Background
Performing the mutagenesis
Next steps
Final Remarks
Site-directed mutagenesis of cell division proteins
for contraction generation in vitro
Don Praveen Amarasinghe
MOAC Doctoral Training Centre
University of Warwick
28th May 2013
References
Background
Performing the mutagenesis
Contents
1
Background
2
Performing the mutagenesis
3
Next steps
4
Final Remarks
Next steps
Final Remarks
References
Background
Performing the mutagenesis
Contents
1
Background
2
Performing the mutagenesis
3
Next steps
4
Final Remarks
Next steps
Final Remarks
References
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Bacterial cell division
A wide body of work has been carried out on the proteins that
make up the division complex (“divisome”) and the
contractile Z-ring.
Background
Performing the mutagenesis
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Final Remarks
References
Bacterial cell division
A wide body of work has been carried out on the proteins that
make up the division complex (“divisome”) and the
contractile Z-ring.
However, very little is known about the mechanism of Z-ring
contraction.
Figures reproduced from Ghigo et al. [2] and [12]
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Three Z-ring proteins
This project focuses on three proteins known to be involved in the
formation of the Z-ring [3, 6–8, 11].
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Three Z-ring proteins
This project focuses on three proteins known to be involved in the
formation of the Z-ring [3, 6–8, 11].
FtsZ – A GTPase that forms the polymer chains. These
chains make up the bulk of the Z-ring.
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Three Z-ring proteins
This project focuses on three proteins known to be involved in the
formation of the Z-ring [3, 6–8, 11].
FtsZ – A GTPase that forms the polymer chains. These
chains make up the bulk of the Z-ring.
ZipA – A membrane protein to which FtsZ oligomers bind to.
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Three Z-ring proteins
This project focuses on three proteins known to be involved in the
formation of the Z-ring [3, 6–8, 11].
FtsZ – A GTPase that forms the polymer chains. These
chains make up the bulk of the Z-ring.
ZipA – A membrane protein to which FtsZ oligomers bind to.
ZapA – A cytosol protein that aids the bundling of FtsZ
protofilaments.
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Aim of this project
The aim of this project is to tag FtsZ, ZipA and ZapA, with a view
to observing their interaction in an “artificial cell”. This will help
to determine their role in the mechanism of Z-ring contraction.
Background
Performing the mutagenesis
Contents
1
Background
2
Performing the mutagenesis
3
Next steps
4
Final Remarks
Next steps
Final Remarks
References
Background
Performing the mutagenesis
Next steps
Why (site-directed) mutagenesis?
Final Remarks
References
Background
Performing the mutagenesis
Next steps
Final Remarks
Why (site-directed) mutagenesis?
We could tag the protein by expressing it with a fluorescent
tag attached (e.g. GFP).
References
Background
Performing the mutagenesis
Next steps
Final Remarks
Why (site-directed) mutagenesis?
We could tag the protein by expressing it with a fluorescent
tag attached (e.g. GFP).
These proteins are bulky (e.g. GFP has 238 resiudes,
compared with 383 and 328 in FtsZ and ZipA respectively)
and their expression in the same a-a sequence as the target
protein could alter secondary structure.
References
Background
Performing the mutagenesis
Next steps
Final Remarks
Why (site-directed) mutagenesis?
We could tag the protein by expressing it with a fluorescent
tag attached (e.g. GFP).
These proteins are bulky (e.g. GFP has 238 resiudes,
compared with 383 and 328 in FtsZ and ZipA respectively)
and their expression in the same a-a sequence as the target
protein could alter secondary structure.
An alternative, more elegant approach is to try to tag the
protein using amino acid side-chains (e.g. Cysteine residues
and their thiol groups).
References
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Why (site-directed) mutagenesis?
We could tag the protein by expressing it with a fluorescent
tag attached (e.g. GFP).
These proteins are bulky (e.g. GFP has 238 resiudes,
compared with 383 and 328 in FtsZ and ZipA respectively)
and their expression in the same a-a sequence as the target
protein could alter secondary structure.
An alternative, more elegant approach is to try to tag the
protein using amino acid side-chains (e.g. Cysteine residues
and their thiol groups).
If we cannot find an appropriate side-chain in a suitable place,
instigate a mutation to provide one.
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Mutation positions
Apologies for the lack of diagrams on this slide!! Crystal structures
of E. coli FtsZ and ZipA cover short fragments – not all of the
protein. The mutations chosen were
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Mutation positions
Apologies for the lack of diagrams on this slide!! Crystal structures
of E. coli FtsZ and ZipA cover short fragments – not all of the
protein. The mutations chosen were
FtsZ – G21C, G36C, G256C and A382C.
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Mutation positions
Apologies for the lack of diagrams on this slide!! Crystal structures
of E. coli FtsZ and ZipA cover short fragments – not all of the
protein. The mutations chosen were
FtsZ – G21C, G36C, G256C and A382C.
ZipA – G62C, A193C, A328C and a mutation of a
phenylalanine residue coded for upstream of the ZipA gene in
pET-52b plasmid (denoted FvecC).
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Mutation positions
Apologies for the lack of diagrams on this slide!! Crystal structures
of E. coli FtsZ and ZipA cover short fragments – not all of the
protein. The mutations chosen were
FtsZ – G21C, G36C, G256C and A382C.
ZipA – G62C, A193C, A328C and a mutation of a
phenylalanine residue coded for upstream of the ZipA gene in
pET-52b plasmid (denoted FvecC).
In ZapA, no mutations were required as there is a cysteine
present in an appropriate place, where a fluorescent tag can
be attached.
Background
Performing the mutagenesis
Primer Design
Next steps
Final Remarks
References
Background
Performing the mutagenesis
Next steps
Final Remarks
Primer Design
The QuikChange primer calculator was used to generate
primers – this has been used in other work ([1, 10]).
References
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Primer Design
The QuikChange primer calculator was used to generate
primers – this has been used in other work ([1, 10]).
Minimise the energy cost of mismatches in upto 3 base pairs.
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Primer Design
The QuikChange primer calculator was used to generate
primers – this has been used in other work ([1, 10]).
Minimise the energy cost of mismatches in upto 3 base pairs.
Melting temperature and GC content are important factors.
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Primer Design
The QuikChange primer calculator was used to generate
primers – this has been used in other work ([1, 10]).
Minimise the energy cost of mismatches in upto 3 base pairs.
Melting temperature and GC content are important factors.
Online tool to see if primers will self-anneal because of
repeated subsequences.
Background
Performing the mutagenesis
Next steps
Final Remarks
Protocol summary I
PCR
Dpn1 Digest
37°C
1 hour
Generate forward and reverse primers to instigate each
mutation.
Create a stock of plasmids which contain the gene for the
wild-type protein.
References
Background
Performing the mutagenesis
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References
Protocol summary II
PCR
Dpn1 Digest
37°C
1 hour
Mutagenesis PCR – For each mutation, run the PCR with the
mixture of the two primers, plasmids, free nucleotides, DNA
polymerase and buffer. This should generate copies of the
plasmid with the desired mutation.
Treat PCR mixtures with Dpn1 to remove the wild-type DNA
template.
Background
Performing the mutagenesis
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Final Remarks
References
Protocol summary III
Grow cells
Mini-prep
Transform Top10 E.coli cells to replicate mutated plasmid.
Grow on agar plates overnight.
Grow up cells picked from plate colonies. Harvest the
plasmids with a mini-prep kit.
Obtain sequence data of the harvested plasmids to check that
the desired mutation is in place.
Background
Performing the mutagenesis
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Final Remarks
References
Protocol summary IV
Grow cells
Induce cells
Cells contain protein with
desired mutation.
Transform BL21(DE3) (for FtsZ) or C43(DE3) (for ZipA)
cells with the mutated plasmids.
Induce cells with IPTG. These cells will then over-express the
mutated gene.
Harvest and purify the mutated protein.
Background
Performing the mutagenesis
Results . . . ?
Mutagenesis using PCR is tricky!
Next steps
Final Remarks
References
Background
Performing the mutagenesis
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References
Results – DNA gels
Mutagenesis of all mutations was tested over a range of annealing
temperatures, annealing times and DNA polymerases. DNA gels
were carried out to indicate the presence of PCR product.
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Results – Sequencing
Sequencing results of transformations that produced colonies
were varied:
Background
Performing the mutagenesis
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References
Results – Sequencing
Sequencing results of transformations that produced colonies
were varied:
Some colonies had no mutation.
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Results – Sequencing
Sequencing results of transformations that produced colonies
were varied:
Some colonies had no mutation.
Others had multiple copies of the primer found at the point of
mutation.
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Results – Sequencing
Sequencing results of transformations that produced colonies
were varied:
Some colonies had no mutation.
Others had multiple copies of the primer found at the point of
mutation.
Work by Edelheit et al. [1] suggests that primer-primer
annealing may cause insertions of multiple repeats of primer
sequence.
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Results – Sequencing
Sequencing results of transformations that produced colonies
were varied:
Some colonies had no mutation.
Others had multiple copies of the primer found at the point of
mutation.
Work by Edelheit et al. [1] suggests that primer-primer
annealing may cause insertions of multiple repeats of primer
sequence.
They propose Single Primer Reactions IN Parallel (SPRINP) –
instead of one PCR reaction with both primers, run two
separate PCRs, each with one of the primers, and combine
them.
Background
Performing the mutagenesis
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Final Remarks
Results – Success!!
In the end, we obtained plasmids that coded for the G21C
mutation in FtsZ and the FvecC and A193C mutations in ZipA.
References
Background
Performing the mutagenesis
Next steps
Final Remarks
Results – Success!!
In the end, we obtained plasmids that coded for the G21C
mutation in FtsZ and the FvecC and A193C mutations in ZipA.
References
Background
Performing the mutagenesis
Contents
1
Background
2
Performing the mutagenesis
3
Next steps
4
Final Remarks
Next steps
Final Remarks
References
Background
Performing the mutagenesis
“If I had more time...”
Protein verification.
Next steps
Final Remarks
References
Background
Performing the mutagenesis
Next steps
Final Remarks
“If I had more time...”
Protein verification.
MALDI to check the amino acid sequence is the one desired
References
Background
Performing the mutagenesis
Next steps
Final Remarks
References
“If I had more time...”
Protein verification.
MALDI to check the amino acid sequence is the one desired
Circular dichroism to check that the secondary structure is still
the same as in the wild-type.
Background
Performing the mutagenesis
Next steps
Final Remarks
References
“If I had more time...”
Protein verification.
MALDI to check the amino acid sequence is the one desired
Circular dichroism to check that the secondary structure is still
the same as in the wild-type.
Linear dichroism to ascertain if the mutated FtsZ will still
polymerise.
Background
Performing the mutagenesis
Next steps
Final Remarks
References
“If I had more time...”
Protein verification.
MALDI to check the amino acid sequence is the one desired
Circular dichroism to check that the secondary structure is still
the same as in the wild-type.
Linear dichroism to ascertain if the mutated FtsZ will still
polymerise.
Attach fluorescent tags to the cysteine residues generated
(one possible method is that of Kim et al. [4]).
Background
Performing the mutagenesis
Next steps
Final Remarks
References
“If I had more time...”
Protein verification.
MALDI to check the amino acid sequence is the one desired
Circular dichroism to check that the secondary structure is still
the same as in the wild-type.
Linear dichroism to ascertain if the mutated FtsZ will still
polymerise.
Attach fluorescent tags to the cysteine residues generated
(one possible method is that of Kim et al. [4]).
Construct “artificial cells” containing the tagged FtsZ and
ZapA in the cytosol and ZipA in the membrane (SMALPs,
researched by Knowles et al. [5]).
Background
Performing the mutagenesis
Next steps
Final Remarks
References
“If I had more time...”
Protein verification.
MALDI to check the amino acid sequence is the one desired
Circular dichroism to check that the secondary structure is still
the same as in the wild-type.
Linear dichroism to ascertain if the mutated FtsZ will still
polymerise.
Attach fluorescent tags to the cysteine residues generated
(one possible method is that of Kim et al. [4]).
Construct “artificial cells” containing the tagged FtsZ and
ZapA in the cytosol and ZipA in the membrane (SMALPs,
researched by Knowles et al. [5]).
Observe the binding of FtsZ polymers to the membrane-bound
ZipA with the aid of ZapA using confocal microscopy.
Background
Performing the mutagenesis
Next steps
“Artificial Cells” and SMALPing
Final Remarks
References
Background
Performing the mutagenesis
Next steps
Final Remarks
References
“Artificial Cells” and SMALPing
In work by Osawa et al. [9], FtsZ was trapped inside tubular
vesicles by replacing the C-terminus with an amphipathic
helix. Upon addition of GTP, Z-ring formation and contractile
forces were observed.
Background
Performing the mutagenesis
Next steps
Final Remarks
References
“Artificial Cells” and SMALPing
In work by Osawa et al. [9], FtsZ was trapped inside tubular
vesicles by replacing the C-terminus with an amphipathic
helix. Upon addition of GTP, Z-ring formation and contractile
forces were observed.
More recently, a technique using Styrene Maleic Acid Lipid
Particles (SMALP) to integrate membrane proteins into the
membrane of giant unilamellar vesicles has been developed [5].
Background
Performing the mutagenesis
Contents
1
Background
2
Performing the mutagenesis
3
Next steps
4
Final Remarks
Next steps
Final Remarks
References
Background
Conclusions
Performing the mutagenesis
Next steps
Final Remarks
References
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Conclusions
Site directed mutagenesis can be used as an alternative
approach to tagging proteins to study their kinetics . . . but
improvements can be made (such as the single-primer method
(SPRINP [1]).
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Conclusions
Site directed mutagenesis can be used as an alternative
approach to tagging proteins to study their kinetics . . . but
improvements can be made (such as the single-primer method
(SPRINP [1]).
Protein verification – it remains to be seen whether the
mutated proteins expressed in this project retain key
properties of the wild-type form.
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Conclusions
Site directed mutagenesis can be used as an alternative
approach to tagging proteins to study their kinetics . . . but
improvements can be made (such as the single-primer method
(SPRINP [1]).
Protein verification – it remains to be seen whether the
mutated proteins expressed in this project retain key
properties of the wild-type form.
Beyond this, the next steps are to tag the proteins (The effect
of the tag will need to be considered), construct an artificial
cell containing these proteins and observe the resulting
interactions and Z-ring contraction.
Background
Performing the mutagenesis
Next steps
Final Remarks
References
References
[1] Oded Edelheit, Aaron Hanukoglu, and Israel Hanukoglu. Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to
generate mutants for protein structure-function studies. BMC Biotechnology, 9(61):1–8, June 2009. ISSN 1472-6750. doi: 10.1186/1472-6750-9-61.
URL http://www.biomedcentral.com/1472-6750/9/61.
[2] Jean-Marc Ghigo, David S. Weiss, Joseph C. Chen, Justin C. Yarrow, and Jon Beckwith. Localization of FtsL to the Escherichia coli septal ring.
Molecular Microbiology, 31(2):725–737, 1999. ISSN 1365-2958. doi: 10.1046/j.1365-2958.1999.01213.x. URL
http://dx.doi.org/10.1046/j.1365-2958.1999.01213.x.
[3] Nathan W. Goehring and Jon Beckwith. Diverse Paths to Midcell: Assembly of the Bacterial Cell Division Machinery. Current Biology, 15(13):
R514–R526, July 2005. ISSN 0960-9822. doi: 10.1016/j.cub.2005.06.038. URL
http://linkinghub.elsevier.com/retrieve/pii/S0960982205006731.
[4] Younggyu Kim, Sam O. Ho, Natalie R. Gassman, You Korlann, Elizabeth V. Landorf, Frank R. Collart, and Shimon Weiss. Efficient Site-Specific
Labeling of Proteins via Cysteines. Bioconjugate Chemistry, 19(3):786–791, 2008. doi: 10.1021/bc7002499. URL
http://pubs.acs.org/doi/abs/10.1021/bc7002499. PMID: 18275130.
[5] Timothy J. Knowles, Rachael Finka, Corinne Smith, Yu-Pin Lin, Tim Dafforn, and Michael Overduin. Membrane Proteins Solubilized Intact in Lipid
Containing Nanoparticles Bounded by Styrene Maleic Acid Copolymer. Journal of the American Chemical Society, 131(22):7484–7485, 2009. doi:
10.1021/ja810046q. URL http://pubs.acs.org/doi/abs/10.1021/ja810046q.
[6] Anuradha Kuchibhatla, Anusri Bhattacharya, and Dulal Panda. ZipA Binds to FtsZ with High Affinity and Enhances the Stability of FtsZ
Protofilaments. PLoS ONE, 6(12):e28262, 12 2011. doi: 10.1371/journal.pone.0028262. URL
http://dx.doi.org/10.1371%2Fjournal.pone.0028262.
[7] Harry H. Low, Martin C. Moncrieffe, and Jan Löwe. The Crystal Structure of ZapA and its Modulation of FtsZ Polymerisation. Journal of Molecular
Biology, 341(3):839–852, 2004. ISSN 0022-2836. doi: 10.1016/j.jmb.2004.05.031. URL
http://www.sciencedirect.com/science/article/pii/S002228360400600X.
[8] William Margolin. FtsZ and the division of prokaryotic cells and organelles. Nature Reviews – Molecular Cell Biology, 6(11):862 – 871, November
2005. ISSN 1471-0072. doi: 10.1038/nrm1745. URL http://dx.doi.org/10.1038/nrm1745.
[9] Masaki Osawa, David E Anderson, and Harold P Erickson. Reconstitution of contractile FtsZ rings in liposomes. Science, 320(5877):792–794, May
2008. ISSN 1095-9203. doi: 10.1126/science.1154520. URL http://dx.doi.org/10.1126/science.1154520.
[10] Sambra D. Redick, Jesse Stricker, Gina Briscoe, and Harold P. Erickson. Mutants of FtsZ Targeting the Protofilament Interface: Effects on Cell
Division and GTPase Activity. Journal of Bacteriology, 187(8):2727–2736, 2005. doi: 10.1128/JB.187.8.2727-2736.2005. URL
http://jb.asm.org/content/187/8/2727.abstract.
[11] Elaine Small, Rachel Marrington, Alison Rodger, David J. Scott, Katherine Sloan, David Roper, Timothy R. Dafforn, and Stephen G. Addinall. FtsZ
Polymer-bundling by the Escherichia coli ZapA Orthologue, YgfE, Involves a Conformational Change in bound GTP. Journal of Molecular Biology, 369
(1):210 – 221, 2007. ISSN 0022-2836. doi: 10.1016/j.jmb.2007.03.025. URL
http://www.sciencedirect.com/science/article/pii/S002228360700366X.
[12] Miguel Vicente and Jan Lowe. Ring, helix, sphere and cylinder: the basic geometry of prokaryotic cell division. EMBO Reports, 4(7):655–660, 2003.
ISSN 1469-221X. doi: 10.1038/sj.embor.embor885. URL http://dx.doi.org/10.1038/sj.embor.embor885.
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Acknowledgements
Claire Dow
All of the Structural Biology Lab (C10) at Gibbet Hill
MOAC DTC staff and fellow students including Liam Messin
for superb bottle labelling
The University of Warwick, EPSRC and BBSRC
Background
Performing the mutagenesis
Next steps
Final Remarks
References
Acknowledgements
Claire Dow
All of the Structural Biology Lab (C10) at Gibbet Hill
MOAC DTC staff and fellow students including Liam Messin
for superb bottle labelling
The University of Warwick, EPSRC and BBSRC
Thank you for listening! :-) Any questions?