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Characterization of the Interaction of Yersinia Type
III Secretion System Chaperone LcrG to Tip
Protein LcrV Mason Wilkinson
Schraidt et al. PLoS Pathog 6(4), 2010
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Outline
• Introduction to the Type III Secretion System• My Project and its Purpose• Methods• Results• Future Directions• Questions
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Gram-Negative Bacteria Require the T3SS for Infection
Bacteria Associated Disease
Yersinia pestis Bubonic Plague
Shigella flexneri Dysentery
Burkholderia pseudomallei Melioidosis
Salmonella typhimurium Salmonellosis
Pseudomonas aeruginosa Pneumonia
Chlamydia trachomatis Trachoma and STD
Escherichia coli Gastroenteritis
These Gram-negative bacteria have become resistant to current antibiotics.
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Wang et al, Mol. BioSyst., 2008
LcrVType III Secretion System
• The Tip Protein LcrV is essential for T3SS function.• The role of LcrV is to detect the host environment and regulate
secretion.• Chaperone LcrG prevents premature assembly of the T3SS apparatus.
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Why Use Nuclear Magnetic Resonance?
• LcrV and LcrG do not co-crystalize.• NMR provides residue-specific information.
Isoleucine
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Isoleucine Methyl Probes for NMR
Isotopically-labeled alpha ketoacids are used to label isoleucine delta methyls with 13C.
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NMR Spectrum of Isoleucine Methyl-Labeled LcrV
1 NMR Peak = 1 Isoleucine Methyl
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Isoleucine Methyl Assignment by Site-Directed Mutagenesis
1. Introduce IL point mutant via Polymerase Chain Reaction (PCR).
2. Transform mutant plasmid into E. coli.
3. Express Mutant LcrV.
Thermo Fisher Scientific. ”Phusion Site-Directed Mutagenesis Protocol.”
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Making an isotopically labeled recombinant protein for NMR Plasmid containing protein sequence +E. coli BL21(DE3) in minimal media
Grow cells, add α-ketoacids (Isoleucine methyl labels)and induce expression with IPTG
Purification by affinity chromatography
Concentrate protein andtransfer to NMR tube
Acquire data using 800 MHz magnet
LcrV
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Assignment of Isoleucine 206 by Site Directed Mutagenesis
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Complete Assignment of LcrV Isoleucine Methyls
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LcrG interacts with specific isoleucines of LcrV.
Kaur, Kawaljit. 2014-2016.
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The LcrV Surface Affected by LcrG
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Conclusions
• PCR was used to introduce point mutations into LcrV and mutant proteins were expressed and purified for NMR.
• All 25 isoleucine methyl NMR peaks of LcrV were assigned.
• These isoleucine methyl assignments were used to map the LcrV surface affected by LcrG.
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Future Directions
• Publish results in the journal, Biochemistry.
• Use our isoleucine methyl assignments to test for interaction between LcrV and small molecules to develop T3SS inhibitors.
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AcknowledgementsDe Guzman Lab:Dr. Roberto N. De GuzmanKawaljit KaurDr. Supratim DeyAndrew McShanPallavi Guha BiswasAmritanshu ChakravartySanjay YadavaSikta Patnaik (Former KU)
Funding:
KU Biotechnology Training Grant:T32-GM008359
NIH Grant:R01-AI074856
Biomolecular NMR KU Core:P30-GM110761
Yersinia pestis says… Thank you for your attention!
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15N Amide-Labeled Wild Type LcrV
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Lactose
Isopropyl-beta-ThiogalactosideNamrata. Biochemistry for Medics. 2016.
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LcrV I309L – First Purification LcrV I309L – After TEV Cleavage
Bears HIS-TagNo HIS-Tag
LcrVHIS6
TEV Cleavage
Site
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HIS-Tag
Imidazole
KPL. “HIS-Tagged Protein Purification” Protocol. 2016.