Binding Analysis of a Novel Peptide to Malaria Knob Protein http://www.nuigalway.ie/cryst/ oscail_tutorial/moilin/ biomolecules/ala_gly.png Jessica Preston Professors C. Takoudis, A. Chisti, and G. Jursich Graduate Research Assistant Lixiao Zeng Postdoctoral Researcher Xeurong Li
Overview Malaria Challenges Project Description BIAcore ELISA Results and Conclusion Future Work
Malaria Over 5,000 Years Old 300-500 Mil. Infections /Year 3 Mil. Deaths /Year http://www.scienceblogs.com/aetiology /2006/01/mechanism_of_malaria_hide_ and.php In Africa: 5,000 Deaths /Day, 1 Child /3 Sec.
Malaria Endemic Regions http://icb.usp.br/~cewinter/biocomp/pics/world.jpg
Malaria Challenges http://www.jhsph.edu/bin/n/v/ 22malaria.jpg Drug Resistance Genetic Complexity Thousands of Antigens Produced Several Life Stages Several Strategies to Confuse Immune System Different Species and Strains Drugs and Mosquito Nets Inaccessible to Many
Malaria Life Cycle http://www.arthurdovermd.com/malaria_lifecycle.gif
Cerebral Malaria Caused by Plasmodium falciparum Red blood cells become covered with knobs Infected cells avoid destruction in spleen Occludes blood vessels http://www.scidev.net/scidev_images/black-and-white.jpg
Malaria Knob Protein (KP) http://www.wehi.edu.au/facweb/faculty/alan_cowman/images/060084-2.jpg Knobs provide elevation for adhesive ligands, allowing parasitized red blood cells to adhere to blood vessels in the brain.
Peptide Binding Project http://www.steve.gb.com/images/science/peptide_backbone.png A 10 amino acid biotinylated peptide (Biotin-AP) has been synthesized which is expected to bind KP and interrupt knob formation.
BIAcore Analysis http://www.dur.ac.uk/proteomics/biacore/biacore.jpg
BIAcore Operation http://www.rci.rutgers.edu/~longhu/biacore/pic/spr.gif The binding of an analyte to an immobilized ligand changes the surface plasmon resonance (SPR) response angle.
BIAcore Materials Sensor Chip: Steptavidin (SA) coated sensor chip Ligand: 300 nm Biotin-AP immobilized on sensor chip Analyte: 3 types of 2000 nm Knob Protein: Thioredoxin (TRX) Glutathione-S-Transferase (GST) Histidine (His) Buffer: HEPES Buffered Saline (HBS) ph 7.4 HEPES= N-2-Hydroxyethylpiperazine-N'-2-Ethanesulfonic Acid
Biotin-AP Immobilization Injection of 200 nm Biotin-AP RU 19650 Immobilization Phase 19600 Resonance Units 19550 19500 Response 19450 19400 19350 19300 19250 0 100 200 300 400 500 600 700 800 Time s Time /s
BIAcore Results Injection of 2000 nm Knob Proteins Resonance Units (RU) RU Flow Cell 2 20100 20000 19900 TRX-N2 HIS-N2 Response 19800 19700 GST-N2 19600 19500 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 Time s Time (s) RU Flow Cell 1 20100 20000 HIS-N2 19900 19800 TRX-N2 Flow Cell 1 (Negative control) Response 19700 19600 19500 GST-N2 19400 19300 0 200 400 600 800 1000 1200 s
The Enzyme-Linked ImmunoSorbent Assay (ELISA) Allows the visualization of a protein binding interaction
ELISA Binding Site GST-KP-C1 Biotin-KP-AP SA-HRP Peroxide KP-C1 = an important segment of KP KP-AP = synthesized peptide SA-HRP = streptavidin + horseradish peroxidase http://en.wikipedia.org/wiki/elisa
ELISA Materials Binding Analysis: 10 μg GST-KP-C1 + various conc. Biotin-KP-AP Negative controls: GST-KP-C1 + Biotin-KP-SP GST + Biotin-KP-AP GST + Biotin-KP-SP
ELISA Results KAHRP and KP-AP Binding Interaction 1 Optical Density 0.8 0.6 0.4 0.2 GST+Biotin-AP GST-C1+Biotin-AP GST+Biotin-SP GST-C1+Biotin-SP 0 125 250 500 1000 2000 3000 4000 Concentration /nm
Estimation of Dissociation Constant K D = [GST-KP][KP-AP] /[complex] When half of GST-KP is complexed with KP-AP, [GST-KP] = [complex] K D = [complex] [KP-AP][ / [complex] K D = [KP-AP] K D 1.2 μm
Summary ELISA results indicate that binding between KP and KP-AP is specific Binding of KAHRP to Biotin-KP-AP is moderately strong (K D 1.2 μm) Binding was not detected with BIAcore, most likely due to restricted flexibility and low diffusion rates
Future Work Alanine Knockout Scan Replace a single amino acid with alanine and compare this peptide s binding affinity with KP- AP Can help determine binding mechanism by identifying important amino acid residues
References Melanie Rug, et al. The Role of KAHRP in knob formation and cytoadherence of P. falciparum-infected human erythrocytes. BLOOD July 2006 108 (1): 370-378 Kirchgatter, K., Del Portillo, HA. Clinical and molecular aspects of severe malaria, Anais Da Academia Brasileria De Ciencias September 2005 77 (3): 455-475
Acknowledgements DoD-ASSURE The Department of Defense- Awards to Stimulate and Support Undergraduate Research Experiences NSF-REU The National Science Foundation- Research Experiences for Undergraduates Grant NSF EEC 0453432
Questions?