Investigating the Folding Properties of Superoxide Dismutase

1. Investigating the Folding Properties of Superoxide Dismutase Jose Hejase, Oakland University Nadia Petlakh, University of Michigan-Dearborn Megan Rost, Michigan State University Kim Keala Williams, Oakland University Oakland University SIBHI Summer Fellowship Program Dr. John Finke, Department of Chemistry, Oakland University Dr. Mohamed Zohdy, Department of Electrical and Computer Engineering, Oakland University

2. Research Goals • To perform parallel chemical experimentation and computer simulation to investigate the folding properties of SOD • To compare and develop new computer software for the same purpose

3. What are proteins? • The ultimate expression of genetic information • All living things are comprised of protein macromolecules • Each cellular function is catalyzed by proteins

4. Amino Acids • All proteins are composed of a sequence of various amino acids • Sequence combinations drawing from only 20 amino acids make up all peptides, and yet there is a great diversity among proteins!

6. The properties of the individual amino acids within the peptide sequence determines how the protein folds

7. Superoxide Dismutase

8. Superoxide Dismutase (SOD) • SOD is a free-radical scavenger that eliminates superoxide 2 O2- + 2 H2O → O2 + H2O2 + 2 OH− • It is abundant in the cytoplasm of most cells • A dimer of two identical domains each containing a Tyrosine residue • The folding pattern has not yet been elucidated • Misfolding can lead to aggregates that are correlated to diseases such as Amyotrophic Lateral Sclerosis (Lou Gehrig’s Disease)

9. Purpose: • The purpose of this experiment is to analyze the folding properties of Superoxide Dismutase (SOD) using Fluorescence Resonance Energy Transfer (FRET) • To develop a Monte Carlo system of protein modeling to compare to the AMBER system

10. Experimental Approach: • Remove the metal center from native SOD using dialysis • Nitrate tyrosine108 on both domains of apoSOD protein • Introduce nitrated protein to unmodified SOD to converge domains, creating a protein containing a nitrated domain and a non-nitrated domain • Create an HPLC protocol by which we will analyze nitrated SOD • Use FRET to study the intermediate folding stages of SOD

11. A process using equilibrium exchange through a porous bag Involves the exchange of various buffers with a dialysis bag containing the dissolved protein Dialysis

12. Dialysis, cont. • Dialysis was performed to remove the Zinc and Copper ions from the native protein • This was accomplished by altering the pH of the solution and disrupting the electrostatic salt bridges holding these ions in place

13. Nitration of Tyrosine108 • ApoSOD was reacted with tetranitromethane in the dark to nitrate the Tyr108 residues on the protein • Reaction was repeated using additional controls and modifications • The reaction was quenched using column chromatography, and UV spectroscopy analysis was performed. +

14. Gravity Filtration • Size-exclusion gravity filtration was used to separate the products of the reaction mixture • It separates mixtures based upon the size/molecular weight of each moiety

15. UV Spectroscopy • Tyrosine residues absorb UV light in the 250-275nm range • Nitrated Tyr residues absorb UV light in the 250-275 nm range as well as at 360 nm at pH 7.0 and at 420 nm at pH 9.0 • This is useful for determining the success of our reaction

16. First and Second Reactions of SOD with TNM

17. SOD with late TNM Addition

18. Separation of Fraction #2 of SOD with late TNM addition

19. Successful Nitration of SOD with TNM

20. Continuation of Experimentation: • Create a heterogeneous donor/acceptor protein • This is necessary to proceed with FRET analysis of protein folding

21. AMBER • Assisted Model Building with Energy Refinement • A computer program which provides Energies and Coordinates for each amino acid residue present in a protein chain at each picosecond of time. • ETotal = EBond + EAngle + EDihedral + ELJ + ERep

22. How Does AMBER Work? • Obtain a protein structure from the protein data bank and modify for our research purposes • Run simulated annealing • Submit various structures for nanosecond lengths of time at different temperatures • Analyze data for the appearance of a flipping point and intermediates

23. What is a Flipping Point? • The flipping point occurs when a protein switches between folded and unfolded states at a given temperature • No Flipping point? No problem! -can sill choose the best temperature of unfolding through extrapolated data • While not preferred equilibrium data, this kinetic analysis is still very useful.

24. Unfolding of SOD

25. Probability Analysis

26. Movie Time!

27. Discrete Molecular Dynamics • DMD is another method that can be used for analyzing protein folding. • Unlike MD models, DMD only calculates forces at collision points leaving time gaps • We have been using a website, ifold.dokhlab.org, to run some simulations in this manner on SOD. • Still in the process of analyzing and comparing this data to that obtained using AMBER MD software

28. DMD

29. Monte Carlo Simulation • Random moves hoping to get to the least energy conformation. • No forces evaluation included. • Whether the altered structure is energetically feasible at a certain temperature. (relative energy, before and after) • Bad cannot provide time dependent (specific) quantities. • Good estimating thermodynamic properties.

30. Metropolis Monte Carlo Algorithm

31. Our Monte Carlo • Based on a 3D lattice model representation. • Three kinds of moves:

32. Monte Carlo Simulation • Random moves hoping to get to the least energy conformation. • No forces evaluation included. • Whether the altered structure is energetically feasible at a certain temperature. (relative energy, before and after) • Bad cannot provide time dependent (specific) quantities. • Good estimating thermodynamic properties.

33. Metropolis Monte Carlo Algorithm

34. Our Monte Carlo(till now) • Based on a 3D lattice model representation. • Three kinds of moves:

35. Our Monte Carlo(till now) • Potential energy depends on the interactions between the amino acids, which are not connected. (topological neighbors) In our program, to calculate the energy, we used the following criteria: *Alike amino acids interaction contribute with -3 *Different amino acids interaction contribute with -1

36. Our Monte Carlo • What is already done? Random generation of moves and the an potential energy calculation. • What is going to be added? Before and after energy comparison and Boltzmann probability implementation.

37. Moves Video • (Matlab video here)

38. Continuing Our Project… • Determine optimal reaction conditions of titration • Proceed to FRET analysis • Additional analysis time permitting • Continue AMBER analysis • Proceed with Monte Carlo program in Matlab • Developing a software approach for the Finke Method

39. The Finke Method • Given: the contacts of the folded protein. • Goal: determining which contacts go first and which ones follow. • Method: virtually decrease the distance between any two amino acids containing the area of contact. First fold Unfolded protein

40. References • Principles of Biochemistry Fourth edition. Lehninger. W. H. Freeman and Company: New York, 2005. • Effect of Nitration on the Activity of Bovine Erythrocyte Cu,Zn-Superoxide Dismutase (BESOD) and a Kinetic Analysis of Its Dimerization-Dissociation Reaction as Examined by Subunit Exchange between the Native and Nitrated BESODs. Oneda, Inouye. J. Biochemistry, 2003, Vol. 134, No. 5 • Solokovsky, M., Riordan, J. F., and L., V. B. (1966) Biochemistry 5, 3582-3589. 49. • 3-Nitrotyrosine as a spectroscopic probe for investigating protein protein interactions, Vincenzo De Filippis, Roberta Frasson and Angelo FontanaDepartment of Pharmaceutical Sciences and CRIBI Biotechnology Center, University of Padua, I-35131 Padua, Italy • http://www.rcsb.org/pdb/explore.do?structureId=1E9P • www.wikipedia.org • Monte Carlo (MC) Simulation, http://cmm.info.nih.gov/intro_simulation/node25.html • Understanding Monte Carlo Simulations, http://thglab.lbl.gov/margaret/Lecture8_MC_HP.pdf • N. Socci & J. Onuchic, “Folding kinetics of proteinlike heteropolymers”, (UCSD, 03/1994)