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 Next steps Final Remarks References Bacterial cell division Figures reproduced from Ghigo et al. [2] and Vicente and Lowe [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 — FtsZ 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 — ZipA 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 — ZapA 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 Aim of this project The aim of this project is to tag FtsZ, ZipA and ZapA. This is with a view to observing these proteins interact in an “artificial cell”, enabling us to determine their role in the mechanism of Z-ring contraction. 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 Why (site-directed) mutagenesis? We could tag the protein by expressing it with a fluorescent tag attached (e.g. GFP) . . . . . . but these proteins are bulky. Protein Number of residues GFP 238 FtsZ 383 ZipA 328 ZapA 109 References Background Performing the mutagenesis Next steps Final Remarks References Why (site-directed) mutagenesis? An alternative approach — tag the cysteine residues using their thiol groups (see Kim et al. [4] for example). O O H2N H2N OH OH SH S Tag 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 No mutations in ZapA. Four mutations in each of FtsZ and ZipA chosen. 7 out of 8 of these are Gly or Ala to Cys. H2N CH C O O O OH H2N CH C OH H2N HS CH C OH CH2 H CH3 Glycine Alanine Cysteine GGA GGC GGG GGT GGA GGC GGG GGT TGC TGT Other mutation — phenylalanine residue coded for upstream of the ZipA gene in pET-52b plasmid. Background Performing the mutagenesis Next steps Final Remarks References Primer Design QuikChange1 primer calculator used to generate primers (protocol used by others [1, 10]). Basic principles behind primer building: Need the primer size to be just right. Too short - cannot overcome energy costs of mismatch 1 Too long and GC rich - melting temperature too high. Trademark of Stratagene / Agilent Technologies. Background Performing the mutagenesis Next steps Final Remarks References Primer Design QuikChange2 primer calculator used to generate primers (protocol used by others [1, 10]). Basic principles behind primer building: Online tool to see if primers will self-anneal because of repeated subsequences. 2 Trademark of Stratagene / Agilent Technologies. Background Performing the mutagenesis Next steps Final Remarks Protocol summary I Methylated DNA plasmids Forward primer with mutation Reverse primer with mutation plus free nucleotides, DNA polymerase and buffer Dpn1 Digest PCR Mixture of plasmids with and without mutation 37°C 1 hour Original methylated DNA plasmids digested by Dpn1 References Background Performing the mutagenesis Next steps Final Remarks Protocol summary II Grow cells Mini-prep Transform Top10 E. coli cells Top10 cells replicate and produce many copies of the mutated plasmid Harvest the mutationcontaining plasmids References Background Performing the mutagenesis Next steps Final Remarks Protocol summary III Induce cells Grow cells Transform over-expression strain E. coli cells Clones of cells and plasmids produced Add IPTG Incubate at 37°C for 4 hours Cells contain protein with desired mutation. References Background Performing the mutagenesis Results . . . ? Mutagenesis PCR is tricky! Next steps Final Remarks References Background Performing the mutagenesis Next steps Final Remarks Results — DNA gels DNA gels were carried out to indicate the presence of PCR product. FtsZ - Gly to Cys at residue 21 Phusion DNA polymerase 61.7 GeneRuler Ladder 66 68.5 70 Annealing Temp / °C References Background Performing the mutagenesis Next steps Final Remarks Results — DNA gels Most gels looked like this: . . . ZipA - Ala to Cys at residue 193 Phusion DNA polymerase GeneRuler Ladder Annealing Temp / °C ZipA - Ala to Cys at residue 328 Phusion DNA polymerase 50.0 51.7 52.8 54.3 56.0 57.4 58.5 60.0 References Background Performing the mutagenesis Next steps Final Remarks Results — DNA gels . . . which meant a lot of this: We did the transformations anyway. Result — lots of colonies! References Background Performing the mutagenesis Next steps Final Remarks References Results — DNA Sequence Validation 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 Results – Success!! We obtained plasmids for three mutations. Protein FtsZ ZipA ZipA Residue Gly Ala Phe Residue Number 21 193 Upstream in plasmid 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 Improving the yield How do we avoid inserting multiple copies of the primer? One way is Single Primer Reactions IN Parallel (SPRINP), proposed by Edelheit et al. [1]. Methylated DNA plasmids PCR Forward primer with mutation Mix and run PCR cycles WITHOUT using a polymerase Methylated DNA plasmids PCR Reverse primer with mutation References Background Performing the mutagenesis Next steps Final Remarks “If I had more time . . . ” SPRINP improvement. Protein verification. MALDI — amino acid sequence check. Circular dichroism — secondary structure check. Linear dichroism — mutated FtsZ polymerisation check. Attach tags. Construct “artificial cells” containing tagged proteins (SMALPs [5]). Observe using confocal microscopy. References Background Performing the mutagenesis Next steps Final Remarks “Artificial Cells” and SMALPs Membrane bound FtsZ — Osawa et al. [9]. Styrene Maleic Acid Lipid Particles (SMALP) — Knowles et al. [5]. 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 Conclusions Site directed mutagenesis can be used as an alternative approach to tagging proteins to study their kinetics . . . but there are limitations. 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 Acknowledgements Claire Dow & David Roper All of the Structural Biology Lab (C10) at Gibbet Hill MOAC DTC staff and fellow students The University of Warwick, EPSRC and BBSRC Thank you for listening! :-) Any questions? References