Heat-sensitive UV-Vis measurements for Phytochrome
Authors
Date
2016Discipline
Solu- ja molekyylibiologiaAccess restrictions
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Fytokromit ovat punaista valoa aistivia proteiineja, jotka säätelevät suurta joukkoa solun
toimintoja kasvi-, bakteeri- ja sienisoluissa. Nämä valoaktiiviset proteiinit muuntavat
konformaatiotaan palautuvasti aktiivisen (Pfr) ja inaktiivisen (Pr) tilan välillä. Fytokromien
aminohappojen väliset heikot vuorovaikutukset - etenkin vetysidokset - vaikuttavat lopulta
laskostumiseen ja proteiinin lämpöstabiiliuteen. Fytokromit esiintyvät homodimeerisinä
rakenteina, joiden monomeerit koostuvat useista domeeneista. Nämä jakautuvat N-
terminaaliseen valosensitiiviseen moduuliin (PSM) ja C-terminaaliseen OutPut moduliin
(OPM). PSM koostuu kolmesta domeenista (PAS-GAF ja PHY). Tässä työssä tutkittiin
PSM:n eri konstruktioita UV-Vis spektroskopialla - kustakin sekä dimeeriä että
monomeeriä sekä Pr, että Pfr tiloissa. Spektrit mitattiin lämmittäen näytteitä viiden asteen
intervalleina 20 C:sta 85 C:een. Absorbanssin muutoksista lämpötilan funktiona päätteltiin
kunkin näytteen lämmönkestävyyttä. Havaittiin, että verrattaessa dimeeriä ja monomeeriä
dimeeri sieti noin viisi astetta enemmän lämmitystä. Kun verrattiin PAS-GAF-PHY:a ja
PAS-GAF:a havaittiin, että PAS-GAF-PHY sietää noin 10 astetta suurempaa lämpötilaa
denaturoitumatta merkittävästi. Mittausten jälkeen näytteet jäähdytettiin
huoneenlämpötilaan ja niiden spektrit mitattiin uudestaan. Jäähdytettyjen näytteiden
spektreissä oli joitakin eroja PAS-GAF-PHY ja PAS-GAF näytteiden välillä. Muutokset
viittasivat PAS-GAF fragmenttien täydellisempään denaturoitumiseen.
...
Phytochromes are red/far-red sensing proteins, which regulates a wide array of cellular
functions in plant, bacterial and fungal cells. These proteins change their conformation
reversibly between active (Pfr) and inactive (Pr) state, in response to lightning conditions.
They were first discovered from plants in 1959, but despite intensive studies, the actual
mechanism of their action still holds some mysteries. Weak interactions between the amino
acid residues of phytochromes, alters in their thermal stability – mainly through hydrogen
bonds. Phytochromes consists from a N-terminal photo sensing module (PSM) and from a
C-terminal output module (OPM). The photo sensing module contains three domains (PAS-
GAF and PHY). In this experiment the heat sensitivity of different photo sensing module
constructs were monitored (PAS-GAF-PHY dimer and monomer, and PAS-GAF dimer and
monomer) by UV-Vis spectroscopy. Measurements were done by heating various PSM
constructs in Pr and Pfr states. Spectra were measured at 5-degree intervals from 20 C to 85
C. The heat durability of samples were determined from the spectral changes. As a result,
comparison between a dimer and monomer showed higher heat tolerance in a dimeric
construct. Comparison between PAS-GAF-PHY and PAS-GAF showed that PAS-GAF-
PHY tolerated approximately 10 degrees higher temperature before denaturation. After
spectral measurements samples were cooled and their spectra were measured. There was
some difference between cooled spectra of PAS-GAF-PHY and PAS-GAF samples. These
spectral variations indicated from more complete denaturation of PAS-GAF fragments.
...




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