Protein Dynamics Studies of Structure-Switching in Lymphotactin By Max Shokhirev Final Project for PHAR201 Fall 2008

Overview What is lymphotactin and why do we care? Coarse-Grained Molecular Dynamics (MD). How can we use MD to study Lymphotactin? Creating an online user-friendly coarse-grained molecular dynamics tool to help the next generation of scientists.

Lymphotactin is a metamorphic protein 3DIn general an amino acid sequence determines the 3D structure of a protein Lymphotactin has evolved to adopt two completely different tertiary folds depending on the environment it is in. Each fold has a different function! It is important to try to understand how and why structural switching has evolved in Lymphotactin.

Protein Folding

Lymphotactin is a metamorphic protein 3DIn general an amino acid sequence determines the 3D structure of a protein Lymphotactin has evolved to adopt two completely different tertiary folds depending on the environment it is in. Each fold has a different function! It is important to try to understand how and why structural switching has evolved in Lymphotactin.

Lets take a look at the two different folds of Lymphotactin…

Overview What is lymphotactin and why do we care? Coarse-Grained Molecular Dynamics (MD). How can we use MD to study Lymphotactin? Creating an online user-friendly coarse-grained molecular dynamics tool to help the next generation of scientists.

Molecular Dynamics (MD) Deterministic –Given initial conditions and parameters it is possible to calculate the conditions at any other point in time. Iterative (Discrete) –Repeat force calculations at each time step and move particles accordingly. –Need to pick Δt such that the particles move continuously

Velocity-Verlet Integrator –Scheme for calculating new position, velocity, and acceleration at each time step: 1.Compute New Position 2.Compute Half Velocity 3.Compute Force 4.Compute Velocity Position Velocity Acceleration Time step

Initial Conditions… Initial Positions –Cα positions extracted from PDB file –Even these coarse-grained models are sufficient for modeling the dynamics of small proteins (Clementi C, Nymeyer H and Onuchic J N (2000) Topological and Energetic Factors: What Determines the Structural Details of the Transition State Ensemble and ``En-route'' Intermediates for Protein Folding? An Investigation for Small Globular Proteins. J. Mol. Biol. 298: ) Bonding Interactions –Bonding information from PDB Direct bonds, angles between 3 residues, dihedral angles. Velocity? –Generated using genVel based on equipartition theory at a specified temperature. Other parameters –Masses, LJ types, Specific LJs, general simulation parameters

Initial Temperature… The temperature is proportional to the average speed of particles in a system. We can assign temperatures based on the Maxwell-Boltzman velocity distribution function: V i = (Normalized Gaussian Random number) * sqrt((Kb*Na*T)/M i )

Temperature Control… System is coupled to a virtual heat bath: –V new =V old *sqrt(1-(ts/tau)*(1- T target /T current )) ts = time step length tau = coupling coefficient

Force Field Force on each particle calculated from components –Direct bond –Angle –Dihedral –Specific LJ –Non-specific LJ

Bond Interactions V = ½k(X i -X 0 ) 2 F i = k*(X i -X 0 )/X i

Angle Interactions

Dihedral Interactions

Lennard-Jones Interactions LJ 6-12 LJ

Results from an example Simulation… Temperature of 350…

Temperature of 800… Results from an example Simulation…

Overview What is lymphotactin and why do we care? Coarse-Grained Molecular Dynamics (MD). How can we use MD to study Lymphotactin? Creating an online user-friendly coarse-grained molecular dynamics tool to help the next generation of scientists.

Determining Melting Temperature 1.Run simulation(s) at different using known structures of Ltn 10(1J8I), and Ltn 40(2JP1) 2.Calculate average total free energy as a function of temperature. 3.Specific Heat is the derivative of this function. 4.The peak of the specific heat curve = Tm 5.Need to scale the simulation to real-world values

Determining Melting Temperature

1.Run simulation(s) at different using known structures of Ltn 10(1J8I), and Ltn 40(2JP1) 2.Calculate average total free energy as a function of temperature. 3.Specific Heat is the derivative of this function. 4.The peak of the specific heat curve = Tm 5.Need to scale the simulation to real-world values

Alanine Scanning in silico Which residues are important for Ltn 10 and Ltn 40 stability? “Mutate” each of the 93 residues in each structure by removing Lennard-Jones interactions. Measure the change in average Q value at the wildtype melting temperature

What is Q value? Q value = Current # of Native Contacts Total # of Native Contacts

Results can be meaningful… Computed: Experimental:

Important Residues from NMR Volkman B F, Kron M A, Elgin E S, Kutlesa S, Peterson F C, Tuinistra R L (2008) Interconversion between two unrelated protein folds in the lymphotactin native state. PNAS 105:

Is it possible to see the switching? Unfortunately, NMR experiments performed by Volkman et al, revealed that switching was happening on the s time scale. In order to observe the switching naturally, approximately 10 0 seconds of simulation time is needed. This is not really feasible, even with coarse- grained molecular dynamics.

Steered Molecular Dynamics We can bias the system to switching by applying an artificial force on each atom in one structure to “steer” the system toward the second form. May or may not provide useful data.

How to simulate Dimerization? Simulate two molecules at the same time… –Extend the forces to include forces between molecules What keeps the molecules together? –Studies from Wolynes and coworkers have used polyglycine linkers with success as seen to the right with arc repressor Levy Y, Papoian G A, Onuchic J N, Wolynes P G (2004). Energy Landscape Analysis of Protein Dimers. Israel Journal of Chemistry. 44:

Two mutants known to stabilize one form over the other… It has been experimentally determined that residue 55, when mutated, results in Ltn 40 being favored. On the other hand, a disulfide bond has been shown to favor Ltn 10. Residue 55 can be “mutated” by removing or attenuating the Lennard-Jones contacts it forms A strong disulfide bond force can be added between two Cysteine residues. The mutants should show a shift in the system’s free energy toward one form or the other. Consequently, it is then possible to elucidate how the mutants are affecting the dynamics of Ltn.

Modeling Charges… It has also been shown that Ltn 10 is favored in higher salt concentration environments, while Ltn 40 is dominant when salt is absent (Volkman et al). Coulombic potential between residues. F coulomb = Q 1 Q 2 r2r2

Significance We live in a protein world –Proteins have evolved particular structural conformations in order to perform specific biological functions –Metamorphic proteins have evolved to adopt two or more different 3D structures depending on environmental conditions. –With the help of molecular dynamics simulation, we can take a very close look at how this structural switching is occurring in a controlled way by studying the contribution of each element in the system to the energetics of the entire system.

Overview What is lymphotactin and why do we care? Coarse-Grained Molecular Dynamics (MD). How can we use MD to study Lymphotactin? Creating an online user-friendly coarse-grained molecular dynamics tool to help the next generation of scientists.

Free Online Molecular- Dynamics Simulations… It is needlessly difficult to study molecular dynamics. –No free user-friendly MD simulators available to the general public and scientific community Steep learning curve Terminal-based user interaction Hard to set up non-standard simulations (mutations, hybrid potentials etc. ) No integrated analysis tools

Free Online Molecular- Dynamics Simulations… JavaMolD: a way to make MD simulations fun, interactive, and relatively easy. –Online and offline versions will be available –Simulate-Visualize-Analyze all in one application (no need to keep 12 applications open at one time!) –Define the potential by adding forces directly to the 3D structure –Built in functions to: mutate residues, merge two potentials into one (a chimera), support for Weighted Histogram Analysis of Free Energy plots. –Free to use as a research or teaching tool.

Thank you… Feel free to me if you have questions/comments: This presentation was created as part of structural biology class taught by Dr. Philip Bourne at UCSD. Sources: Volkman B F, Kron M A, Elgin E S, Kutlesa S, Peterson F C, Tuinistra R L (2008) Interconversion between two unrelated protein folds in the lymphotactin native state. PNAS 105: Clementi C, Nymeyer H and Onuchic J N (2000) Topological and Energetic Factors: What Determines the Structural Details of the Transition State Ensemble and ``En-route'' Intermediates for Protein Folding? An Investigation for Small Globular Proteins. J. Mol. Biol. 298: Shokhirev M N, Miyashita O. Molecular Dynamics Simulations using a Go-like Model to study folding of Cro protein families. (to be published in 2009) Chodera, JD, Swope, W C, Pitera, J W, Seok, C, and Dill, K A (2007). Use of the Weighted Histogram Analysis Method for the Analysis of Simulated and Parallel Tempering Simulations. J. Chem. Theory Comput. 1: Levy Y, Papoian G A, Onuchic J N, Wolynes P G (2004). Energy Landscape Analysis of Protein Dimers. Israel Journal of Chemistry. 44: