Biochemical characterization of LOV domain proteins from protist-SK
1. 1
Biochemical characterization of LOV domain proteins from Protist
Submitted by
Hari Mohan
M.Sc. (Final) Goa University
May –June, 2016
As
Summer Research Project Report
To
Jawaharlal Nehru University
Under supervision of
Dr. Suneel Kateriya
Associate Professor
School of Biotechnology
JNU, New Delhi-110067
India
2. 2
Acknowledgement
I am deeply grateful to the School Of Biotechnology, Jawaharlal Nehru University for giving
me an opportunity to working in their lab. I express my gratitude to Dean Prof. Uttam K. Pati
for enabling me to work. I take great honour to thank my mentor Dr. Suneel Kateriya, for his
invaluable guidance and advice during the course of my summer training. Under his guidance I
learned a lot from my mistakes and overcame the difficulty smoothly. I am also thankful to Dr.
S. K. Veetil for her advises and support. I am highly thankful to Ms. Komal Sharma for her
incomparable patience in guiding me through my work, her presence of mind and her cheerful
approach in solving those problems I encountered. She made me strong in concepts and was
always ready to help me with opening up a different approach to any situation at hand. I
gratefully acknowledge my labmates Ms. Sushmita Kumari, Ms. Shivanika Soni and Ms. Ayushi
Mishra for their understanding, encouragement, and cooperation. They have always motivated
me at various stages and have been a constant moral support. I warmly thank Mr. Deepak Kumar
for his regular support. I am deeply thankful to Ms.Vidya Natrajan and Ms. Preeti for their
support and for making the ambience a very friendly one.
I am thankful to my classmates who shared the same position as mine, and understood my
feelings very well. Talking to them always made me smile, and that fun filled, pleasant moments
in class will be treasured.
Lastly, I would like to give my special thanks to my parents and my sisters and brother whose
love enabled me to complete this work.
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Table of Contents
S.No Title Page No.
1. Introduction 5
2. Objectives 9
3. Material & methods 10
4. Results and discussion 13
5. Future plan 18
6. Reference 19
4. 4
Introduction
Light is a non-ionising electromagnetic radiation with three main characteristic factor-
wavelength, speed/velocity, and frequency. Light is a prime source of energy and study of its
interaction with living organisms is termed as Photobiology. The cells that respond to light in a
living organism are known as a photoreceptor. There are various kinds of photoreceptor
classified on the basis of a range of light spectrum they sense i.e. red/far-red light receptor
phytochrome (Phy), various blue light receptors such as cryptochrome (Cry) and phototropin
(phot), a UV-B photoreceptor. Photoreceptors consist of two part, the protein part and non-
protein, chromophore part. The non-protein part can respond to light through photoisomerization
or photo excitation. The protein omnipresence in higher plant ranges from 114 to 130 kDa
depending upon species. This was reported as blue – light photoreceptor phototropin (phot)
which activates phototransduction (4, 18). Phot usually represents the proteinceous part of the
photoreceptor. There is six class of photoreceptor protein such as light oxygen-voltage (LOV)
sensors, xanthopsins, phytochromes, blue-light sensors using flavin adenine dinucleotide
(BLUF), cryptochromes, and rhodopsins.
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Figure 1. Chromophore and simplified photochemistry of the six photoreceptor classes. (a)
Light-oxygen-voltage (LOV). (b) Xanthopsins. (c) Phytochrome. (d) Blue-light sensors using
FAD (BLUF). (e) Cryptochrome. (f) Rhodopsin. (M¨ oglich et al.)
The blue light receptor which sense Light along with Oxygen and Voltage are called LOV
domain which belongs to PAS domain family (8).LOV domain is present on the N terminus of
phototropin and also are present in families such as ZTL/FKF1/LKP2 (specific to land plants),
aureochrome (in photosynthetic stramenopiles) etc.
Figure 2. Schematic alignment of the LOV blue light. receptor protein families, phototropin,
ZTL/FKF1/LKP2 (both from A.thaliana) and aureochrome(from Vaucheria frigida)(Japanese
Society of Plant Physiologists).
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Phototropin
Phototropin has first reported photoreceptor protein in etiolated pea seedling (1) localised in the
plasma membrane. Phototropin was originally named as NPH1 (non-phototropic hypocotyl)
protein based on its function of phototropism. Arabidopsis contains two phototropin designated
as phot1 and phot2. Apart from phototropism, phot1 and phot2 also regulate photosynthetic
efficiency through opening stomatal pore and regulating chloroplast movement. Under low light
conditions, phot1 and phot2 induce chloroplast accumulation movement to the upper cell surface
to promote light capture for photosynthesis. Both phot1 and phot2 play significantly different
roles. Phototropin is composed of N terminus photosensory domain and C terminus
serine/therionine kinase domain .Phototropin belongs to AGC family of the kinase (cAMP-
dependent protein kinase, cGMP-dependent protein kinase G and phospholipid dependent
protein kinase C) .The N terminus domain consists of two structurally and functionally related
photosensory domain of ~110amino acid. LOV domains are a member of large PAS (Period
domains associated with cofactor binding mediating protein. LOV domains bind the cofactor
flavin mononucleotide (FMN)
Figure 3. Showing a typical structure of phototropin containing two PAS domain at N-terminal
and serine/threonine kinase at C-terminal.
LOV domain
LOV domain is discovered as tandem sensor domains in the plant photoreceptor phototropin (2).
LOV domains are yellow in colour (collected from expressed E.coli) and emit a strong green
fluorescence when irradiated with UV/blue light.
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Figure 4. Three structures of representatives of the LOV1 and LOV2. Chromophores are
shown as space-filling models in cyan, chromophore-binding domains in green. (a) LOV: A.
sativa phototropin 1 LOV2 domain (PDB entry -dimensional 2V0U) (11).
The Jα-helix is located at the C terminus of LOV2 and is amphipathic in nature, consisting of
polar and a polar sides, he latter of which docks onto the β–sheet
strands of the LOV2-core (17). It is shown that unfolding of the Jα-helix results in activation of
the C-terminal kinase domain (6). LOV domains utilize flavin nucleotide cofactors to detect blue
light. After absorption of a photon in the blue spectral region around 450 nm by the dark-adapted
D450 state, the flavin nucleotide cofactor undergoes efficient intersystem crossing in
picoseconds to yield a triplet L660 state (10). Within microseconds a covalent, thioether bond
between atom C(4a) of the flavin ring and a conserved, spatially proximal cysteine residue is
formed, the S390 state (11). Despite closely similar sequence and structure, individual LOV
domains differ markedly in the kinetics and quantum yield of their photocycle (3, 9).
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Figure 5. Light-induced structural changes in the A. sativa phototropin 1 LOV2 domain (7). The
LOV core domain is shown in white and N and C-terminal α-helical extensions, A’α and Jα, in
green. (a) In the dark the active-site cysteine 450 adopts two conformations. Hydrogen bonds are
formed between Q513 and atom O4 of the FMN ring, and from N414 to D515. (b) Upon blue-
light absorption, a thioether bond (yellow) forms and induces a slight tilt in the FMN ring. Q513
presumably flips its side chain to form new hydrogen bonds to the FMN ring and N414.
Conformational changes could thus be propagated to the terminal helices and could cause
unfolding of the Jα helix (5).
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Objective
1. Data mining for LOV domain containing proteins in protist.
2. Culturing algae
3. Transformation of BL21 with CVG2 and protein expression.
4. Protein isolation.
5. Protein purification using Immobilized Metal Affinity Chromatography.
6. Estimation of protein content in the protein sample using Bradford Assay.
7. Detection of protein using protein specific antibody.
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Material and Methods
Identification of LOV domain containing proteins using freeware from protist genome databases
4.1 Sequence mining of LOV domain containing protein from C.subellipsodea
Putative phototropin sequence of free living C.subellipsodea was identified using
BLAST with phototropin of C.reinhardtii as a query sequence. All relevant protein
sequences retrieved were further analyzed by using Prosite. Sequence alignment
between Crphot and Csphot was carried out using
ClustalW.(http://www.genome.jp/tools-bin/–clustalw)
4.2 Construction of Phylogenetic tree
A phylogenetic tree was constructed using CLUSTALW programme
(http://www.genome.jp/toolsbin/clustalwtree?tree_upgma+160427023910osThk).Predic
tion of 2D & 3D structure. The secondary structure of protein was predicted by using
Psipred programme (http://bioinf.cs.ucl.ac.uk/psipred/result/f9280c66-0916-11e6-8e16-
00163e110593).Tertiary structure of protein was predicted by using the program Swiss
model. (http://swissmodel.expasy.org/workspace/index.php?func=
modelling_simpl).
4.3 Culture the selected species with LOV domain in suitable medium in optimum
conditions
Tris-Acetate-Phosphate And Bold's Basal Medium medium used for C.subelliopsida
and Terrific Broth medium used for BL21 CVG2 bacteria
4.4 Purification of LOV protein
Primary culture :
Take 10ml of LB in an autoclave falcon, add 10µl of kan and inoculate bacterial
colonies from the plate with the help of tip and pour into the falcon. Incubate at 37°c for
overnight in the shaker.
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Secondary culture :
Take 500ml of TBM in the 2lt flask (remove 2ml blank for O.D)Add 500µl of
Kanamycin.Add 5ml of primary culture (G2 Sumo CV BL21) in the flask.Keep in the
shaker at 37°c, 220rpm till the O.D reach 0.5 to 0.6 @750nmIncubate on ice for
40minutes.Add 0.3mM IPTG.Keep in the shaker at 16°c, 220rpm for 2 days.
Protein purification :
Collect the secondary growth in the oak ridge tube. Centrifuge at 6000rpm for 10
minutes. Discard the supernatant.Resuspend pellet in 1XPBS (10ml)+ Lysozyme
(working concentration- 50µg/µl) + PMSF (working concentration-
200µMS).Centrifuge at 200rpm for 20 minutes at 37°C.Sonicate the dissolved pellet,
amplitude- 45% ; Pulse 30 sec ON 30 sec OFF for 5cycles. Take the total cell lysate and
then centrifuge at 13000rpm for 50 minutes at 4°C.Take the soluble fraction for further
purification with affinity column chromatography which contains Co2+ talon
beads.Collect the flow through. Wash the column 3 times with 1XPbs.Elute the sample
in elution buffer and aliquot of 300µl.
4.5 Estimation of protein by Bradford assay
Quantification of protein was did via Bradford assay. In this, first sample was prepared
by taking 98µl of water in 2µl of sample and then add 900µl of Bradford reagent. After
that O.D was taken at 595nm.Analysed the result from the BSA standard curve to
calculate the concentration of protein in sample.
4.6 SDS Page analysis
Protein samples were purified by affinity chromatography and Samples were prepared
under reducing condition in 4X laemmeli's buffer. Heated at 95°C for 5 minutes and
analyzed on 12% gel.
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4.7 Spectral analysis
The absorption spectra was recorded from 250 nm – 600 nm in Perkin with the scan
speed of 2500 nm/ min. The dark adapted spectrum was recorded without blue light.
The data obtained was plotted using IGOR PRO software (3.1.4 version) and Origin
software.
4.8 Algal Cell Extract Preparation:
To prepare total cell extract of an algal culture following protocol was used:
Take secondary growth of well grown algal culture (OD730 -0.6). Spin down the culture at
5000rpm for 10 min in falcon tubes. Discard the supernatant. Resuspend the pellet in 5ml
1xPBS. Sonicate the sample at 40% amplitude; Pulse 30 sec ON 30 sec OFF and 5-6
cycles.
Take out 1 ml from the cell lysate. Spin down at 13000rpm for 5min. Store the supernatant and
re-suspend the pellet in 1ml 1xPBS.
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Result and Discussion
The sequence of Crphot retrieved from NCBI and BLAST it in Phytozome database
Figure: 6 showing the BLAST result with query as Crphot sequence
The Phototropin sequence of six different organisms retrieved from Phytozome database.
>Cs-169
MPAQTGQAEKQQKDVQLHPELQRPGQKVPGPAPQLTKVLAGLRHTFVVADATLP
DCPLVFASEGFLSMTGYSAEEVLGHNCRFLQGEGTDPKEVAIIRDAVKKGEGCSV
RLLNYRRDGTPFWNLLTMTPIKTEDGKVSKFVGVQVDVTSKTEGRAFSDATGVPL
LVKYDTRLRENVAKNIVQDVTLQVQEAEEEDSGAASEAARVSSLKGFNKLWHKM
GNKVTRPQCLGGPPSAPLGDPKAQASAHDPQLQKQGERVGKKMTAPKTFPRVAM
DLATTVERIQQNFCICDPNLPDNPIVFASDGFLEMSQYDRFEVLGRNCRFLQGPDT
DPKAISIIRDAIKSQSEATVRILNYRKSGQPFWNMLTIAPMADVDGTSRFFIGVQVD
VTAEDVPMTGGIPQVDAKAVKAADPMGSVLGMAQRQMGAGWAVHDPWAAIHA
GVASLKPHKAQEKVWAALRENDRKNGRLALSQFRRLKQLGTGDVGLVDMVELQ
DGSGRYAMKTLEKAEMLERNKVMRVLTEAKILSVVDHPFLASLYGTIVTDTHLHF
LMQICEGGELYALLTSQPSKRFKESHVRFYTAEVLIALQYLHLMGFVYRDLKPENI
LLHSSGHILLTDFDLSFCQGSTKVKFEKKKNGHANSSQPGATQVSPAEEIMMIAVP
EARANSFVGTEEYLAPEVINGVGHGAGVDWWSFGILIYELLYGFTPFRGKKRDETF
NNILKRPLSFPELPEVSDECKDLISQLLERDPAKRLGAHAGAEEIKAHPFYESINWA
LLRNTRPPYIPRRSALRKANKPSPAAQAQFDDF*
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The phototropin sequence showing zero E-value undergone further alignment.
Figure: 7 Showing Amino acid sequence alignment of Phototropin. Multiple sequence
alignment, pairwise alignment and their scores
Figure: 8 Showing Phylpgenetic analysis of closely related species with photropin Rooted
phylogenetic tree with branch length (UPGMA) Of closely related species
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Conserved domain in closly related organisms
Figure: 9 showing conserved domains in related protein sequence
(http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi?cdsid=QM3-qcdsearch-
B4B260BF69CA98F&tdata=qopts)
Structural analysis of phototropin
Secondary structure of Crphot and Csphot.
A
B.
Figure :10 Secondary structure prediction of Crphot (fig A), and Csphot (fig b), sequence using
GOR IV secondary structure prediction method (https://npsapreabi.ibcp.fr). Α-helix indicated by
the blue line and random coil by light red and the extended strand is indicated as red light.
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Sec structure of proteins α-helix (%) Extended strand (%) Random coil
(%)
Csphot 37.03 15.74 42.23
CrPhot 36.53 16.13 47.33
Table: 1 Comparative analysis of Csphot and Crphot. The overall conclusion of this table is that
Crphot have fewer α-helices than the Csphot.
Tertiary structure prediction
(a) (b)
Figure: 11 Predicted tertiary structure of Phototropin fig. (a) Csphot and fig. (b) Crphot. The
tertiary structure of phototropin was done by utilizing SWISS-model.
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Protein Oligo-state method seq
similarity
Csphot hetero-oligomer X-ray, 2.20 A 0.40
Crphot monomer X-ray, 1.74 A 0.39
Table: 2 Comparative analysis of Tertiary structure on the basis of Swiss Model
Spectrophotometric analysis
Figure: 11 Absorption spectra of pure FMN (Sigma). Spectra shows two characteristic peaks of
FMN i.e. 370 nm and 450 nm
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Figure: 13 Absorption spectra of protein in dark showing characteristic vibronic peaks of FMN
in protein- bound form
Isolated protein from transformed BL21 CVG2 bacteria found to be yellow colored pallet
whereas dark green colored in case of C-169.Baterial protein sample undergone further
purification through Immobilized metal chromatography method collected in three
elution(E1,E2,andE3).It was found E2 contained maximum protein concentration followed by E3
and E1.
Elute-2 Elute 3 C-169(Total cell lysate)
Figure: 14 showing color density of recombinant LOV protein and algal total protein sample
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SDS page analysis of algal protein sample showing various band whereas bacterial purified
protein sample showed band belonging to size 50-70 kDa concluded with the help of protein
ladder.
4X 2X 2X(37) 4XS 2XS 2XS(37) E2 E3 M
Figure: 15 Image showing protein bands of characteristic size
It was quantified the protein concentration in sample through Bradford Assay @ 595nm
Sample Abs@595nm Protein conc.
E2 (CVG2) 0.325 15.65ug/ul
E3 (CVG2) 0.191 6.84ug/ul
Table: 3 showing concentration of protein in samples
Future Plan
The characterization of LOV domain proteins will be done using modern methods of the UV-
visible spectroscopy.
22. 22
Reference
1. Crosson S, Rajagopal S, Moffat K. 2003. The LOV domain family: Photoresponsive
signaling modules coupled to diverse output domains. Biochemistry 42:2–10
2. Christie JM, Reymond P, Powell GK, Bernasconi P, Raibekas AA, et al. 1998.
Arabidopsis NPH1: a flavoprotein with the properties of a photoreceptor for
phototropism. Science 282:1698–701
3. Christie JM, Swartz TE, Bogomolni RA, Briggs WR. 2002. Phototropin LOV domains
exhibit distinct roles in regulating photoreceptor function. Plant J. 32:205–19
4. Gallagher S, Short TW, Ray PM, Pratt LH, Briggs WR. 1988. Light-mediated changes in
two proteins found associated with plasma membrane fractions from pea stem sections.
Proc. Natl. Acad. Sci. USA 85:8003–7
5. Harper SM, Neil LC, Gardner KH. 2003. Structural basis of a phototropin light switch.
Science 301:1541–44
6. Harper SM, Christie JM, Gardner KH. 2004. Disruption of the LOV-J alpha helix
interaction activates phototropin kinase activity. Biochemistry 43:16184–92
7. Halavaty AS, Moffat K. 2007. N- and C-terminal flanking regions modulate light-
induced signal transduction in the LOV2 domain of the blue light sensor phototropin 1
from Avena sativa. Biochemistry 46:14001–9
8. Huala E, Oeller PW, Liscum E, Han IS, Larsen E, Briggs WR. 1997. Arabidopsis NPH1:
a protein kinase with a putative redox-sensing domain. Science 278:2120–23
9. Kasahara M, Swartz TE, Olney MA, Onodera A, Mochizuki N, et al. 2002.
Photochemical properties of the flavin mononucleotide-binding domains of the
phototropins from Arabidopsis, rice, and Chlamydomonas reinhardtii. Plant Physiol.
129:762–73
10. Swartz TE, Corchnoy SB, Christie JM, Lewis JW, Szundi I, et al. 2001. The photocycle
of a flavin binding domain of the blue light photoreceptor phototropin. J. Biol. Chem.
276:36493–500
11. Salomon M, Eisenreich W, Durr H, Schleicher E, Knieb E, et al. 2001. An opto
mechanical transducer ¨in the blue light receptor phototropin from Avena sativa. Proc.
Natl. Acad. Sci. USA 98:12357–61
12. Short TW, Porst M, Palmer J, Fernbach E, Briggs WR. 1994. Blue light induces
phosphorylation at seryl residues on a Pea (Pisum sativum L.) plasma membrane
protein.Plant Physiol. 104:1317–24
