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![Page 1: Towards Systematic Identification of cdiGMP Binding Proteins Kevin Roelofs Laboratory of Dr. Vincent Lee University of Maryland, College Park.](https://reader030.fdocuments.net/reader030/viewer/2022032706/56649dfe5503460f94ae675e/html5/thumbnails/1.jpg)
Towards Systematic Identification of cdiGMP Binding Proteins
Kevin RoelofsLaboratory of Dr. Vincent Lee
University of Maryland, College Park
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What is the total regulatory potential of a second messenger?
Nucletoide Second Messengers:cAMPppGpp(p)cdiGMPcdiAMPc-AMP/GMP
Membrane Proteins
Cytosolic Enzymes
Transcription Factors
Phenotypes
Receptors:
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CdiGMP in Vibrio cholerae
• In V. cholerae cdiGMP regulates:– Biofilm Formation– Host Colonization– Virulence– Environmental dissemination
• 51 proteins with domains for synthesis and degradation of cdiGMP
• 4 Currently Identified Receptors
Tamayo Ann Rev Microbiol 2007
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Current approaches in second messenger receptor identification
Identify target genes required for phenotype regulated by second messenger
Systematically purify and test target proteins for binding to second messenger
Pull down proteins with second messenger as bait
Identify second messenger in crystal structures of purified proteins
Identify receptors with conserved binding domains
1) Genetics 2) Biochemistry 3) Bioinformatics
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Problems with current approaches
1. Genetics – Receptors not always associated with observable phenotype
2. Biochemistry – Receptor must be expressed
3. Bioinformatics – Limited to identification of previously described binding motifs
None of these approaches systematically identify all genomically encoded receptors
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Differential Radial Capillary Action of Ligand Assay (DRaCALA)
Protein and bound ligand are immobilized on nitrocellulose membrane while free ligand is distributed uniformly by capillary action
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DRaCALA detects specific binding interactions
Proof of Principle: Purify 10 µM protein
Mix with nM amounts of radiolabeled nucleotide
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Quantitative Detection of Binding Interactions in E. coli Whole Cell Lysate
Mix with 32P cdiGMP:1. Purified Alg442. Purified Alg44
added to BL21 E. coli cell lysate
3. BL21 E. coli cell lysate with endogenously expressed Alg44
Plot Fraction Bound vs Protein Concentration
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Design of DRaCALA ORFeome Screen
1. Obtain Open Reading Frame (ORF) librarya. Contains all annotated ORFs from individual organismb. V. cholerae ORF library contains 3866 unique clones
2. Express individual V. cholerae ORFs in E. coli T7IQa. 2 libraries: N-terminal His- or HisMBP-
3. Screen whole cell lysates in 96-well format for increased cdiGMP binding
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Results of Partial (938 ORFs) Screen
Positive Clone Positive Control
Buffer control
-0.10.00.10.20.30.40.50.6
VCA0681
VCA0593
VCA0753
VCA0042
VC2681
VC1641
His-ORF Library
Frac
tion
Boun
d
-0.1
0.1
0.3
0.5
0.7
0.9 VCA0681 VCA0593
VCA0753
VCA0042
VC1641
HisMBP-ORF Library
Frac
tion
Boun
d
Other
PilZ domain
EAL domain
3 standard deviation positive cutoffAverage Fraction Bound
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1 mM Competitor
His-PlzE
His-VC1641
NC NC cdiGMP cGMP cAMP GMP GDP GTP CTP ATP UTP
Vectorcontrol ORF expressed
Biochemical Classification of Positive Hits
His-PlzE
His-VC1641
2x Serial Dilutions of E. coli WCL into binding buffer
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DRaCALA ORFeome Screens
• A screen of Staph aureus ORFeome has identifed 4 novel cdiAMP receptors
• Systematically identify genomically encoded receptors of metabolites
• Identify processes regulated by second messenger systems
• Systems-level understanding of regulatory potential of second messenger
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FundingUniversity of MarylandNIH R21NIH T32 Training GrantDOD DTRACystic Fibrosis Foundation
Lee Lab Members
Acknowledgements
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DRaCALA detects specific binding of soluble and insoluble proteins in WCLPAGE of Whole Cell Lysate (W) and Soluble Fraction (S)
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Nucleotide Second Messengers Regulate Bacterial Metabolism
Nucletoides:cAMPcdiGMPpGpGcdiAMPc-AMP/GMPppGpp(p)
Receptors:ProteinRNA
What is the total regulatory potential of a second messenger?
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What is the biological function of any small molecule?
Glucose cAMP
Alternative Carbohydrate Utilization
CRP?
E. coli whole cell lysates from strains lacking CRP still bind cAMP
Mona Wu“Intracellular cAMP levels affect bacterial chemotaxis responses by inhibiting CheA”Poster # _______________
cAMP has one known protein receptor in E. coli: CRP
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SM – less story (use other slide)Detect – put this in comparisonsDRaCALA – no animationExample - ORFStaph (use figure that was made remove names thank grundling lab) - Vibrio
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1. CdiGMP Binding ProteinsReceptors, Diguanylate Cyclases, Phosphodiesterases
Mills et al. 2012 Cell Micro
ORFORF
Differentiating ORFs that directly and indirectly increase cdiGMP binding
2. Conserved non-binding regulators of cdiGMP signaling Transcription factors