CBMS research booklet
2016
PROTEIN BIOCHEMISTRY
Professor Robert Willows
[email protected]
E8A302 T: (02) 9850 8146
BIOCHEMISTRY: FROM PROTEIN COMPOSITION TO ENZYME STRUCTURE
AND FUNCTION
Proteins are the main functional components of life, with functions including storage of nitrogen, structural
rigidity, transport, signaling, binding, and enzymatic catalysis. The projects offered in my lab span the broad
area of protein biochemistry including: proteomic analysis of cereals, enzyme functional analysis, structural
studies of molecular machines, regulation of pigment biosynthesis and protein-protein interaction studies.
BIOSYNTHESIS OF CHLOROPHYLLS
Chlorophyll is a dangerous molecule as it is a potent photosensitizer and producer of reactive oxygen species.
The organisms which make chlorophyll go to great lengths to ensure that it or its coloured precursors don’t
accumulate to reek oxidative devastation on the cell. The projects on offer aim to understand the mechanism
by which chlorophyll is made by enzymes in the pathway and identify the regulatory mechanisms for
chlorophyll synthesis.
PURIFICATION OF A REGULATORY KINASE
Characterisation of a kinase that phosphorylates the GENOMES UNCOUPLED 4 protein in plants or algae.
This project builds on our recent paper in FEBS Letters and our recently published crystal structure which
both show the algal and plant GUN4 differs significantly from the cyanobacterial GUN4.
MAGNESIUM CHELATASE COMPLEXES (with ANSTO)
We have all five magnesium chelatase subunits expressed and
are able to reconstitute the large protein complexes in vitro.
The stoichiometry of the subunits is variable as some subunits
act as substrates with corresponding substrate like properties.
X-ray and neutron scattering at ANSTO and the Australian
Synchrotron allow us to probe the mechanism of assembly of
these complexes as shown in the figure.
We also use synthetic biology techniques to examine the
assembly of the complex in vivo by controlling expression of
each subunit and monitor the magnesium protoporphyrin
formation.
SYNTHETIC BIOLOGY: CHLOROPHYLL SYNTHESIS IN E. COLI
The 2013-2015 iGEM teams have cloned all of the known genes involved in chlorophyll biosynthesis from a
eukaryotic organism, Chlamydomonas reinhardtii, into E .coli. Some of these genes have been assembled
into operons. This project will assemble the different operons into super operons to test the effectiveness of
chlorophyll biosynthesis in an organism that normally does not make chlorophyll. Optimisation of expression
and testing of individual operons will allow for evaluation of how chlorophyll synthesis may be regulated in
the absence of other potentially competing regulatory factors.
44
Department of Chemistry and Biomolecular Sciences
CBMS research booklet
2016
We have a complete genome sequence of this organism and have extensive proteomic data which indicate
light dependent changes in protein expression.
There are numerous projects available on this novel organism, ranging from genomic analysis to light quality
regulation of photosynthetic genes and structural studies of phycobilisomes (see articles 2, 7, 9, 11). One
particularly interesting project is in identifying the light regulatory mechanisms for gene synthesis in this
organism.
PROTEOMICS OF CEREALS PROTEOMICS OF CEREALS
The cereal research is associated with Grain Growers Pty Ltd looking at proteomics of barley and wheat
grains. We are interested in determining the extent to which environmental and genetic variation determines
protein composition in wheat, rice and barley. The protein profile between varieties grown at the same
location and varieties grown at different locations is being determined. Initial trial scale analyses have found
numerous differences between the varieties of barley grain grown at the same time and location. The reason
for these variations may shed light on how cereals cope with environmental factors such as nutrient
availability, drought, salinity and temperature as well as biological stresses. In addition, many cereal proteins
have distinct impacts on quality; taste and performance in baking and malting are key examples.
Understanding these factors are very important for optimizing quality in the food industry.
Selected publications
1.
Schliep M., Crossett B., Willows R. D., Chen M. “18O-Labelling of chlorophyll d in Acaryochloris marina reveal chlorophyll a and
molecular oxygen are precursors” J. Biol. Chem. 2010, 285 (37), 28450-28456.
2.
Chen, Min, Schliep, Martin, Willows, Robert D, Cai, Zheng-Li, Neilan, Brett A, Scheer, Hugo “A Red- Shifted Chlorophyll” Science,
2010, 329 (5997), 1318-1319.
3.
Lundqvist J., Elmlund H., Peterson Wulff R., Berglund L., Elmlund D., Emanuelsson C., Hebert H., Willows R.D., Hansson M.,
Lindahl M. and Al-Karadaghi S. “ATP-induced conformational dynamics in the AAA+ motor unit of magnesium chelatase”
Structure, 2010, 18, 354-365.
4.
Meinecke L., Alawady A., Schroda M., Willows R. D., Kobayashi M.C., Niyogi K.K., Grimm B., and Beck C. F. “Chlorophyll-deficient
mutants of Chlamydomonas reinhardtii that accumulate magnesium protoporphyrin IX” Plant Molecular Biology, 2010, 72 (6),
643-658.
5.
Jerkovic A., Kriegel A. M., Bradner J. R., Atwell B. J., Roberts T. H., Willows R. D. “Strategic distribution of protective proteins
within bran layers of wheat (Triticum aestivum L.) protects the nutrient-rich endosperm” Plant Physiol., 2010, 152, 1459-1470.
6.
Zhou, S., Sawicki, A., Willows, R.D., Luo, M. (2012) C-terminal residues of Oryza sativa GUN4 are required for the activation of the
ChlH subunit of magnesium chelatase in chlorophyll synthesis. FEBS Letters 586, 205-210
7.
Li, L., Scales, N., Blankenship, R.E., Willows, R.D., Chen, M. (2012) Extinction coefficient for re-shifted chlorophylls: chlorophyll d
and chlorophyll f. Biochimica et Biophysica Acta - Bioenergetics 1817 1292–1298
8.
Chen M, Li Y., Birch D. & Willows R.D. (2012) A cyanobacterium that contains chlorophyll f - a red-absorbing photopigment. FEBS
Letters, 586(19), 3249-3254
9.
Robert D. Willows, Yaqiong Li , Hugo Scheer , and Min Chen (2013) Structure of Chlorophyll f Org. Lett., 2013, 15 (7), pp 1588–
1590
10. Müller, A. H., Sawicki, A., Zhou, S., Tabrizi, S. T., Luo, M., Hansson, M., & Willows, R. D. (2014). Inducing the oxidative stress
response in Escherichia coli improves the quality of a recombinant protein: Magnesium chelatase ChlH. Protein Expression and
Purification. Protein Expression and Purification, 101(C), 61–67
11.
Yaqiong Li, Yuankui Lin, Christopher J. Garvey, Debra Birch, Robert W. Corkery, Patrick C Loughlin, Hugo Scheer, Robert D
Willows, Min Chen (2015) Characterization of red-shifted phycobilisoms isolated from the chlorophyll f-containing cyanobacterium
Halomicronema hongdechloris. Biochim. Biophys. Acta. Bioenergetics Accepted Oct 2015
12. Tarahi Tabrizi, S, Langley D.B., Harrop, S.J. Duff A.P., Willows R.D. (2015) Structure of GUN4 from Chlamydomonas reinhardtii
ACTA Crystalographica Section F 71 (8), 1094-1099
Department of Chemistry and Biomolecular Sciences
45
PROTEIN BIOCHEMISTRY
ANALYSIS OF HALOMICRONEMA: A CHLOROPHYLL F CONTAINING ORGANISM ISOLATED
FROM STROMATOLITES