Transcript Electrochemical Studies of our Beloved Nitrophorins

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
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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
Nitrophorin Environment
Yummy
Blood!
Salivary glands:
pH ~5.5; NO bound
NO displaced by Hm,
which is present at a
higher concentration at
the site of the bite
Host tissues:
pH 7.3; Hm bound
General Nitrophorin Function
• Nitrophorins are NO 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 released in the host tissues causing
vasodilation.
Other Nitrophorin Functions
• Anticoagulant properties of NP2 and NP3
– NP2 was found to bind to factor IX of the Xase
coagulation system2.
• Platelet aggregation inhibition by NP7
– Through an NO mediated mechanism3
• Histamine binding serves an
anti-inflammatory function
Nitrophorin Similarity
• Nitrophorins 1-4 share 38% sequence
identity
• NP2 and NP3 have 79% sequence identity
• NP1 and NP4 have 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 Loop
AB Loop
EF Loop
His 57
Heme
Center
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 potential,
ligand 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:
Example Measurement
NP2 D1A D89A pH5.5 Im binding
NP2 D1A D89A pH5.5 Im binding
0.16
0.15
Absorbance at 430 nm
Absorbance
0.3
0.25
0.2
0.14
0.13
0.12
0.15
0.11
0
2e-5
4e-5
6e-5
[Im]
0.1
0.05
325
375
425
475
Wavelength (nm)
525
575
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 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
Spectrophotometer:
Records the spectrum of
the protein in the
electrochemical cell
Potentiostat:
Provides a steady
measurable electrical
potential
Physical Setup
Argon Gas:
Helps prevent oxygen
contamination of the
protein solution
The Electrochemical Cell
Argon Gas
Reference
Electrode
Auxiliary
Electrode
Working Gold
electrode reduces
the protein at the
cell window.
Light
Electrochemical Data
0.48
Absorbance vs Applied Potential
0.47
0.5
0.46
A
A430nm
0.45
0.44
0.43
0.42
0.41
0.4
0.40
-600
-500
-400
-300
-200
mV (vs. silver electrode)
0.3
400
500
Wavlength (nm)
600
-100
0
The Electrochemical Cell
Thank you…
References
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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:
Dr. Walker, Dr. Berry, Dr. Shokhireva, Honjun
Zhang, and the rest of the Walker Lab