Step-by-Step introduction to Simulating Dynamic and Equilibrium Behavior on the example of alanine zwitterions salvation. This Lab further explores HyperChem’s molecular mechanics functionality, and demonstrates how to use molecular dynamics with HyperChem. Using the AMBER force field you will optimize an alanine zwitterions in both isolation and solution to determine the effect of solvent on the optimal structure. You will also perform molecular dynamics in solution to simulate the behavior of this species in biological systems This Lab has 5 sections: In section 1, you will create an optimized alanine zwitterion. In section 2, you will use a molecular solvent model with periodic boundary conditions to solvate the zwitterion. In section 3, you will learn how to superpose molecular structures on each other to visually compare the isolated molecule and the solvated molecule. In section 4, you will use molecular dynamics to anneal the system to obtain a lower energy minimum. In section 5, you will use Langevin dynamics to simulate the presence of a solvent during annealing, and Monte Carlo to sample configurations from a Boltzmann-weighted distribution. Advanced exercise- playback dynamics Section 1 Creating the Isolated Alanine Zwitterion and measuring it properties a. Sketching the Alanine Zwitterion Although you could easily use the HyperChem amino acid library to display alanine, as a practice exercise you will build it from scratch, using the chemical formula: C'O -CH-NH 2 3 | CH 3 Before drawing: Turn on Allow Arbitrary Valence and Explicit hydrogens in Built menu Turn off Show Aromatic Rings as Circles in the Display menu. 1 b. Assigning Atomic Charges The next step is to place charges on some atoms to approximately represent the charge distribution in the zwitterion. In this exercise, you make a simple approximation of a charge of +1.0 on the nitrogen atom and charges of –0.5 on the oxygens (why?). In order to do this you need to assign nonzero values for the charges on these three atoms. To assign atomic charges: 1. Get into selection mode and set the select level to atoms. 2. Select the nitrogen atom. 3. Choose Set Charge on the Build menu and assign a charge of +1. 4. R-click in an empty area to clear the selection. 5. Select both oxygen atoms and assign a charge of –0.5. This sets a charge of –0.5 for each of the oxygen atoms. 6. R-click in an empty area to deselect the atoms. c. Choosing the Force Field The next step is to choose a force field. You should choose a force field before you invoke the Model Builder because the Model Builder assigns atom types to each atom according to the force field that you specify. Alternatively, you could explicitly assign atom types any time by using Calculate Types on the Build menu. To choose the force field: 1. Select Molecular Mechanics on the Setup menu. The Molecular Mechanics Force Field dialog box opens. 2 2. Select AMBER, then L-click on Options. The Force Field Options dialog box opens. 3. Use the following default values, then choose OK. d. Building, Exploring and calculating the alanine zwitterion properties 1. Using the Model Builder build and display a first approximation of the alanine zwitterion. 2. Rotate and translate the structure to look at its conformation. 3. Open the Labels dialog box from the Display menu and choose Chirality for atoms. If the central carbon is labelled R rather than S, then switch to the drawing tool, hold down the [Shift] key and click on the central carbon. You can use [Shift]+L-click with the drawing tool to rotate two neighbors, thus changing chirality. You can also draw wedges to specify conformation or chirality. 4. Measure and report main geometry parameters (the planar O-C’-O angle, the O-C’-C -N torsion angle, and the bound lengths). 5. Save the structure as ala_vac.hin. 3 e. Performing a Single Point Calculation Now, perform a single point calculation to compute the energy. Report the Energy value, the gradient and the geometry. The large energy and gradient indicate that the model-built structure is far from optimal. The principal strain is due to the C-N and C-O distances. f. Optimizing the Isolated Molecule 1. Perform geometry optimization, using following parameters in the Molecular Mechanics Optimization dialog box: 2. What are the optimal geometry, energy, gradient? Report all the calculated values. Which parameters changed the most? Later in this lesson, you use the N-C -C’-O angle to demonstrate molecular dynamics features of HyperChem, so save this angle as a named selection. 3. With the angle still selected, choose Name Selection on the Select menu. •Select Other, enter ncco, then choose OK (you may choose the second NCCO torsion and save it as nooc_2). 4 4. Save the optimized isolated alanine structure as ala_vac_opt.hin file so that you can compare it with the solvated structure you create next. Section 2 Solvating the Structure HyperChem lets you place a molecular system in a box of water molecules with periodic boundary conditions to simulate behavior in aqueous solution, as in a biological system. In this part of the project, you solvate the alanine zwitterions you created in such model water. a. Before you continue, follow these steps: 1. Remove labels from the display by using the default settings in the Labels dialog box. 2. Choose Periodic Box on the Setup menu to open the Periodic Box Options dialog box. b. Setting up Periodic Boundary Conditions 5 To solvate a system with HyperChem you specify a rectangular box or cube of equilibrated water molecules. You define the dimensions of the box, place the solute in the center, and define the minimum distance between the solvent and solute atoms. HyperChem eliminates all water molecules with atoms that come closer to a solute atom than the specified distance. Specifying the Periodic Box Size There are several factors to consider when you define the size of the periodic box. Most importantly, it must be large enough to accommodate the solute. To take care of that look up the parameter Smallest box enclosing solute (this box is called the reference cube), appears automatically in the Periodic Box Options dialog box. In addition, the solute should not see its image in the next box. Box dimensions at least twice the largest solute dimension avoid solute-solute interactions when using the default cutoffs. A smaller box can be used if the cutoff radius that controls the range of the solute potential is made smaller. (so that the largest solute dimension plus the cutoff radius and range is less than the smallest box dimension). The standard reference cube of water is formed from an equilibrated cube of 18.70136Å and its nearest images, so you shouldn’t use dimensions larger than 56.10408Å. Dimensions that are multiples of 18.70136Å minimize initial bad contacts. To choose the periodic box size: 1. Specify a box size of 12.0 by 10.0 by 12.0 Ångstroms, as shown in the following illustration. 2. Choose OK. This places the structure in the center of the box surrounded by 40 water molecules. 6 Note: Because the orientations of the water molecules in the box are not symmetrical, you may end up with fewer than 40 water molecules after the ones that are too close to your structure are deleted. If this happens, the results that you get from calculations may not be quite the same as those given in this lesson. This will depend on how your structure is oriented before you solvate it. c. Displaying the Solvated System 1. Choose Rendering on the Display menu. 2. Turn on Perspective in the Vector and Line Options dialog box 3. Set the select level to Molecules. 4. Select a bond or atom of the alanine molecule, and rotate the whole system to look like this: d. Adjusting Cutoffs and Dielectric Options When you use periodic boundary conditions, you need to check the options for cutoffs and dielectric in the Force Field Options dialog box. To check the options for cutoffs and dielectric: 1. Choose Molecular Mechanics on the Setup menu. 2. Choose Options to open the Force Field Options dialog box. 3. Look at the Cutoffs options at the right of the dialog box. When you choose the Periodic box menu option, HyperChem automatically changes the options for cutoffs to Inner and Outer options, which are more appropriate for the solvated system with the nearest-image periodic boundary conditions. The Outer cutoff is set to one-half of the smallest box 7 dimension, and the Inner cutoff is set to 4Å less to ensure that there are no discontinuities in the potential surface. 4. Now look at options for Dielectric, which are at the top of the dialog box. When you use explicit solvent, you should use a constant dielectric. 5. Select Constant instead of Distance dependent. Note: The dielectric constant is not automatically changed because the new setting is stored in the chem.ini file. 6. Choose OK to close the Force Field Options dialog box. 7. Choose OK to close the Molecular Mechanics Force Field dialog box. The Force Field Options dialog box should look like: e. Optimizing the Solvated Molecule The next step is to optimize the solvated system using the chosen periodic boundary conditions. Since the alanine molecule was set in its optimum (isolated state) geometry, the optimization primarily relaxes the solvent, but also, to some extent, the alanine is changed. Changes in alanine reflect the differences between the structures of a single isolated alanine molecule and the same molecule in solution. To optimize the molecule in solution: 1. Get into selection mode and R-click in an empty area to deselect the alanine. When there is an active selection, an optimization is performed on the selection only. Therefore if you started the optimization with alanine selected, only the position of the alanine atoms is allowed to change and the water molecules are constrained. To optimize the complete system and allow reorganization of the solvent structure in the presence of alanine, you must clear the selection. 2. Using Geometry Optimization on the Compute menu, start the optimization of the solvated alanine, including the water molecules. (Set the 8 Molecular Mechanics Optimization parameters as in step 1.). If the calculation hasn't converged, increase the number of maximum cycles in the Molecular Mechanics diagonal box to 800 and perform the calculation again. 3. When the calculation finishes, report the energy and gradient values, which appear on the status line. The optimized structure for the solvated system might only be a local minimum. In a system with many degrees of freedom, such as this one, there might be many minima and it can be very difficult to locate the global minimum. When there are enough degrees of freedom, it is possible that any single static conformation is insignificant, and that only a statistical treatment of many low-energy conformations is appropriate. 4. Save the structure as ala_sol_opt.hin. Section 3 Superposition The system currently displayed is a minimized structure for the alanine zwitterion in water. It is instructive to compare the structure with the corresponding isolated optimized structure. Instead of comparing the molecules by measuring individual structural properties, you use HyperChem’s superposition feature to visually compare the two structures by superimposing. Deleting the Water Molecules Before superposing, delete the water molecules from the isolated structure. a. To delete the water molecules: 1. Get into selection mode and set the select level to Molecules. 2. L-click on the alanine. This selects the alanine. 9 3. Choose Complement Selection from the Selection menu. This selects only the water molecules. 4. Choose Clear on the Edit menu. 5. If a dialog box appears asking if you want to delete the selected atoms, choose Yes. 6. Choose Show Periodic box in Display menu so that it is not set and only the alanine molecule is shown. 7. Save the file as ala_sol_opt_NoSol.hin . Measure and report the geometry parameters, the energy, and the gradient. b. Merging the Two Systems The next step is to merge this structure with the isolated structure. Before you merge the two systems, you should select the current system so that you can distinguish it on the workspace after the merge. To merge the two systems: 1. Select the alanine. 2. Choose Name Selection on the Select menu and save the selection as solvated. This makes it convenient for you to display this selection later if you want to recover it. 3. Choose Merge on the File menu and open ala-gas.hin. Both the isolated and the solvated structures should now be on screen. The solvated structure should still be selected. 10