the photoactive yellow protein (PYP) photocycle is examined via ab initio vibrational self-consistent field (VSCF) calculations and time-resolved resonance Raman spectroscopy. For the first time, anharmonicity is considered explicitly in calculating the vibrational spectra of an ensemble consisting of the PYP chromophore surrounded by model compounds used as mimics of the important active-site residues. Predictions of vibrational frequencies on an ab initio corrected semiempirical potential energy surface show remarkable agreement with experimental frequencies for all three states, thus shedding light on the potential along the reaction path. For example, calculated frequencies for vibrational modes of the red-shifted intermediate, PYPL, exhibit an overall average error of 0.82% from experiment. Upon analysis of anharmonicity patterns in the PYP modes we observe a decrease in anharmonicity in the C8-C9 stretching mode nu29 (trans-cis isomerization marker mode) with the onset of the cis configuration in PYPL. This can be attributed to the loss of the hydrogen-bonding character of the adjacent C9-O2 to the methylamine (Cys69 backbone). For several of the modes, the anharmonicity is mostly due to mode-mode coupling, while for others it is mostly intrinsic. This study shows the importance of the inclusion of anharmonicity in theoretical spectroscopic calculations, and the sensitivity of experiments to anharmonicity. The characterization of protein active-site residues by small molecular mimics provides an acceptable chemical structural representation for biomolecular spectroscopy calculations.     

Probing the primary event in the photocycle of photoactive yellow protein using photochemical hole-burning technique

Photochemical hole-burning spectroscopy was used to study the excited-state electronic structure of the 4-hydroxycinnamyl chromophore in photoactive yellow protein (PYP). This system is known to undergo a trans-to-cis isomerization process on a femtosecond-to-picosecond time scale, similar to membrane-bound rhodopsins, and is characterized by a broad featureless absorbance at 446 nm. Resolved vibronic structure was observed for the hole-burned spectra obtained when PYP in phosphate buffer at pH 7 was frozen at low temperature and irradiated with narrow bandwidth laser light at 431 nm. The approximate homogeneous width of 752 cm-1 could be calculated from the deconvolution of the hole-burned spectra leading to an estimated dephasing time of approximately 14 fs for the PYP excited-state structure. The resolved vibronic structure also enabled us to obtain an estimated change in the C=C stretching frequency, from 1663 cm-1 in the ground state to approximately 1429 cm-1 upon photoexcitation. The results obtained allowed us to speculate about the excited-state structure of PYP. We discuss the data for PYP in relation to the excited-state model proposed for the photosynthetic membrane protein bacteriorhodopsin, and use it to explain the primary event in the function of photoactive biological protein systems. Photoexcitation was also carried out at 475 nm. The vibronic structure obtained was quite different both in terms of the frequencies and Franck-Condon envelope. The origin of this spectrum was tentatively assigned.     

Impact of Strand Number on Parallel β‐Sheet Stability

We have examined whether parallel β‐sheet secondary structure becomes more stable as the number of β‐strands increases, via comparisons among peptides designed to adopt two‐ or three‐stranded parallel β‐sheet conformations in aqueous solution. Our three‐strand design is the first experimental model of a triple‐stranded parallel β‐sheet. Analysis of the designed peptides by nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy supports the hypothesis that increasing the number of β‐strands, from two to three, increases the stability of the parallel β‐sheet. We present the first experimental evidence for cooperativity in the folding of a triple‐stranded parallel β‐sheet, and we show how minimal model systems may enable experimental documentation of characteristic properties, such as CD spectra, of parallel β‐sheets.     

Low-temperature spectroscopy of Met100Ala mutant of photoactive yellow protein

The trans-to-cis photoisomerization of the p-coumaroyl chromophore of photoactive yellow protein (PYP) triggers the photocycle. Met100, which is located in the vicinity of the chromophore, is a key residue for the cis-to-trans back-isomerization of the chromophore, which is a rate-determining reaction of the PYP photocycle. Here we characterized the photocycle of the Met100Ala mutant of PYP (M100A) by low temperature UV-visible spectroscopy. Irradiation of M100A at 80 K yielded a 380 nm species (M100A(BL)), while the corresponding intermediate of wild type (WT; PYP(BL)) is formed above 90 K. The amounts of redshifted intermediates produced from M100A (M100A(B') and M100A(L)) were substantially less than those from WT. While the near-UV intermediate (PYP(M)) is not formed from WT in glycerol samples at low temperature, M100A(M) was clearly observed above 190 K. These alterations of the photocycle of M100A were explained by the shift in the equilibrium between the intermediates. The carbonyl oxygen of the thioester linkage of the cis-chromophore in the photocycle intermediates is close to the phenyl ring of Phe96 (<3.5 A), which would be displaced by the mutation of Met100. These findings imply that the interaction between chromophore and amino acid residues near Met100 is altered during the early stage of the PYP photocycle.     

UV-visible and 1H or 13C NMR spectroscopic studies on the specific interaction between lithium ions and the anion from tropolone or 4-isopropyltropolone (hinokitiol) and on the formation of protonated tropolones in acetonitrile or other solvents

The specific interaction between lithium ions and the tropolonate ion (C(7)H(5)O(2)-: L-) was examined by means of UV-visible and 1H or 13C NMR spectroscopy in acetonitrile and other solvents. On the basis of the electronic spectra, we can propose the formation of not only coordination-type species (Li+(L-)2) and the ion pair (Li+L-) but also a "triple cation" ((Li+)2L-) in acetonitrile and acetone; however, no "triple cation" was found in N,N-dimethylformamide (DMF) and in dimethylsulfoxide (DMSO), solvents of higher donicities and only ion pair formation between Li+ and L- in methanol of much higher donicity and acceptivity. The 1H NMR chemical shifts of the tropolonate ion with increasing Li+ concentration verified the formation of (Li+)2L- species in CD3CN and acetone-d6, but not in DMF-d6 or CD(3)OD. With increasing concentration of LiClO(4) in CD(3)CN, the 1H NMR signals of 4-isopropyltropolone (HL') in coexistence with an equivalent amount of Et(3)N shifted first toward higher and then toward lower magnetic-fields, which were explained by the formation of (Li+)(Et(3)NH+)L'- and by successive replacement of Et(3)NH+ with a second Li+ to give (Li+)2L'-. In CD(3)CN, the 1,2-C signal in the 13C NMR spectrum of tetrabutylammnium tropolonate (n-Bu(4)NC(7)H(5)O) appeared at an unexpectedly lower magnetic-field (184.4 ppm vs TMS) than that of tropolone (172.7 ppm), while other signals of the tropolonate showed normal shifts toward higher magnetic-fields upon deprotonation from tropolone. Nevertheless, with addition of LiClO(4) at higher concentrations, the higher and lower shifts of magnetic-fields for 1,2-C and other signals, respectively, supported the formation of the (Li+)2L- species, which can cause redissolution of LiL precipitates. All of the data with UV-visible and 1H and 13C NMR spectroscopy demonstrated that the protonated tropolone (or the dihydroxytropylium ion), H(2)L+, was produced by addition of trifluoromethanesulfonic or methanesulfonic acid to tropolone in acetonitrile. The order of the 5-C and 3,7-C signals in 13C NMR spectra of the tropolonate ions was altered by addition of less than an equivalent amount of H+ to the tropolonate ion in CD(3)CN. Theoretical calculations satisfied the experimental 13C NMR chemical shift values of L-, HL, and H(2)L+ in acetonitrile and were in accordance with the proposed reaction schemes.     

Simple Ligand Effects Switch a Hydrogenase Mimic between H2 and O2 Activation

Herein, we report a [NiRu] biomimetic system for O₂‐tolerant [NiFe]hydrogenases and demonstrate that electron donation to the [NiRu] center can switch the system between the activation of H₂ and O₂ through simple ligand effects by using hexamethylbenzene and pentamethylcyclopentadienyl ligands, respectively. Furthermore, we present the synthesis and direct observations of a [NiRu]–peroxo species, which was formed by the oxygenation of a Ni‐SIa model [NiRu] complex, that we propose as a biomimetic analogue of O₂‐bound species (OBS) of O₂‐tolerant [NiFe]hydrogenases. The [NiRu]–peroxo complex was fully characterized by X‐ray analysis, X‐ray photoelectron spectroscopy (XPS), mass spectrometry, and ¹H NMR spectroscopy. The OBS analogue was capable of oxidizing p‐hydroquinone and sodium borohydride to turn back into the Ni‐SIa model complex.