Rhodnius Nitrophorins: Binding Constant and Electrochemcial Measurements Max Shokhirev University of...
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Transcript of Rhodnius Nitrophorins: Binding Constant and Electrochemcial Measurements Max Shokhirev University of...
Rhodnius Nitrophorins:Binding Constant and
Electrochemcial Measurements
Max Shokhirev
University of Arizona
Chemistry Department
Walker Lab
The kissing bug…• Nitrophorins are found in the salivary glands of
Rhodnius Prolixus which is commonly known as the kissing bug.
• Rhodnius is native to the Amazon River Delta and has spread as far north as Arizona and Texas
• Rhodnius also helps spread Chagas disease throughout Central and South America, which can be fatal.
Nitrophorins
• Belong to the lipocalin family.
• A Histidine residue coordinates the heme
• Bind NO and often other ligands
• Nitrophorins 1-4 found in the saliva of fifth instar Rhodnius. Nitrophorin 5 and 6 found only during the first instar. NP7 has been expressed in a laboratory setting, but hasn’t been identified in the bug itself1
Yummy Blood!
Yummy Blood!
Nitrophorin EnvironmentNitrophorin Environment
Salivary glands:Salivary glands:pH ~5.5; NO boundpH ~5.5; NO bound
Host tissues:Host tissues:pH 7.3; Hm boundpH 7.3; Hm bound
NO displaced by Hm, NO displaced by Hm, which is present at a which is present at a higher concentration at higher concentration at the site of the bitethe site of the bite
General Nitrophorin Function
• Nitrophorins are NNOO transport proteins
• Use a heme cofactor to bind and release the NO
• The Fe(III) heme is stabilized in order to prevent auto reduction of the Fe center – NO binding to Fe(II) is irreversible!NO binding to Fe(II) is irreversible!
• NO released in the host tissues causing vasodilation.
Other Nitrophorin Functions
• AnticoagulantAnticoagulant properties of NP2 and NP3– NP2 was found to bind to factor IX of the Xase
coagulation system2.
• Platelet aggregation inhibitionPlatelet aggregation inhibition by NP7– Through an NO mediated mechanism3
• Histamine binding serves an anti-inflammatoryanti-inflammatory function
Nitrophorin Similarity
• Nitrophorins 1-4 share 38%38% sequence identity
• NP2 and NP3 have 79%79% sequence identity
• NP1 and NP4 have 90%90% sequence identity
• All share the same structural motifs:– Eight stranded beta barrel with a b-type heme
within the barrel
Structure of NP2
GH LoopGH LoopGH LoopGH Loop
AB LoopAB LoopAB LoopAB Loop
His 57His 57His 57His 57
Heme Heme CenterCenterHeme Heme CenterCenter
EF LoopEF Loop
From PDB 1T684
One way to study a protein…
• Site-directed mutagenesis studies– Synthesize NP2 with a select amino acid
changed.
– Observe the changes in redox potentialredox potential, ligand bindingligand binding constants, NMR, EPR…
– Rationalize the observed trends.
– Publish a paper.
What I do…
• Mostly Spectroscopy
– Binding Constant Measurements– Electrochemical Measurements – A little Cyclic Voltometry
Binding Constant Measurements• The UV-vis spectrum of nitrophorins
changes upon ligand binding. • The ratio of ligand-bound to ligand-free can
be measured by observing the change in absorbance for one species as a function of changing ligand concentration
• We can fit the resulting data using the following tight ligand binding equation:
NP2 D1A D89A pH5.5 Im binding
0.05
0.1
0.15
0.2
0.25
0.3
325 375 425 475 525 575
Wavelength (nm)
Ab
sorb
ance
Example Measurement
NP2 D1A D89A pH5.5 Im binding
[Im]
0 2e-5 4e-5 6e-5
Ab
so
rba
nce
at 4
30
nm
0.11
0.12
0.13
0.14
0.15
0.16
Binding Constant Measurements
• This is a powerful method for the study of a protein such as a nitrophorin:– Allows us to observe how pH affects ligand
binding– Allows us to compare different ligands with
respect to nitrophorin affinity for those ligands– Each mutant studied in this way reveals
something about the importance of the mutated amino acid to protein function.
Electrochemistry• NO tends to auto reduceauto reduce Fe(III) to Fe(II) if the
Fe(III) is not protected.– NO dissociation constants from Fe(II) in picomolar
range
• Nitrophorins stabilize the Fe(III) form of the heme center through both heme ruffling and negatively charged residues near the heme center.
• It is possible to measure the potential at which half of the nitrophorins in solution are reduced by observing the spectrum of the nitrophorin solution at different applied potentials
Electrochemical Studies
• Measuring redox potential:– Different pH values
– With ligands bound (NO, Hm, Im) or just H2O
• Redox potential related to concentration through the Nernst Equation:
Physical Setup
Potentiostat:Potentiostat:Provides a steady Provides a steady measurable electricalmeasurable electrical potentialpotential
Spectrophotometer:Spectrophotometer:Records the spectrum ofRecords the spectrum ofthe protein in the the protein in the electrochemical cellelectrochemical cell
Physical Setup
Argon Gas:Argon Gas:Helps prevent oxygen Helps prevent oxygen contamination of the contamination of the protein solutionprotein solution
The Electrochemical Cell
LightLight
Argon GasArgon GasArgon GasArgon Gas
Working GoldGold electrode reduces the protein at the cell window.
Working GoldGold electrode reduces the protein at the cell window.
Auxiliary Electrode
Auxiliary Electrode
Reference Electrode
Reference Electrode
Electrochemical DataElectrochemical Data
400 500 600
0.3
0.4
0.5
A
Wavlength (nm)
-600 -500 -400 -300 -200 -100 0
0.40
0.41
0.42
0.43
0.44
0.45
0.46
0.47
0.48
A4
30
nm
mV (vs. silver electrode)
-600 -500 -400 -300 -200 -100 0
0.40
0.41
0.42
0.43
0.44
0.45
0.46
0.47
0.48
A4
30
nm
mV (vs. silver electrode)
Absorbance vs Applied Potential
The Electrochemical Cell
Thank you…Thank you…ReferencesReferences
• 1) Moˆnica F. Moreira; Heloisa S.L. Coelho; Russolina B. Zingali; Pedro L. Oliveira; Hatisaburo Masuda (2003) Insect Biochemistry and Molecular Biology 33, 23-28.
• 2) Nanda P. Gudderra; Jose´ M. C. Ribeiro; John F. Andersen. (2005) J. Biological Chemistry 280, 25023-28.
• 3) John F. Andersen; Nanda P. Gudderra; Ivo M. B. Francischetti; Jesus G. Valenzuela; and Jose´ M. C. Ribeiro. (2004) Biochemistry 43, 6987-96
• Ribeiro, J. M. C.; Hazzard, J. M. H.; Nussenzveig, R.; Champagne, D.; Walker, F. A. (1993) Science 260, 539-541.
• Shokhireva, T. Kh.; Berry, R. E.; Uno, E.; Balfour, C. A.; Zhang, H.; Walker, F. A. (2003) Proc. Natl. Acad. Sci. USA 100, 3778-3783.
• Andersen, J. F.; Montfort, W. R. (2000) J. Biol. Chem. 275, 30496-30503.• Roberts, S. A.; Weichsel, A.; Qiu, Y.; Shelnutt, J. A.; Walker, F. A.; Montfort,
W. R. (2001) Biochemistry 40, 11327-11337.
Acknowledgements:Acknowledgements:Dr. Walker, Dr. Berry, Dr. Shokhireva, Honjun Dr. Walker, Dr. Berry, Dr. Shokhireva, Honjun
Zhang, and the rest of the Walker Lab Zhang, and the rest of the Walker Lab