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Heat-sensitive UV-Vis measurements for Phytochrome

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Authors
Mäkelä, Sami
Date
2016
Discipline
Solu- ja molekyylibiologia
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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. ...
 
Keywords
phytochrome photoswitchable protein photo-sensing module stability denaturation temperature
URI

http://urn.fi/URN:NBN:fi:jyu-201606042874

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