Photodynamics of BLUF domain proteins [Elektronische Ressource] : a new class of the biological blue-light photoreceptors / vorgelegt von Peyman Zirak Yousefabadi

Sea

Sujets

• Biologie

Informations

• Publié par: universitat_regensburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 27
• Poids de l'ouvrage: 4 Mo

Extrait

Photodynamics of
BLUF Domain Proteins
a New Class of the Biological Blue-Light Photoreceptors




Dissertation

Zur Erlangung des Doktorgrades
der Naturwissenschaften
(Dr. rer. nat.)
der Fakultät Physik
der Universität Regensburg

vorgelegt von
Peyman Zirak Yousefabadi
aus Tabriz, Iran


Regensburg 2007
Photodynamics of
BLUF Domain Proteins
a New Class of the Biological Blue-Light Photoreceptors



Dissertation


Zur Erlangung des Doktorgrades
der Naturwissenschaften
(Dr. rer. nat.)
der Fakultät Physik
der Universität Regensburg

vorgelegt von
Peyman Zirak Yousefabadi
aus Tabriz, Iran



Regensburg 2007































Diese Arbeit wurde angeleitet von Prof. Dr. A. Penzkofer


Prüfungsausschuss:

Vorsitzender: Prof. Dr. I. Morgenstern
Erster Gutachter: Prof. Dr. A. Penzkofer
Zweiter Gutachter: Prof. Dr. C. Schüller
Prüfer: Prof. Dr. D. Weiss


Regensburg, 16 Mai 2007 Table of Contents I
Table of Contents


1 Introduction ..............................................................................................................................1
1.1 Photoreceptors..................................................................................................................1
1.2 Aims .................................................................................................................................3
2 An overview of the physical and chemical properties of free flavins ......................................5
2.1 Physics and chemistry of flavins in oxidized form ..........................................................5
2.2 istry of flavins in different redox states ...............................................8
3 BLUF proteins........................................................................................................................11
3.1 AppA protein from Rhodobacter sphaeroides ...............................................................11
3.1.1 Physiological function............................................................................................11
3.1.2 AppA Crystal structure...........................................................................................14
3.2 Slr1694 from synechocystis sp. PCC6803......................................................................16
3.2.1 16
3.2.2 Crystal structure .....................................................................................................16
3.3 BlrB from Rhodobacter sphaeroides .............................................................................18
3.3.1 Physiological function18
3.3.2 Crystal structure19
4 Fundamentals..........................................................................................................................20
4.1 Absorption......................................................................................................................20
4.2 Intrarmolecular interactions ...........................................................................................21
4.2.1 Energy level scheme and relaxation processes.......................................................21
4.2.2 Fluorescence lifetime and fluorescence quantum yield .........................................23
4.2.3 Fluorescence anisotropy and degree of fluorescence polarization.........................26
4.3 Intermolecular interactions.............................................................................................27
4.3.1 Electron transfer .....................................................................................................27
4.3.2 Excitation energy transfer ......................................................................................29
5 Experimental methods............................................................................................................31
5.1 Absorption measurements ..............................................................................................31
5.2 Spectral fluorescence measurements..............................................................................32
5.3 Temporal fluorescence measurements ...........................................................................34
5.3.1 Real time fluorescence measurements ...................................................................34
5.3.2 Fluorescence up-conversion...................................................................................35
6 Results ....................................................................................................................................38
6.1 AppA ..............................................................................................................................38
6.1.1 Chromophore composition.....................................................................................38
6.1.2 Absorption studies..................................................................................................39
6.1.3 Fluorescence studies...............................................................................................41
6.1.4 Photo-cycle dynamics.............................................................................................46
6.2 AppAH44R mutant.........................................................................................................54
6.2.1 Chromophore composition54
6.2.2 Absorption studies55
6.2.3 Fluorescence studies56
6.2.4 Photo-cycle dynamics58
6.3 BlrB ................................................................................................................................69 Table of Contents II
6.3.1 Chromophore composition.....................................................................................69
6.3.2 Absorption studies..................................................................................................70
6.3.3 Fluorescence studies...............................................................................................71
6.3.4 Photo-cycle dynamics.............................................................................................74
6.4 Slr1694 ...........................................................................................................................90
6.4.1 Chromophore composition90
6.4.2 Absorption studies91
6.4.3 Fluorescence studies92
6.4.4 Photo-cycle dynamics95
7 Discussion ............................................................................................................................106
7.1 Photo induced electron transfer....................................................................................106
7.2 signaling state formation ..............................................................................................107
7.3 AppA, BlrB and Slr1694 photo-cycles.........................................................................109
8 Summary ..............................................................................................................................112
9 References114
10 Appendix121
11 Acknowledgement................................................................................................................122

1.Introduction 1
1 Introduction
1.1 Photoreceptors
For centuries, poets, philosophers, artists and scientists have noted and studied the phototrophic
movement of plants. In one of the earliest depictions of plant phototropism, Venus the ancient
goddess of love, transforms Clytie, a water nymph, into a plant because of her infatuation with
Apollo, the sun god. Associated with her metamorphosis into a green plant, Clytie turns and
follows the movement of Apollo [Ovi98]. This tale of unrequited love is based on believes of
early classical philosophers (mostly Aristotelians) that plants exhibit passive responses to the
environment. Accordingly the phototrophic (and solar tracking) tendencies of plant is attributed
to the sun activity in removing fluid from the illuminated side of the plant [Whi06].
thThis simple explanation of phototropism persisted until 17 century, where experimental
observations (which were downplayed due to Aristotelians) began to recognize plant sensitivity
[Web66b].
As due to these observations it became more and more accepted that phototropism is stimulated
by light, the focus returned to the property of light response where it was revealed that blue light
is more effective at orienting the plants [Whi06].
For many years the only proof for the existence of blue light photoreceptors was this sensitivity
to blue light. These photoreceptors are called cryptochrome due to the difficulty for isolating
them (crypto is taken from the Greek word Kryptos that means “hidden”) [Hor03]. However
nowadays the name cryptochrome is only used for the first identified protein of blue light
photoreceptors family. Eventually a 120-kD membrane bound flavin based protein called “Phot”,
was identified as the key e