Papers by Janne Ihalainen
Ubiquitous Structural Signaling in Bacterial Phytochromes
The Journal of Physical Chemistry Letters, 2015
The phytochrome family of light-switchable proteins has long been studied by biochemical, spectro... more The phytochrome family of light-switchable proteins has long been studied by biochemical, spectroscopic and crystallographic means, while a direct probe for global conformational signal propagation has been lacking. Using solution X-ray scattering, we find that the photosensory cores of several bacterial phytochromes undergo similar large-scale structural changes upon red-light excitation. The data establish that phytochromes with ordinary and inverted photocycles share a structural signaling mechanism and that a particular conserved histidine, previously proposed to be involved in signal propagation, in fact tunes photoresponse.
Ubiquitous Structural Signaling in Bacterial Phytochromes
The Journal of Physical Chemistry Letters, 2015
The phytochrome family of light-switchable proteins has long been studied by biochemical, spectro... more The phytochrome family of light-switchable proteins has long been studied by biochemical, spectroscopic and crystallographic means, while a direct probe for global conformational signal propagation has been lacking. Using solution X-ray scattering, we find that the photosensory cores of several bacterial phytochromes undergo similar large-scale structural changes upon red-light excitation. The data establish that phytochromes with ordinary and inverted photocycles share a structural signaling mechanism and that a particular conserved histidine, previously proposed to be involved in signal propagation, in fact tunes photoresponse.
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Design, Synthesis, and Biological Evaluation of NonsteroidalCycloalkane[d]isoxazole-Containing Androgen Receptor Modulators
Journal of Medicinal Chemistry
A series of 6,7-disubstituted-4-phenoxyquinoline derivatives bearing 4-oxo-3,4-dihydrophthalazine... more A series of 6,7-disubstituted-4-phenoxyquinoline derivatives bearing 4-oxo-3,4-dihydrophthalazine-1-carboxamide moieties were designed, synthesized and evaluated for their c-Met kinase inhibition and cytotoxicity against H460, MKN-45, HT-29 and MDA-MB-231 cancer cell lines in vitro. Most compounds displayed good to excellent potency against four tested cancer cell lines as compared with foretinib. The SAR analyses indicated that compounds with halogen groups, especially fluoro groups at 4-position on the phenyl ring (moiety B) were more effective than those with nitro groups or methoxy groups. In this study, a promising compound 33 (c-Met IC50=1.63nM) was identified, which showed the most potent antitumor activities with IC50 values of 0.055μM, 0.071μM, 0.13μM, and 0.43μM against H460, MKN-45, HT-29 and MDA-MB-231 cell lines, respectively.
Journal of Biological Chemistry, 2015
Background: Bacteriophytochromes are dimeric histidine kinases, but the functional role of their ... more Background: Bacteriophytochromes are dimeric histidine kinases, but the functional role of their dimerization interfaces is unclear. Results: The phytochrome from Deinococcus radiodurans has two dimerization interfaces, which are critical for thermal back reversion and are altered by illumination. Conclusion: The dimerization interfaces cause strain in the structure. Significance: A functional role for the dimerization interfaces is proposed.
The minor capsid protein VP11 of thermophilic bacteriophage P23-77 facilitates virus assembly using lipid-protein interactions
Journal of Virology, 2015
Thermus thermophilus bacteriophage P23-77 is the type member of a new virus family of icosahedral... more Thermus thermophilus bacteriophage P23-77 is the type member of a new virus family of icosahedral, tailless, inner membrane-containing dsDNA viruses infecting thermophilic bacteria and halophilic archaea. Those viruses have a unique capsid architecture consisting of two major capsid proteins assembled in various building blocks. We analyzed the function of minor capsid protein VP11, which is the third known capsid component in bacteriophage P23-77. Our findings show that VP11 is a dynamically elongated dimer with predominantly α-helical secondary structure and a high thermal stability. A high proportion of basic amino acids in the protein enable electrostatic interaction with negatively charged molecules including nucleic acid and large unilamellar lipid vesicles (LUVs). The plausible biological function for VP11 is elucidated by demonstrating the interactions of VP11 with Thermus derived LUVs and with the major capsid proteins by means of dynamic light scattering technique. In particular, the major capsid protein VP17 was able to link VP11-complexed LUVs into larger particles, whereas the other P23-77 major capsid protein, VP16, was unable to link VP11-comlexed LUVs. Our results rule out a previously suggested penton function for VP11. Instead, the electrostatic membrane association of VP11 triggers binding of major capsid protein VP17, thus facilitating a controlled incorporation of the two different protein species into the assembling capsid. The study of thermophilic viruses with inner membranes provides valuable insights into the mechanisms used for stabilization and assembly of protein-lipid systems at high temperatures. Our results reveal a novel way by which an internal membrane and outer capsid shell are linked in a virus that uses two different major protein species for capsid assembly. We show that a positive protein-charge is important to form electrostatic interactions with the lipid surface, thereby facilitating the incorporation of other capsid proteins on the membrane surface. This implies an alternative function for basic proteins present in the virions of other lipid-containing thermophilic viruses, whose proposed role in genome packaging is based on their capability to bind DNA. The unique minor capsid protein of bacteriophage P23-77 resembles in its characteristics the scaffolding proteins of tailed phages, though it constitutes a substantial part of the mature virion.
Biochemistry, Jan 18, 2014
Phytochromes consist of several protein domains and a linear tetrapyrrole molecule, which interac... more Phytochromes consist of several protein domains and a linear tetrapyrrole molecule, which interact as a red-light-sensing system. In this study, size-exclusion chromatography and light-scattering techniques are combined with UV-vis spectroscopy to investigate light-induced changes in dimeric Deinococcus radiodurans bacterial phytochrome (DrBphP) and its subdomains. The photosensory unit (DrCBD-PHY) shows an unusually stable Pfr state with minimal dark reversion, whereas the histidine kinase (HK) domain facilitates dark reversion to the resting state. Size-exclusion chromatography reveals that all phytochrome fragments remain as dimers in the illuminated state and dark state. Still, the elution profiles of all phytochrome fragments differ between the illuminated and dark states. The differences are observed reliably only when the whole UV-vis spectrum is characterized along the elution profile and show more Pfr-state characteristics at later elution volumes in DrBphP and DrCBD-PHY fr...
Origins of fluorescence in evolved bacteriophytochromes
The Journal of biological chemistry, Jan 14, 2014
Use of fluorescent proteins to study in vivo processes in mammals requires near-infrared (NIR) bi... more Use of fluorescent proteins to study in vivo processes in mammals requires near-infrared (NIR) biomarkers that exploit the ability of light in this range to penetrate tissue. Bacteriophytochromes (BphPs) are photoreceptors that couple absorbance of NIR light to photoisomerization, protein conformational changes, and signal transduction. BphPs have been engineered to form NIR fluorophores, including IFP1.4, Wi-Phy, and the iRFP series, initially by replacement of Asp-207 by His. This position was suggestive because its main chain carbonyl is within hydrogen-bonding distance to pyrrole ring nitrogens of the biliverdin chromophore, thus potentially functioning as a crucial transient proton sink during photoconversion. To explain the origin of fluorescence in these phytofluors, we solved the crystal structures of IFP1.4 and a comparison non-fluorescent monomeric phytochrome DrCBDmon. Met-186 and Val-288 in IFP1.4 are responsible for the formation of a tightly packed hydrophobic hub arou...
The structural diversity of benzofuran resorcinarene leads to enhanced fluorescence
Chemistry, an Asian journal, 2014
An unexpected and previously unknown resorcinarene mono-crown with a fused benzofuran moiety in i... more An unexpected and previously unknown resorcinarene mono-crown with a fused benzofuran moiety in its macrocyclic core was obtained as a byproduct from a bridging reaction of tetramethoxy resorcinarene with tetraethylene glycol ditosylate. The formation of the fused benzofuran moiety in the resorcinarene macrocycle resulted in a unique rigid and puckered boat conformation, as shown by XRD studies in the solid state. Modification of the macrocycle was also observed to affect the photophysical properties in solution by enhancing the fluorescence brightness compared with a conventional resorcinarene macrocycle. The fluorescent properties enabled unique detection of structural features, that is, the rigid boat conformation with the conjugated benzofuran system and the more flexible crown bridge part, in solution.
Journal of Virology, 2014
In recent decades, Raman spectroscopy has entered the biological and medical fields. It enables n... more In recent decades, Raman spectroscopy has entered the biological and medical fields. It enables nondestructive analysis of structural details at the molecular level and has been used to study viruses and their constituents. Here, we used Raman spectroscopy to study echovirus 1 (EV1), a small, nonenveloped human pathogen, in two different uncoating states induced by heat treatments. Raman signals of capsid proteins and RNA genome were observed from the intact virus, the uncoating intermediate, and disrupted virions. Transmission electron microscopy data revealed general structural changes between the studied particles. Compared to spectral characteristics of proteins in the intact virion, those of the proteins of the heat-treated particles indicated reduced ␣-helix content with respect to -sheets and coil structures. Changes observed in tryptophan and tyrosine signals suggest an increasingly hydrophilic environment around these residues. RNA signals revealed a change in the environment of the genome and in its conformation. The ionized-carbonyl vibrations showed small changes between the intact virion and the uncoating intermediate, which points to cleavage of salt bridges in the protein structure during the uncoating process. In conclusion, our data reveal distinguishable Raman signatures of the intact, intermediate, and disrupted EV1 particles. These changes indicate structural, chemical, and solute-solvent alterations in the genome and in the capsid proteins and lay the essential groundwork for investigating the uncoating of EV1 and related viruses in real time.
The Journal of Physical Chemistry B, 2013
Fluorescent proteins are versatile tools for molecular imaging. In this study, we report a detail... more Fluorescent proteins are versatile tools for molecular imaging. In this study, we report a detailed analysis of the absorption and fluorescence properties of the chromophore-binding domain from Deinococcus radiodurans and its D207H mutant. Using single photon counting and transient absorption techniques, the average excited state lifetime of both studied systems was about 370 ps. The D207H mutation slightly changed the excited state decay profile but did not have a considerable effect on the average decay time of the system or the shape of the absorption and emission spectra of the biliverdin chromophore. We confirmed that the fluorescence properties of both samples are very similar in vivo and in vitro. However, we found that the paraformaldehyde fixation of the Escherichia coli cells containing the recombinant phytochrome protein significantly changed the fluorescence properties of the chromophore-binding domain. The biliverdin fluorescence was diminished almost completely, and the fluorescence originated only from the protoporphyrin molecules. Our results emphasize that the effect of protoporphyrin IXa should not be ignored in the fluorescence experiments with phytochrome systems while designing better red fluorescence markers for cellular imaging.
The Journal of Physical Chemistry B, 2010
A series of photoswitchable, R-helical peptides were studied using two-dimensional infrared spect... more A series of photoswitchable, R-helical peptides were studied using two-dimensional infrared spectroscopy (2D-IR). Single-isotope labeling with 13 C 18 O at various positions in the sequence was employed to spectrally isolate particular backbone positions. We show that a single 13 C 18 O label can give rise to two bands along the diagonal of the 2D-IR spectrum, one of which is from an amide group that is hydrogen-bonded internally, or to a solvent molecule, and the other from a non-hydrogen-bonded amide group. The photoswitch enabled examination of both the folded and unfolded state of the helix. For most sites, unfolding of the peptide caused a shift of intensity from the hydrogen-bonded peak to the non-hydrogen-bonded peak. The relative intensity of the two diagonal peaks gives an indication of the fraction of molecules hydrogen-bonded at a certain location along the sequence. As this fraction varies quite substantially along the helix, we conclude that the helix is not uniformly folded. Furthermore, the shift in hydrogen bonding is much smaller than the change of helicity measured by CD spectroscopy, indicating that non-native hydrogen-bonded or mis-folded loops are formed in the unfolded ensemble.
The Journal of Physical Chemistry B, 2002
Fluorescence quantum yield and fluorescence lifetime measurements were performed on trimeric ligh... more Fluorescence quantum yield and fluorescence lifetime measurements were performed on trimeric lightharvesting complex II (LHCII) from spinach in the temperature range 7-293 K. From the results the radiative rate was calculated, which is related to the amount of delocalization of excitations over different pigments because of intermolecular interactions. The emitting dipole strength of LHCII is very similar to that of unbound Chl a, and it appears to be almost independent of temperature. The apparent increase of the radiative rate upon lowering the temperature can largely be explained by the shrinking of the sample. It is concluded that at all temperatures the amount of exciton delocalization in LHCII is small. † Abbreviations.
Synthesis, characterization and applicability of three isotope labeled azobenzene photoswitches
Organic & Biomolecular Chemistry, 2008
We describe a short, efficient approach for the synthesis of three novel isotope labeled azobenze... more We describe a short, efficient approach for the synthesis of three novel isotope labeled azobenzene photoswitches. The synthesis is based on commercially available fully isotope labeled precursors. The target molecules have been obtained in good yields, checked for purity, and identified by NMR and IR spectroscopy and a variety of standard analytical methods (UV-vis, mp, ESI-MS, elemental analysis). Using conventional coupling techniques the three isotope labeled photoswitches can be incorporated very easily in biomacromolecules.
Strong coupling between surface plasmon polaritons and β-carotene in nanolayered system
The Journal of Chemical Physics, 2013
In this article we experimentally demonstrate the strong coupling between surface plasmon polarit... more In this article we experimentally demonstrate the strong coupling between surface plasmon polaritons (SPP) and the S(2) state of β-carotene. The SPPs are excited by prism coupling technique on a thin silver film with β-carotene embedded in a polymer layer on top of that. Rabi splittings with energies 80 and 130 meV are observed in the recorded dispersion relations. Both coupled oscillator model and transfer matrix method are used to fit the experimental results. The scattered radiation of the propagating strongly coupled SPP-S(2) hybrids is collected and an increase of the low energy splitting to 120 meV is observed compared to the reflectivity data. In addition, we performed molecule excitation by laser and analyzed the emission patterns revealing clear surface plasmon coupled fluorescence of β-carotene. By increasing the concentration of β-carotene we are able to collect also surface plasmon coupled Raman scattering. This study substantially extends the SPP-molecular excitation strong coupling studies to biomolecules, and energy transfer and coupling properties of excited states of carotenoids.
Photoprotective function of IsiA in Synechocystis PCC 6803
... Titel, Photoprotective function of IsiA in Synechocystis PCC 6803. Boek/bron titel, Genomics,... more ... Titel, Photoprotective function of IsiA in Synechocystis PCC 6803. Boek/bron titel, Genomics, Proteomics and Structure for Functional Understanding. Auteur/Editor, HCP Matthijs. Uitgever, ESF (European Science Foundation). Plaats, Uva Amsterdam NL. Jaar, 2005. Pagina's, 90 ...
Biochemistry, 2005
The flavoprotein AppA from Rhodobacter sphaeroides contains an N-terminal domain belonging to a n... more The flavoprotein AppA from Rhodobacter sphaeroides contains an N-terminal domain belonging to a new class of photoreceptors designated BLUF domains. AppA was shown to control photosynthesis gene expression in response to blue light and oxygen tension. We have investigated the photocycle of the AppA BLUF domain by ultrafast fluorescence, femtosecond transient absorption, and nanosecond flashphotolysis spectroscopy. Time-resolved fluorescence experiments revealed four components of flavin adenine dinucleotide (FAD) excited-state decay, with lifetimes of 25 ps, 150 ps, 670 ps, and 3.8 ns. Ultrafast transient absorption spectroscopy revealed rapid internal conversion and vibrational cooling processes on excited FAD with time constants of 250 fs and 1.2 ps, and a multiexponential decay with effective time constants of 90 ps, 590 ps, and 2.7 ns. Concomitant with the decay of excited FAD, the rise of a species with a narrow absorption difference band near 495 nm was detected which spectrally resembles the long-living signaling state of AppA. Consistent with these results, the nanosecond flashphotolysis measurements indicated that formation of the signaling state was complete within the time resolution of 10 ns. No further changes were detected up to 15 µs. The quantum yield of the signalingstate formation was determined to be 24%. Thus, the signaling state of the AppA BLUF domain is formed on the ultrafast time scale directly from the FAD singlet excited state, without any apparent intermediate, and remains stable over 12 decades of time. In parallel with the signaling state, the FAD triplet state is formed from the FAD singlet excited state at 9% efficiency as a side reaction of the AppA photocycle.
Functional flexibility of light harvesting in cyanobacteria
Journal of Organic Chemistry, 2005
Regulation of light harvesting is a matter of high importance in photosynthetic organisms. High p... more Regulation of light harvesting is a matter of high importance in photosynthetic organisms. High plants and algae involve mechanism of state transitions which is regulated by the redox state of the plastoquinone pool and leads to the reversible redistribution of excitation energy between the two photosystems through a reorganization of the antennae and to an overall increase in photosynthetic quantum
A time-resolved study of the photocycle of AppA, a flavin-binding photoreceptor that regulates photosynthesis gene expression in Rhodobacter sphaeroides
Tetrahedron Letters, 2005
We measured,picosecond,time-resolved fluorescence of intact Photosystem,I complexes,from,Chlamydo... more We measured,picosecond,time-resolved fluorescence of intact Photosystem,I complexes,from,Chlamydomonas,reinhardtii and Arabidopsis thaliana. The antenna system of C. reinhardtii contains about 30–60 chlorophylls more than that of A. thaliana, but lacks the so- called red chlorophylls, chlorophylls that absorb at longer wavelength than the primary electron donor. In C. reinhardtii, the main lifetimes of excitation trapping are about 27 and 68 ps.
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Papers by Janne Ihalainen