Rhodnius Nitrophorins: Binding Constant and Electrochemcial Measurements

1. Rhodnius Nitrophorins:Binding Constant and Electrochemcial Measurements Max Shokhirev University of Arizona Chemistry Department Walker Lab

2. 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.

3. 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

4. Nitrophorin Environment Yummy Blood! NO displaced by Hm, which is present at a higher concentration at the site of the bite Salivary glands: pH ~5.5; NO bound Host tissues: pH 7.3; Hm bound

5. 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.

6. 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

7. 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

8. AB Loop His 57 Heme Center Structure of NP2 GH Loop EF Loop From PDB 1T684

9. 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.

10. What I do… • Mostly Spectroscopy • Binding Constant Measurements • Electrochemical Measurements • A little Cyclic Voltometry

11. 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:

12. Example Measurement

13. 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.

14. 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

15. 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:

16. Spectrophotometer: Records the spectrum of the protein in the electrochemical cell Potentiostat: Provides a steady measurable electricalpotential Physical Setup

17. Argon Gas: Helps prevent oxygen contamination of the protein solution Physical Setup

18. Auxiliary Electrode Working Goldelectrode reduces the protein at the cell window. Reference Electrode Light Protein The Electrochemical Cell Argon Gas

19. Absorbance vs Applied Potential Electrochemical Data

20. The Electrochemical Cell

21. Thank you… References • 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 Chemistry280, 25023-28. • 3) John F. Andersen; Nanda P. Gudderra; Ivo M. B. Francischetti; Jesus G. Valenzuela; and Jose´ M. C. Ribeiro. (2004) Biochemistry43, 6987-96 • Ribeiro, J. M. C.; Hazzard, J. M. H.; Nussenzveig, R.; Champagne, D.; Walker, F. A. (1993) Science260, 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) Biochemistry40, 11327-11337. Acknowledgements: Dr. Walker, Dr. Berry, Dr. Shokhireva, Honjun Zhang, and the rest of the Walker Lab