Determination of a conformational change in filamin A with Förster resonance energy transfer

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dc.contributor.author Seppälä, Jonne
dc.date.accessioned 2011-05-19T10:44:22Z
dc.date.available 2011-05-19T10:44:22Z
dc.date.issued 2011
dc.identifier.uri http://urn.fi/URN:NBN:fi:jyu-2011051910885 en
dc.identifier.uri http://hdl.handle.net/123456789/27046
dc.description.abstract Filamins are large rod-like proteins that cross-link actin filaments into three-dimensional networks. They also bind to a plethora of proteins with distinct functions showing that they have a versatile role in cells. Functional filamins are dimers consisting of an N-terminal actin binding domain followed by 24 immunoglobulin-like domains. The most C-terminal domains mediate the dimerization. Two hinge regions are located between the domains 15 and 16 and 23 and 24, respectively, and produce structural flexibility that is essential for the protein function. The domains 18-19 and 20-21 are folded in a pairwise manner in which the first β strands of the even numbered domains are folded along with the odd numbered domains. Recent study implies that a focal adhesion protein migfilin binding replaces the β strand of the domain 20 inducing a conformational change in filamin A that makes its structure more flexible. Previously only stretching force induced conformational changes have been reported and they are thought to function as a mechanism to sense tension. The aim of this study was to demonstrate the migfilin induced conformational change using Förster resonance energy transfer (FRET). Enhanced green fluorescent protein (EGFP) was cloned to the C-terminus of a four domain fragment of filamin A which was first mutated to have a unique site for the acceptor fluorophore conjugation. Then, the recombinant fusion protein was expressed overnight and subsequently purified with affinity chromatography and gel filtration. Finally, the purified protein was labeled with Alexa Fluor 532 C5 maleimide to produce a FRET pair. The change in the energy transfer efficiency upon migfilin addition was measured with steady-state and time-resolved fluorescence methods. No statistically significant change in the amount of energy transfer was observed. The reason is unclear, though it is possible that the EGFP disrupted the dynamics of filamin A or that the migfilin binding was abolished. However, it is also possible that the used method was not sensitive for the possible conformational change in the construct. Changes in the construct are required in further studies.
dc.format.extent 46 s.
dc.language.iso eng
dc.rights This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited. en
dc.rights Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty. fi
dc.subject.other EGFP
dc.subject.other filamin A
dc.subject.other FRET
dc.title Determination of a conformational change in filamin A with Förster resonance energy transfer
dc.type Book en
dc.identifier.urn URN:NBN:fi:jyu-2011051910885
dc.subject.ysa proteiinit
dc.type.dcmitype Text en
dc.type.ontasot Pro gradu fi
dc.type.ontasot Master’s thesis en
dc.contributor.tiedekunta Matemaattis-luonnontieteellinen tiedekunta fi
dc.contributor.tiedekunta Faculty of Mathematics and Science en
dc.contributor.laitos Bio- ja ympäristötieteiden laitos fi
dc.contributor.laitos Department of Biological and Environmental Science en
dc.contributor.yliopisto University of Jyväskylä en
dc.contributor.yliopisto Jyväskylän yliopisto fi
dc.contributor.oppiaine solu- ja molekyylibiologia fi

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