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[{"key": "dc.contributor.advisor", "value": "Ihalainen, Janne", "language": "", "element": "contributor", "qualifier": "advisor", "schema": "dc"}, {"key": "dc.contributor.advisor", "value": "Rumfeldt, Jessica", "language": "", "element": "contributor", "qualifier": "advisor", "schema": "dc"}, {"key": "dc.contributor.author", "value": "Kurttila, Moona", "language": "", "element": "contributor", "qualifier": "author", "schema": "dc"}, {"key": "dc.date.accessioned", "value": "2019-09-27T06:36:46Z", "language": null, "element": "date", "qualifier": "accessioned", "schema": "dc"}, {"key": "dc.date.available", "value": "2019-09-27T06:36:46Z", "language": null, "element": "date", "qualifier": "available", "schema": "dc"}, {"key": "dc.date.issued", "value": "2019", "language": "", "element": "date", "qualifier": "issued", "schema": "dc"}, {"key": "dc.identifier.uri", "value": "https://jyx.jyu.fi/handle/123456789/65667", "language": null, "element": "identifier", "qualifier": "uri", "schema": "dc"}, {"key": "dc.description.abstract", "value": "Fytokromit ovat kasveista, sienist\u00e4 ja bakteereista l\u00f6ytyvien valoa aistivien proteiinien perhe, jotka toiminnallaan s\u00e4\u00e4telev\u00e4t n\u00e4iden organismien kasvua ja kehityst\u00e4. Fytokromeilla on kaksi palautuvaa rakenteellista konformaatiota, punaista valoa absorboiva Pr ja kaukopunaista valoa absorboiva Pfr. Valosignaalin aiheuttama isomerisaatio proteiinin sis\u00e4\u00e4n hautautuneessa bilin-kromoforissa laukaisee konformaation muutoksen tilasta toiseen, mutta n\u00e4iden suuren kokoluokan rakenteellisten muutosten havainnointi liuoksessa tai reaaliajassa on haastavaa. T\u00e4ss\u00e4 tutkimuksessa liitimme geneettisesti v\u00e4r\u00e4htelij\u00e4leiman, ep\u00e4luonnollisen aminohapon p-atsidofenyylialaniinin (pAzF), yhdeks\u00e4\u00e4n keskeiseen paikkaan Deinococcus radiodurans bakteerifytokromissa (DrBphP). Leima on ihanteellinen hy\u00f6dynnett\u00e4v\u00e4ksi Fourier-muunnosinfrapuna (FTIR) spektroskooppisessa analyysissa, sill\u00e4 atsido-ryhm\u00e4n (\u2013N3) asymmetriset v\u00e4r\u00e4hdykset sijaitsevat proteiinin IR-spektrin signaalittomalla taajuudella. Ymp\u00e4r\u00f6iv\u00e4n s\u00e4hk\u00f6kent\u00e4n ja vetysitoutumisen herk\u00e4sti aistivan leiman avulla voidaan siksi havaita paikallisia kemiallisia ja rakenteellisia muutoksia Pr- ja Pfr-tilojen v\u00e4lill\u00e4. Leiman taajuuden siirtym\u00e4 paljasti selv\u00e4sti havaittavia muutoksia nelj\u00e4n paikan l\u00e4hiymp\u00e4rist\u00f6ss\u00e4. Odotettu muutos havainnollistettiin rakenteellisesti joustavassa hiuspinniulokkeessa sijaitsevassa Y472, miss\u00e4 leima siirtyi vahvoja vetysidoksia muodostavasta ymp\u00e4rist\u00f6st\u00e4 eli liuottimesta Pr-tilassa heikkoja tai ei ollenkaan vetysidoksia muodostavaan ymp\u00e4rist\u00f6\u00f6n, eli kohti proteiinia, Pfr:ss\u00e4. Samanlainen, melko radikaali muutos havaittiin l\u00e4hell\u00e4 isomerisoituvaa biliverdiinin D-rengasta paikassa Y176, mink\u00e4 perusteella leiman vetysidospari voisi olla proteiinin sis\u00e4inen vesimolekyyli. Hiuspinni\u00e4 kohti osoittavassa F203:ssa leima aisti ulokkeen uudelleen laskostumisen Pr- ja Pfr-tilojen v\u00e4liss\u00e4 ja raportoi luultavasti hydrofobisesta ymp\u00e4rist\u00f6st\u00e4 \uf103-heliksin vieress\u00e4 Pfr:ss\u00e4. Kauempana biliverdiinist\u00e4 ja hiuspinniulokkeesta sijaitsevassa R228 leiman ymp\u00e4rist\u00f6ss\u00e4 havaittiin ainoastaan pienest\u00e4 muutoksesta, jonka havaittujen taajuuksien ja paikan perusteella arvioitiin raportoivan jatkuvasta liuottimelle altistumisesta. FTIR- ja UV-Vis -spektroskopia paljastivat, ett\u00e4 kaikissa yhdeks\u00e4ss\u00e4 paikassa leima vaikutti fytokromin absorptio-ominaisuuksiin jonkin verran. Kuitenkin ainoastaan kolmessa paikassa leima h\u00e4iritsi selv\u00e4sti proteiinin laskostumista tai valomuutosominaisuuksia, mik\u00e4 havaittiin pienen\u00e4 puhdistussaantona tai poikkeavina UV-Vis absorptiospektrein\u00e4. Onnistuneesti liitettyjen leimojen todellinen vahvuus piilee niiden potentiaalissa paljastaa signaalin eteneminen reaaliajassa aikaerotteisen FTIR-spektroskopian avulla ja siten ratkaista valosignaloinnin mysteeri bakteerifytokromissa.", "language": "fi", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.abstract", "value": "Phytochromes are a family of photosensory proteins found in plants, fungi and bacteria, which function to support the growth and development of these organisms. Phytochromes populate two reversible structural conformations: red light absorbing Pr and far-red light absorbing Pfr. A photosignal causes the isomerization of the bilin chromophore buried inside the protein, which triggers the conversion from one state to the other, but site-specific observation of these large-scale changes in solution or in real time is challenging. In this study, a vibrational probe, an unnatural amino acid p-azidophenylalanine (pAzF), was genetically incorporated into nine key positions of the Deinococcus radiodurans bacteriophytochrome (DrBphP). This probe is ideal for Fourier-transform infrared (FTIR) spectroscopy analysis as the asymmetric vibrations of the azide group (\u2013N3) locate in signal-free region of the protein IR spectrum. Furthermore, the probe is sensitive to its local electric environment, especially the H-bonding strength, and therefore allows the detection of site-specific structural changes between Pr and Pfr. In four positions, the frequency shift revealed clearly detectable changes in the local environment. The expected change in the structurally flexible hairpin extension was demonstrated in position Y472 where the probe moved from strongly H-bonding environment in Pr, i.e. solvent, to non- or weakly H-bonding environment in Pfr, i.e. toward the protein. Similar, quite extreme but opposite change in the local environment of the label was observed in Y176 near the biliverdin isomerizing D-ring, which suggested that the H-bonding partner of the label in Pfr is an intramolecular water molecule. In position F203 that points towards the hairpin extension, the label sensed the refolding of the extension between Pr and Pfr and reported probably on a hydrophobic environment next to the \uf103-helix in Pfr. Further from the D-ring and the hairpin, the label in R228 revealed a minor change, which, based on the observed frequencies and the position, was addressed to a continuous solvent exposure of the residue. FTIR and UV-Vis spectroscopy indicated that the label affected the DrBphP absorption properties to a certain degree in all nine positions, but only in three positions it severely impeded the protein folding or photoswitchability revealed by a low purification yield or aberrant UV-Vis absorption spectra. The true power of the successfully incorporated probes lies in their potential to reveal the signal propagation in real time by using time-resolved FTIR and, in this way, unveiling the mystery of the signal transduction in bacteriophytochromes.", "language": "en", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.provenance", "value": "Submitted by Miia Hakanen (mihakane@jyu.fi) on 2019-09-27T06:36:46Z\nNo. of bitstreams: 0", "language": "en", "element": "description", "qualifier": "provenance", "schema": "dc"}, {"key": "dc.description.provenance", "value": "Made available in DSpace on 2019-09-27T06:36:46Z (GMT). No. of bitstreams: 0\n Previous issue date: 2019", "language": "en", "element": "description", "qualifier": "provenance", "schema": "dc"}, {"key": "dc.format.extent", "value": "61", "language": "", "element": "format", "qualifier": "extent", "schema": "dc"}, {"key": "dc.format.mimetype", "value": "application/pdf", "language": null, "element": "format", "qualifier": "mimetype", "schema": "dc"}, {"key": "dc.language.iso", "value": "eng", "language": null, "element": "language", "qualifier": "iso", "schema": "dc"}, {"key": "dc.rights", "value": "In Copyright", "language": "en", "element": "rights", "qualifier": null, "schema": "dc"}, {"key": "dc.subject.other", "value": "amber codon suppression", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "bacteriophytochrome", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "Deinococcus radiodurans", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "FTIR", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "photoswichable protein", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "site-specific", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "vibrational probe", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.title", "value": "Azido IR-probes : a novel way to study phytochromes", "language": "", "element": "title", "qualifier": null, "schema": "dc"}, {"key": "dc.type", "value": "master thesis", "language": null, "element": "type", "qualifier": null, "schema": "dc"}, {"key": "dc.identifier.urn", "value": "URN:NBN:fi:jyu-201909274275", "language": "", "element": "identifier", "qualifier": "urn", "schema": "dc"}, {"key": "dc.type.ontasot", "value": "Pro gradu -tutkielma", "language": "fi", "element": "type", "qualifier": "ontasot", "schema": "dc"}, {"key": "dc.type.ontasot", "value": "Master\u2019s thesis", "language": "en", "element": "type", "qualifier": "ontasot", "schema": "dc"}, {"key": "dc.contributor.faculty", "value": "Matemaattis-luonnontieteellinen tiedekunta", "language": "fi", "element": "contributor", "qualifier": "faculty", "schema": "dc"}, {"key": "dc.contributor.faculty", "value": "Faculty of Sciences", "language": "en", "element": "contributor", "qualifier": "faculty", "schema": "dc"}, {"key": "dc.contributor.department", "value": "Bio- ja ymp\u00e4rist\u00f6tieteiden laitos", "language": "fi", "element": "contributor", "qualifier": "department", "schema": "dc"}, {"key": "dc.contributor.department", "value": "Department of Biological and Environmental Science", "language": "en", "element": "contributor", "qualifier": "department", "schema": "dc"}, {"key": "dc.contributor.organization", "value": "Jyv\u00e4skyl\u00e4n yliopisto", "language": "fi", "element": "contributor", "qualifier": "organization", "schema": "dc"}, {"key": "dc.contributor.organization", "value": "University of Jyv\u00e4skyl\u00e4", "language": "en", "element": "contributor", "qualifier": "organization", "schema": "dc"}, {"key": "dc.subject.discipline", "value": "Solu- ja molekyylibiologia", "language": "fi", "element": "subject", "qualifier": "discipline", "schema": "dc"}, {"key": "dc.subject.discipline", "value": "Cell and molecular biology", "language": "en", "element": "subject", "qualifier": "discipline", "schema": "dc"}, {"key": "yvv.contractresearch.funding", "value": "0", "language": "", "element": "contractresearch", "qualifier": "funding", "schema": "yvv"}, {"key": "dc.type.coar", "value": "http://purl.org/coar/resource_type/c_bdcc", "language": null, "element": "type", "qualifier": "coar", "schema": "dc"}, {"key": "dc.rights.accesslevel", "value": "restrictedAccess", "language": null, "element": "rights", "qualifier": "accesslevel", "schema": "dc"}, {"key": "dc.type.publication", "value": "masterThesis", "language": null, "element": "type", "qualifier": "publication", "schema": "dc"}, {"key": "dc.subject.oppiainekoodi", "value": "4013", "language": "", "element": "subject", "qualifier": "oppiainekoodi", "schema": "dc"}, {"key": "dc.format.content", "value": "fulltext", "language": null, "element": "format", "qualifier": "content", "schema": "dc"}, {"key": "dc.rights.url", "value": "https://rightsstatements.org/page/InC/1.0/", "language": null, "element": "rights", "qualifier": "url", "schema": "dc"}, {"key": "dc.rights.accessrights", "value": "The author has not given permission to make the work publicly available electronically. Therefore the material can be read only at the archival workstation at Jyv\u00e4skyl\u00e4 University Library (https://kirjasto.jyu.fi/en/workspaces/facilities).", "language": "en", "element": "rights", "qualifier": "accessrights", "schema": "dc"}, {"key": "dc.rights.accessrights", "value": "Tekij\u00e4 ei ole antanut lupaa avoimeen julkaisuun, joten aineisto on luettavissa vain Jyv\u00e4skyl\u00e4n yliopiston kirjaston arkistoty\u00f6semalta. Ks. https://kirjasto.jyu.fi/fi/tyoskentelytilat/laitteet-ja-tilat..", "language": "fi", "element": "rights", "qualifier": "accessrights", "schema": "dc"}, {"key": "dc.type.okm", "value": "G2", "language": null, "element": "type", "qualifier": "okm", "schema": "dc"}]
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