Ivana
Kuta Smatanova1, Tanis Hogg2, Rolf Hilgenfeld3,
Rita Grandori4, Jannette Carey5, Frantisek Vacha6
and Dalibor Stys1
1Institute of Physical Biology USB CB & Institute of Landscape Ecology AS CR, Zamek 136, 373
33 Nove Hrady, Czech Republic, e-mail: ivas@bf.jcu.cz, 2JenaDrugDiscovery
GmbH, Löbstedter Str. 78, 07749 Jena, Germany, e-mail:
tanis.hogg@jenadrugdiscovery.com, 3Institute of Biochemistry, University
of Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany, e-mail: hilgenfeld@biochem.uni-luebeck.de,
4Organische Chemie, Johannes Kepler University, Altenbergerstrasse
69, 4040 Linz, Austria, e-mail: rita.grandori@jku.at,
5Department of Chemistry, Princeton University, Washington Rd. and
William St., Princeton, NJ 08544-1009, USA, e-mail: jcarey@princeton.edu, 6Institute of Physical Biology USB CB, Zamek 136, 373 33
Nove Hrady, & Institute of Plant Molecular Biology
AS CR, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic, e-mail:
vacha@jcu.cz
Non-membrane
proteins such as the pokeweed antiviral protein from Phytolacca acinosa (PAP-Saci)
and the tryptophan (W)-repressor binding protein A (WrbA) and also membrane protein, the five-chlorophyll reaction
center of photosystem II from Pisum
sativum, have been crystallized in our laboratory.
Keywords: pokeweed antiviral protein, flavodoxin-like
protein, photosystem II reaction center protein, crystallization experiments
A.
Non-membrane proteins
The antiviral protein,
PAP-Saci
The antiviral protein, PAP-Saci, isolated from seeds of the Chinese pokeweed plant, Phytolacca acinosa, was crystallized.
Interestingly, of two bands seen close to one another in the SDS-PAGE
(molecular masses of approximately 29kDa and 30kDa), only one, the 30kDa form,
was retrieved from re-dissolved PAP-Saci
crystals. The diffraction data colorless PAP-Saci crystals with dimensions of about 0.5 x 0.2 x 0.2 mm were
collected using synchrotron radiation at the IMB Jena - University of Hamburg -
EMBL Beamline X13, DESY (Hamburg) to a resolution of 1.7 Å. The crystal
structure of PAP-Saci was solved by
molecular replacement, using the atomic coordinates of Phytolacca americana PAP-I (PDB ID: 1PAF [1]) as a search model
[2]. The excellent map quality allowed for an ‘X-ray sequencing’ approach (with
the exception of the Asn/Asp and Gln/Glu ambiguities) as several amino acid
exchanges with respect to the sequence of PAP-I from Ph. americana were clearly evident. The full sequence of PAP-Saci was determinated using MALDI-MS and
tandem mass spectrometry techniques. According to the known sequence of PAP-Saci the protein structure was rebuild.
The refined structure includes 261 residues, one N-acetyl-D-glucosamine
monosaccharide (GlcNAc) moiety and 383 water molecules, yielding an R factor of 18.1% and free R factor of 22.3%. PAP-Saci contains a canonical RIP fold
consisting of eight a-helices
and a six-stranded b-sheet.
One GlcNAc residue was found to play a critical role in crystal lattice
formation, forming a packing interface across a crystallographic two-fold with
the identical sequon of an adjacent monomer [3,
4].
Tryptophan (W)-repressor binding protein A, WrbA
Sequence analysis and homology modeling identified the tryptophan
(W)-repressor binding protein A
(WrbA), the polypeptide that specifically binds tryptophan repressor protein
(TrpR) [5], as a member of the new class of flavodoxin-like proteins with
typical a/b twisted
open-sheet fold. The protein binds flavinemononucleotide (FMN) specifically and
weaker than many flavodoxins. The WrbA has no influence on the affinity or form
of DNA binding by the TrpR; its physiological role is still unclear [6]. The
protein WrbA was overexpressed in E. coli
and purified. 5-mg/ml WrbA protein has been used for crystallization
experiments. Crystallization trials were performed in “Cryschem” plates (Hampton
Research, Laguna Niguel, CA, USA) for sitting
drops, in capillary tubes and in dialysis button at room temperature. Within 6
weeks, colorless WrbA crystals with dimensions of about 0.3 x 0.2 x 0.1 mm were
grown in capillaries and in dialysis button from reservoir solution containing
3.0M ammonium sulfate and 0.1M Tris pH 7.50. Other crystals of WrbA were grown
in sitting drops from the B5 solution of JBScreen Crystal Screening Kit 5
(JenaBioscience GmbH, Jena, Germany). The WrbA protein crystals grown in
capillary were measured directly in the
capillary at the EMBL Beamline X13, DESY
(Hamburg) to a resolution of 2.2 Å. Structure solution of the WrbA
apo-protein is in the progress.
Five-chlorophyll reaction center
of photosystem II
Photosystem II (PSII) is a multisubunit pigment-protein complex located
in the photosynthetic membranes of green plants, algae and cyanobacteria. It
contains many cofactors, which together trap, transfer and modulate the
utilization of solar energy to drive the water splitting reaction. These
reactions are being responsible for the production of atmospheric oxygen and
indirectly for almost all the biomass on the planet [7]. For the central role
of PSII in bio-energetics, PSII has been studied using different experimental
techniques [8, 9].
The higher plant’s photosystem II consists of the reaction center
proteins D1 and D2, a- and b-subunits of cytochrome b-559, two
chlorophyll-binding internal antenna proteins CP43 and CP47 and the complex of
manganese-stabilizing proteins of 33, 23, and 16 kDa sizes. The
five-chlorophyll reaction center of photosystem II was isolated from green pea (Pisum
sativum) and purified according to Vacha [10]. 15-mg/ml (1.3‑mg/ml
chlorophyll a) protein has been used
for crystallization experiments. JBScreen Crystal
Screening Kits (JenaBioscience GmbH, Jena, Germany), MembFacTM
crystallization screen for membrane proteins (Hampton Research, Laguna Niguel,
CA, USA) and MemStartTM sparce matrix (Molecular Dimensions
Limited, Soham, UK) were used as a starting point for screening and optimizing
crystallization conditions for the five-chlorophyll reaction center of
photosystem II using vapour diffusion methods. Crystallization solutions
prepared in-house were used as well. Different
types of precipitants and detergents and different pH values were tested
experimentally. Optimal values (pH around 7.00 and PEG4-6K as a precipitant)
have been already found. N-dodecyl-b-D-maltoside (DM) was
found as acceptable detergent. It was found that amphiphile 1,2,3-heptanetriol
[11] does not promote protein denaturation, small pH changes have no effect on
protein crystallization. Crystallization experiments on the PSII membrane
proteins are still in the progress.
Acknowledgements:
This
work is supported by the Ministry of Education of the Czech Republic (grants
LN00A141 and MSM12310001) and by the Grant Agency of the Czech Republic (grant
206/00/D007). The stay of IKS at the Centre
for Design and Structure in Biology (CDSB), a European Research Infrastructure
located at the IMB, Jena, was supported by the European Commission within its
IHP program, under contract no. HPRI-1999-00038. IKS thanks
Jeroen Mesters for his help with WrbA structure measurement.
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