CooA, a CO-sensing heme protein, acts as a transcriptional activator of CO-metabolizing proteins in bacteria such as Rhodospirillum rubrum and Carboxydothermus hydrogenoformans through sequence-specific DNA binding. Previous research indicated a reduced iron center and CO gas were necessary for CooA to achieve its active conformation and bind DNA. To determine if other reaction conditions facilitate CooA activation, the role of pH on CooA function was tested. Specifically, a fluorescence anisotropy assay was employed to measure possible Fe(III) CooA DNA binding from pH 3 - 12. Interestingly, CooA was observed to bind DNA without CO at acidic conditions, with optimal binding observed at pH ~3. These results are discussed in light of the normal CO-dependent activation mechanism of CooA proteins

Clark, Robert W.

DeVries, Rachael

Gunter, Amy

Weaver, Brian R

English

Valparaiso University

Valparaiso UniversityValpoScholarFall Interdisciplinary Research Symposium Office of Sponsored and Undergraduate ResearchFall 10-28-2016The Influence of pH Variation on CooA ActivityBrian R. WeaverValparaiso University, brian.weaver1@valpo.eduRachael DeVriesValparaiso UniversityAmy GunterValparaiso UniversityRobert W. ClarkValparaiso UniversityFollow this and additional works at: http://scholar.valpo.edu/firesThis Poster Presentation is brought to you for free and open access by the Office of Sponsored and Undergraduate Research at ValpoScholar. It has beenaccepted for inclusion in Fall Interdisciplinary Research Symposium by an authorized administrator of ValpoScholar. For more information, pleasecontact a ValpoScholar staff member at scholar@valpo.edu.Recommended CitationWeaver, Brian R.; DeVries, Rachael; Gunter, Amy; and Clark, Robert W., "The Influence of pH Variation on CooA Activity" (2016).Fall Interdisciplinary Research Symposium. Paper 19.http://scholar.valpo.edu/fires/19● What is CooA?○ A CO-sensing heme protein that acts as a transcriptional activator of CO metabolizing proteins● CooA Heme Coordination Structure:○ CooA has a dynamic heme coordination structure that responds to changes in local environment● In vivo CooA DNA-Binding Requires CO Binding“Inactive” state     +        gas      → “Active” stateMETHODSCONCLUSIONS & FUTURE WORKBACKGROUNDCooA, a CO-sensing heme protein, acts as a transcriptional activator of CO-metabolizing proteins in bacteria such as Rhodospirillum rubrum and Carboxydothermus hydrogenoformans through sequence-specific DNA binding. Previous research indicated a reduced iron center and CO gas were necessary for CooA to achieve its active conformation and bind DNA. To determine if other reaction conditions facilitate CooA activation, the role of pH on CooA function was tested. Specifically, a fluorescence anisotropy assay was employed to measure possible Fe(III) CooA DNA binding from pH 3 - 12. Interestingly, CooA was observed to bind DNA without CO at acidic conditions, with optimal binding observed at pH ~3. These results are discussed in light of the normal CO-dependent activation mechanism of CooA proteins.RESULTS & DISCUSSION● Fe(III) CooA exhibits tight DNA binding at pH<7 and modest binding at elevated pH = 12 (both in the absence of CO)● pH cycling studies suggest DNA-binding does not result from protein denaturation● All observed pH-dependent DNA binding is: ○ correlated with loss of N-terminal heme ligand○ consistent with an activation mechanism which requires disruption of a key salt bridge that stabilizes inactive state● Future work: 1) mutagenesis studies to identify salt-bridge AAs; 2) circular dichroism to rule out denaturation2. DNA-Binding Studies Performed at Basic pH3. Link Between Activity & Heme Coordination ● Based on Fig. 10 & 11, DNA-binding at pH 12 required an induction period likely correlated with the loss of the N-terminal heme ligand (where CO normally binds)ACKNOWLEDGEMENTS● Valparaiso University Chemistry Department● Phillip DeLassus Memorial Chemistry Fellowship● Dr. Tom Goyne, Dr. Laura Rowe & Dr. David Scupham ● Josh Wagoner, Teryn Gehred, Jessica Lyza & CHEM 317 studentsABSTRACT1. Protein Purification: Isolated recombinantly-expressed WT CooA from E. coli2. DNA Binding Assay: Probed CooA DNA-binding using fluorescence anisotropy.Step 1: Prepared CooA solutions (1 - 2000 nM)Step 2: Added DNA oligonucleotide with fluorophore attachedStep 3: Measured fluorescence anisotropy using fluorimeterFluorescence Anisotropy: measure of the difference of parallel- and perpendicularly-polarized fluorescence intensityThe Influence of pH Variation on CooA ActivityBrian R. Weaver, Rachael M. DeVries, Amy Gunter, and Robert W. ClarkValparaiso University Department of ChemistryFigure 4. Diagram of Protein Purification including results from SDS-PAGE gel analysisFigure 5. Diagram of inactive CooA in the fluorescence anisotropy assay. ● Unbound DNA, faster tumbling, perpendicular≅parallel fluorescence● lower anisotropy valueFigure 6. Diagram of active CooA in the fluorescence anisotropy assay. ● Bound DNA, slower tumbling, parallel>perpendicular fluorescence● higher anisotropy valueDNAFeHisFeHis1. DNA-Binding Studies Performed at Acidic pH Values● Fe(III) CooA samples exposed to acidic pH values (but with no CO) showed [CooA]-dependent changes in anisotropy consistent with high-affinity DNA binding; behavior was like that observed in CO-containing assays performed at pH 8 ● pH cycling: 1) tight binding, pH <7; 2) weak binding, pH ~7● Addition of CO gas at low pH values did not improve DNA binding (data not shown)RESULTSFigure 7. Anisotropy curves of WT CooA activation at different pH values.Figure 8. Anisotropy curves of WT CooA pH 3 to pH 7 cycling studies.ProFeHisProFeHisDNAFluorophoreFigure 1. CooA has three different heme states in which two protein-derived ligands bind to the heme Fe.Figure 2. Crystal structure of inactive wild-type CooA.COFigure 3. Crystal structure of active homolog, CRPHYPOTHESIS & MOTIVATIONAll published literature has indicated that CO binding to the heme is critical to forming an active CooA structure. Although this allosteric step is important, we hypothesize it may be bypassed entirely if other key interactions that stabilize the active state can be identified. In this study, we tested the role of pH on the in vitro activation of CooA.Rethinking CooA Activation: 1) loss of N-terminal ligand 2) CO binding3) break salt bridgeFigure 9. Anisotropy curves of CooA pH 12 to pH 7 cycling studies.● Although Fe(III) CooA did not bind DNA tightly at pH 8, reversible binding was observed at pH 12 after an induction period Figure 10. Anisotropy curves showing time-dependent activation at pH 12.Figure 11. UV-Vis Spectra showing loss of ligand over time at pH 12.HisCOFeGasProFeHisProFeHisFeHisCOLanzilotta,W., et al.; Nat. Struct. Biol. 2000, 876-883.Passner J., et al.; J. Biol. Chem. 2000, 847-859.Youn, H., et al.; J. Bacteriol. 2004, 1320–1329.Inagaki, S., et al.; J. Biol. Chem., 2005 3269-3274.J Biological Chemistry, 275, 39332-39338.

The Influence of pH Variation on CooA Activity

ValpoScholar

Valparaiso University
ValpoScholar
Fall Interdisciplinary Research Symposium Office of Sponsored and Undergraduate Research
Fall 10-28-2016
The Influence of pH Variation on CooA Activity
Brian R. Weaver
Valparaiso University, brian.weaver1@valpo.edu
Rachael DeVries
Valparaiso University
Amy Gunter
Valparaiso University
Robert W. Clark
Valparaiso University
Follow this and additional works at: http://scholar.valpo.edu/fires
This Poster Presentation is brought to you for free and open access by the Office of Sponsored and Undergraduate Research at ValpoScholar. It has been
accepted for inclusion in Fall Interdisciplinary Research Symposium by an authorized administrator of ValpoScholar. For more information, please
contact a ValpoScholar staff member at scholar@valpo.edu.
Recommended Citation
Weaver, Brian R.; DeVries, Rachael; Gunter, Amy; and Clark, Robert W., "The Influence of pH Variation on CooA Activity" (2016).
Fall Interdisciplinary Research Symposium. Paper 19.
http://scholar.valpo.edu/fires/19
● What is CooA?
○ A CO-sensing heme protein that acts as a transcriptional 
activator of CO metabolizing proteins
● CooA Heme Coordination Structure:
○ CooA has a dynamic heme coordination structure that 
responds to changes in local environment
● In vivo CooA DNA-Binding Requires CO Binding
“Inactive” state     +        gas      → “Active” state
METHODS
CONCLUSIONS & FUTURE WORK
BACKGROUND
CooA, a CO-sensing heme protein, acts as a transcriptional activator 
of CO-metabolizing proteins in bacteria such as Rhodospirillum 
rubrum and Carboxydothermus hydrogenoformans through 
sequence-specific DNA binding. Previous research indicated a 
reduced iron center and CO gas were necessary for CooA to achieve 
its active conformation and bind DNA. To determine if other 
reaction conditions facilitate CooA activation, the role of pH on 
CooA function was tested. Specifically, a fluorescence anisotropy 
assay was employed to measure possible Fe(III) CooA DNA binding 
from pH 3 - 12. Interestingly, CooA was observed to bind DNA 
without CO at acidic conditions, with optimal binding observed at 
pH ~3. These results are discussed in light of the normal CO-
dependent activation mechanism of CooA proteins.
RESULTS & DISCUSSION
● Fe(III) CooA exhibits tight DNA binding at pH<7 and modest 
binding at elevated pH = 12 (both in the absence of CO)
● pH cycling studies suggest DNA-binding does not result from 
protein denaturation
● All observed pH-dependent DNA binding is: 
○ correlated with loss of N-terminal heme ligand
○ consistent with an activation mechanism which requires 
disruption of a key salt bridge that stabilizes inactive state
● Future work: 1) mutagenesis studies to identify salt-bridge 
AAs; 2) circular dichroism to rule out denaturation
2. DNA-Binding Studies Performed at Basic pH
3. Link Between Activity & Heme Coordination 
● Based on Fig. 10 & 11, DNA-binding at pH 12 required an 
induction period likely correlated with the loss of the N-
terminal heme ligand (where CO normally binds)
ACKNOWLEDGEMENTS
● Valparaiso University Chemistry Department
● Phillip DeLassus Memorial Chemistry Fellowship
● Dr. Tom Goyne, Dr. Laura Rowe & Dr. David Scupham 
● Josh Wagoner, Teryn Gehred, Jessica Lyza & CHEM 317 students
ABSTRACT
1. Protein Purification: Isolated recombinantly-expressed WT CooA from E. coli
2. DNA Binding Assay: Probed CooA DNA-binding using fluorescence anisotropy.
Step 1: Prepared CooA solutions (1 - 2000 nM)
Step 2: Added DNA oligonucleotide with fluorophore attached
Step 3: Measured fluorescence anisotropy using fluorimeter
Fluorescence Anisotropy: measure of the difference of parallel- and 
perpendicularly-polarized fluorescence intensity
The Influence of pH Variation on CooA Activity
Brian R. Weaver, Rachael M. DeVries, Amy Gunter, and Robert W. Clark
Valparaiso University Department of Chemistry
Figure 4. Diagram of Protein Purification including results from SDS-PAGE gel analysis
Figure 5. Diagram of inactive CooA in the 
fluorescence anisotropy assay. 
● Unbound DNA, faster tumbling, 
perpendicular≅parallel fluorescence
● lower anisotropy value
Figure 6. Diagram of active CooA in the 
fluorescence anisotropy assay. 
● Bound DNA, slower tumbling, 
parallel>perpendicular fluorescence
● higher anisotropy value
DNA
Fe
His
Fe
His
1. DNA-Binding Studies Performed at Acidic pH Values
● Fe(III) CooA samples exposed to acidic pH values (but with no CO) showed [CooA]-
dependent changes in anisotropy consistent with high-affinity DNA binding; behavior 
was like that observed in CO-containing assays performed at pH 8 
● pH cycling: 1) tight binding, pH <7; 2) weak binding, pH ~7
● Addition of CO gas at low pH values did not improve DNA binding (data not shown)
RESULTS
Figure 7. 
Anisotropy 
curves of 
WT CooA 
activation 
at different 
pH values.
Figure 8. 
Anisotropy 
curves of 
WT CooA 
pH 3 to pH 
7 cycling 
studies.
Pro
Fe
His
Pro
Fe
His
DNA
Fluorophore
Figure 1. CooA has 
three different 
heme states in 
which two protein-
derived ligands 
bind to the heme 
Fe.
Figure 2. Crystal structure 
of inactive wild-type CooA.
CO
Figure 3. Crystal structure 
of active homolog, CRP
HYPOTHESIS & MOTIVATION
All published literature has indicated that CO binding to the heme is 
critical to forming an active CooA structure. Although this allosteric 
step is important, we hypothesize it may be bypassed entirely if other 
key interactions that stabilize the active state can be identified. In this 
study, we tested the role of pH on the in vitro activation of CooA.
Rethinking CooA 
Activation: 
1) loss of N-
terminal ligand 2) 
CO binding
3) break salt bridge
Figure 9. Anisotropy curves of 
CooA pH 12 to pH 7 cycling 
studies.
● Although Fe(III) CooA 
did not bind DNA 
tightly at pH 8, 
reversible binding was 
observed at pH 12 after 
an induction period 
Figure 10. Anisotropy curves showing 
time-dependent activation at pH 12.
Figure 11. UV-Vis Spectra showing 
loss of ligand over time at pH 12.
His
CO
Fe
Gas
Pro
Fe
His
Pro
Fe
His
Fe
His
CO
Lanzilotta,W., et al.; Nat. Struct. Biol. 2000, 876-883.
Passner J., et al.; J. Biol. Chem. 2000, 847-859.
Youn, H., et al.; J. Bacteriol. 2004, 
1320–1329.
Inagaki, S., et al.; J. Biol. Chem., 
2005 3269-3274.
J Biological Chemistry, 275, 39332-39338.


https://scholar.valpo.edu/cgi/viewcontent.cgi?article=1056&amp;context=fires

The Influence of pH Variation on CooA Activity

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