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