Experiment I, Part III - Tripeptide and Protein Modeling 1

Biochemistry 30 Spring, 2008 Experiment #I, Part III - Tripeptide and Protein Modeling Secondary Structure of Peptides Tertiary Structure of Dogfish Lactate Dehydrogenase INTRODUCTION The function of a biological molecule is intimately linked to its structure. Therefore, one goal of biochemistry has been to develop an understanding of the three-dimensional structure of proteins. Over the past three decades, the combined powers of x-ray crystallography, multinuclear multidimensional NMR, computational chemistry, and an impressive array of physical techniques have resulted in detailed models for the structure of many proteins. Structures have been determined for over 50,000 proteins, primarily by x-ray crystallography or NMR, with the vast majority of these having been determined in the last decade. This work is time consuming and tedious, and x-ray structures of crystallized protein do not always reveal all of the structures which can be adopted by a protein in solution. Given the recent, rapid advances in DNA-sequencing techniques and hence the enormous number of known gene sequences (cf., for example, the human genome project), the ability to predict a protein's tertiary structure directly from its amino-acid sequence would be a monumental advance. Today, you will use the molecular modeling program HyperChem to build a replica of the tripeptide that you sequenced last week. The tripeptide will be constructed with standard bond lengths and angles, but you will then vary the dihedrals phi () and psi (). By calculating the energies of 36 different conformations, you will obtain the data needed to construct a small Ramachandran plot. You will then construct a polypeptide by repeating the basic unit of your tripeptide six times. By selecting the appropriate dihedrals, this octadecamer will be constrained into an helical as well as a -strand conformation. You will obtain a picture of your octadecamer in each of these configurations, reporting molecular dimensions, dihedral angles, and energies. Finally, using the program RasMol, you will view one subunit of the tetrameric protein lactate dehydrogenase (an enzyme you will purify from chickens later in the semester) and will study various aspects of its tertiary structure. The write-up for this part of the experiment goes directly in your lab notebook and will comprise answers to all of the questions posed throughout this handout. 13 About HyperChem HyperChem is a computer program that displays and manipulates images of threedimensional molecular models. From these images you can precisely measure bond lengths, bond angles, and interatomic distances. (Although these data may sometimes also be measured on actual models--for example, of the ball and stick variety--the values obtained are only approximate.) HyperChem can also performs complex calculations which are so lengthy as to be impossible without a computer. In order to use the program, you must know the following: what the program can do; what the basis for its calculations is; and how to make the computer do what you want. This introduction contains only enough information to enable you to do the exercises which follow and to learn something from them. More information is contained in the HyperChem manuals. There are two different approaches to calculating the energy of a molecule. The first approach uses pure quantum mechanics, and is termed the ab initio calculation method. The equations associated with this method--originating with the Schrodinger Equation--are complex and time-consuming to solve, and we shall consider this method no further. The second approach focuses on reproducing experimental data and employs many mathematical approximations. These approximations are of two types: semi-empirical quantum mechanical and force field. Semi-empirical quantum mechanical calculations use quantum equations, but tailor them to reproduce chemical data. Both ab initio and semi-empirical quantum mechanical calculations are better suited for obtaining molecular orbitals, which is not our interest here. Instead, you will use the force field approach, which has been developed to answer the types of questions you will be addressing today. Atomic parameters for four different force fields are in the program's memory. Three of these (BIO+, OPAL, and AMBER) were designed for use on large biological molecules such as proteins and DNA. The fourth, which you will use for these exercises, is MM+, a force field designed by N.L. Allinger et al. to reproduce experimental physical data for smaller molecules, such as your tripeptide. A force field energy calculation relies on a set of equations which approximate the potential energy of various intramolecular interactions. The potential energy equations contain variable force constants. The value of the force constants in each calculation depends upon the elements involved in the calculation and their bonding arrangements. Not only does the program recognize that the force constants for bonds to carbon and to oxygen differ, but it also assigns different parameters to different bonding arrangements of the same atom. Thus, the atomic force constants differ for an oxygen atom in for example, formaldehyde (H2C=O), ethanol (CH3CH2OH), an water (H2O). Each force field has its own equations and set of atomic parameters. 14 The parameters of MM+ are plugged into seven equations which approximate the following interactions: bond stretching (the electrostatic energy associated with the distance between the nuclei); bond dipoles (the energy of charge separation due to differing electron density); angle bending (energy associated with changing the angle between two atoms attached to a central atom); linked angle bending and bond stretching (the two often occur at the same time); out-of-plane bending (energy associated with loss of planarity in sp2 hybridized atoms); torsional bending (energy of the interaction of dihedral angles); and van der Waals interactions (long-range attraction and short range repulsion of non-bonded atoms). HyperChem energy calculations are of two types. On the simple side, the program can assign an energy to a geometry. Much more sophisticated is HyperChem's ability to bring the molecule to an optimal, lowest-energy state--to "minimize" the structure. This is done by computationally searching the mathematical surface which the potential energy equation represents, each point of which is the energy of a different geometry. A computational search can be done in four different ways on HyperChem. The essential idea is that the computer explores the geometry and finds the minimum by calculating what sort of changes in geometry (bond lengths, torsion angles, etc.) will lower the energy of the molecule or system of molecules. It then makes those changes, and then calculates what modifications of the new geometry will decrease the energy once again. The minimum is the point at which the computer cannot calculate a change which will further lower the energy. By this method, HyperChem minimizes the energy of a molecule, thus finding a stable conformation, a process called geometry optimization, which for the mathematically literate is discussed below. WARNING: It is critical to appreciate that the conformation obtained after minimization may be, and typically is, one of a myriad of local minima, rather than the true global minimum. The reason is that the energy-minimization algorithms search, in each step of the calculation, for a similar structure of lower energy. When no such lower-energy structure can be found, an energy minimum has been reached, and the calculation ends. Thus, if a molecular conformation must pass through a higher energy state to reach a lower-energy conformation--i.e. a transition state--this lower-energy structure will not be found. The Energy Minimization Procedure The computer finds a minimum energy by generating an N-dimensional surface. A three dimensional surface is like the surface of the ocean at a given point in time, where every point is described by three coordinates (x,y,z, or latitude, longitude, height above sea level). A point on an N-dimensional surface is described by N coordinates. For a molecule of A atoms, the energy can be described by a system of 3A Cartesian coordinates. 15 The direction of greatest change is what the computer uses to narrow in on the minimum of the surface. This comes from a mathematical entity called the gradient. On a two dimensional surface (for instance, a parabola: y = x2 + 2), the gradient is simply the derivative of the equation for the curve (dy/dx = 2x). With three dimensions or more, partial derivatives are taken and these can become mathematically very ugly. So you have the computer to do it for you. There are four search methods available on HyperChem, which are, in order of increasing sophistication: Steepest Descent, Fletcher-Reeves, Polak-Ribiere, and Block-Diagonal Newton-Raphson. Today, we will use the Steepest Descent algorithm, which gives good results relatively quickly. THE TOOLS NOTE--in the following instructions, if neither right nor left click is specified, use left click. The Building Tool - This vital tool has several functions. Left click places atoms in the work space. Left drag places bonds in the work space. Right click erases whatever is under the cursor. Double clicking on the building tool icon in the tool bar at the left of the screen brings up the element palette, from which you choose the element which will be placed on the screen, as described above. NOTE--if you switch your element choice on the palette and then click on an atom already on screen, it will change to the new type. The Select Tool - Anything you left click on will become highlighted and be the subject of any subsequent action (geometry optimization, for instance). To select more than one item, left click on each of your choices in succession. Left click on an empty part of the work space selects everything in the work space. If one atom is selected, its type is displayed on the line at the bottom of the screen. If one bond is selected, the bond length is displayed. Selection of two non-connected atoms gives the distance between them. Selection of three connected atoms gives the relevant bond angle, while selection of four connected atoms gives the torsion angle. Right clicking removes from selection whatever is underneath the cursor. Right clicking on the empty work space deselects the entire selection. 16 Double-clicking on the select tool icon in the tool bar on the left of the screen converts any two-dimensional sketches in the work space (made with the building tool) to threedimensional models. When you go from 2D to 3D, the program automatically fills the unoccupied valence of any atoms on the screen with hydrogens, so you need not draw them out. HyperChem assumes standard geometries and average bond lengths in creating the models, so it does not matter how crudely you draw the 2D sketch. Once a 2D drawing has been converted to a 3D model, you may further alter it, adding 2D sketches and rebuilding until you have your end product. Note that building a model does not necessarily give the lowest energy conformation, but rather a good guess as to what the geometry may resemble. The XY-Rotation Tool - This tool is very useful for changing the view of the molecule. Left drag rotates the molecule around the x and y axes. Dragging horizontally controls the y-axis, and vertically, the x. Use this tool whenever you need to change the view and bring different parts of the molecule clearly into perspective. This is the best tool to have chosen prior to a geometry optimization, as it continues to function while the computer calculates. This allows you to align molecules so that the interesting changes in their geometries during the optimization are visible. The Z Rotation and XY-Translation Tools - These two tools also change the view of the molecule. The second moves the molecule in the plane of the screen, the first rotates it in the plane of the screen. The Zoom Tool - Left dragging with this tool increases and decreases the magnification of a molecule. Space Bar - The space bar centers and fills the screen with the selection (or the whole molecule if there is no selection). It is useful for getting a good look at things. Other Tools - The other two tools on the screen are also for zooming and are of little use for smaller molecules. Play with them if you like, and if your molecule disappears, you can always hit space bar and have it return. 17 BEFORE YOU START HyperChem is a very flexible program, and can be customized to the user's taste and requirements. You must check to make sure that the format you are using is the same as the format of the instructions. Choose Preferences... under the File menu. Set the Window to Violet. Set the Bond to By Element. Set the Selection to Green. Click on OK. Open the Build menu, and make sure that there are no checks by any of the options. Under the Select menu there should be checks by Atoms and Multiple Selections only. Make sure the Display menu has a check by Show Hydrogens, Show Multiple Bonds, and Show Hydrogen Bonds. Choose Element Color... from the Display menu. When the dialog box opens, choose Hydrogen as White, Carbon as Black, Nitrogen as Blue, and Oxygen as Red. Click on OK. Finally, choose Molecular Mechanics... from the Setup menu. When the dialog box opens, choose MM+ as the force field. Click OK, and now you are ready to begin the experiment. PART I: CONSTRUCTION OF YOUR TRIPEPTIDE Note: If not specified, use the left mouse button for all clicking! After recording each value in your notebook, DESELECT ALL ATOMS before moving on to the next step. Note further: all HyperChem energies are reported in kcal/mol; also, the minus sign in front of negative energies is very small... don’t miss it! --Open the Databases menu and select Amino Acids. A conversation window with the three letter names of the 20 amino acids will appear on the screen. Notice that you have a choice of secondary structure (Alpha helix, Beta sheet, or Other). Select Alpha helix for starters. Click on the names of the amino acids from N- to C-terminus following the amino acid sequence of the tripeptide you sequenced last week (or your best recreation of that sequence). Close the conversation window by clicking twice on the top left corner. You will then be able to see the tripeptide model on the screen in front of you rendered in colored sticks. Notice that the tripeptide does not contain a carboxylate group at the C-terminus or an ammonium group at the N-terminus. To add these groups, open the Databases Menu and select: Make zwitterions. Close the Menu and view the Tripeptide in all its glory. Use the XY-Rotation Tool and the Zoom Tool to get a clearer picture of the model. Note the two peptide bonds and the six atoms in the peptide plane. See what the side groups look like, and how they are positioned relative to the peptide bond. --You should also change the rendering of the model by clicking on the Display Menu and choosing Rendering. You will have a choice of sticks, discs, spheres, dots, dots and sticks. Try all of them and see what you will get. --Measure the phi and psi angles. Find the -carbon of the second amino acid of the 18 tripeptide. To measure phi, use the selection tool to highlight the following four atoms: the peptide carbonyl carbon of the amino acid number one, the adjacent peptide nitrogen, the -carbon of amino acid number two, and the peptide carbonyl carbon of amino acid number two. On the bottom of the working space you will see the value of this torsion angle, which is just the phi angle. To measure psi, use the selection tool to highlight the following four atoms: the peptide nitrogen from the second amino acid, the -carbon and peptide carbonyl carbon of the second amino acid, and the peptide nitrogen derived from the third amino acid. Record these values of phi and psi in your notebook. Remember, your tripeptide is in the alpha-helix conformation: verify that the phi and psi angles you have measured are correct. (That is, phi = -58o and psi = -47o.) --Now repeat the above exercise, this time building your tripeptide in the beta-strand conformation. Select New under File Menu., and do not "Save current changes: (untitled)." Your previously constructed tripeptide will disappear. Open the Databases window and select Amino Acid. Select Beta sheet, build your tripeptide, etc., etc. Note, as you will explicitly see in Part III, the structure in front of you is NOT the pleated -strand--it is the completely extended strand, which does not form stable sheets with any polypeptide sequence save polyglycine. PART II: CONSTRUCTION OF A RAMACHANDRAN PLOT --Now, using the computational power of HyperChem, you will construct a low resolution Ramachandran plot. Team up with a neighboring group: following the procedure below, one group should work with glycine, the other with alanine. --Select New under File Menu, and do not save the current file. Open the Databases window and select Amino Acids. When the window opens for choosing secondary structure, choose Other for conformation, and set the phi and psi angles each to 180 degrees (note: they may already be set to these values). Build the peptide Ace Gly Nme or Ace Ala Nme. (Ace is N-acetyl and Nme is N-methyl carboxamide--these groups provide the flanking peptide bonds for the gly or ala -carbon, as well as neighboring -carbon equivalents.) Determine the energy for this conformation by pulling down the Compute Menu and selecting Single Point. (You need not concern yourself with the "Gradient," a measure of the conformational distance between the current geometry and the potential energy minimum.) Now measure the energy of thirty five additional conformations (yes, 35!) by varying phi and psi in 60 degree increments (remember, a dihedral of -180o is the same as +180o), rebuilding your peptide (Ace Gly Nme or Ace Ala Nme), and determining the Single Point energy for the new conformation. To expedite matters, note that you can simply leave the Amino Acids window open the entire time, and click on New under the File menu (do NOT save current changes!) to begin building each new conformation. Once you get the hang of this, the entire process should take approximately 15 minutes. Finally, determine the energy of your derivative with the phi and psi angles for each of the conformations 19 below: -strand in antiparallel -sheet -strand in parallel -sheet right-handed -helix  -139o -119o -58o  +135o +113o -47o --When you are finished determining the energies of all of these conformations, choose the highest-energy one and view it in the spheres mode. Can you see the unfavorable van der Waals contacts?? Sketch this high-energy conformation in your notebook. --On a piece of graph paper, construct a Ramachandran plot of your data. Note that the two -strands and the -helix are obviously allowed conformations--hence, the corresponding energies will give you a range of allowed values with which to interpret the energies of the other 36 conformations. Plot psi on the y-axis, phi on the x with 180o,-180o in the lower left corner, +180o,+180o in the upper right. From a neighboring group, obtain a copy of the Ramachandran plot for the other amino acid: glycine if you did alanine or vice versa. Are the plots as expected? (That is, do they look like those in your handout or in Stryer?) Discuss. Finally, on your Ramachandran plot for glycine, indicate the position and energy for a left-handed -helix--hint: you needn't use HyperChem to calculate these values! PART III: PROBING THE SECONDARY STRUCTURE OF AN OCTADECAMER You will construct a 18 amino acid polymer from six repeats of your tripeptide, in both the -helix and beta strand conformations. --Alpha Helix: Select New under File Menu., and do not "Save current changes: (untitled)." Your previously constructed molecule will disappear. Open the Databases window and select Amino Acids. Select alpha helix, begin with Ace, then six repeats of your tripeptide, culminating with Nme. (Again, Ace is N-acetyl and Nme is N-methyl carboxamide; these groups provide the flanking peptide bonds for the terminal carbons.) Under display menu select Recompute H bonds. They should appear as white dashed bonds up and down the helix. Verify that there are 11 main-chain atoms between H-bonding partners--13 atoms counting the carbonyl oxygen and amino hydrogen participating in the H-bond. Measure the distance from the amino-terminus to the carboxy-terminus by choosing the Select Tool and clicking on the N-terminal nitrogen atom and the C-terminal capping carboxamide nitrogen atom. (This latter nitrogen atom is chosen, rather than the C-terminal carbonyl carbon, so that the distanced spanned includes 18 amino acids and 18 peptide bonds.) The distance is displayed on the bottom of the working space. Note the distance in your notebook. What is the distance spanned per amino acid? Rotate the image of your helix to view down the helical axis and note the position of the side chains. Notice how the N20 terminal nitrogen atom is directly above the C-terminal capping carboxamide nitrogen atom. Is this merely a coincidence?? Explain. --You will now partially minimize the energy of your helix to eliminate any particularly bad interactions. First, deselect all atoms. Open the Compute Menu and select Geometry Optimization. Select Steepest Descent algorithm on the left part of the dialogue window. Set the termination conditions for the calculation to be an RMS gradient of 0.1 kcal/? mol or 10 maximum cycles, and Screen refresh on the bottom to 1 cycle. Click on OK. The computer will start the calculation and show the energy in kcal/mole, gradient in kcal/o mol, and whether or not the gradient converges on the bottom of the screen. It will stop the calculation when either of the termination conditions is fulfilled: i.e., the gradient converges or the ten iterations have been performed. Undoubtedly, this calculation will not yet have converged (a complete minimization will take quite a long time), but particularly unfavorable, high-energy van der Waals repulsions will have been relieved. Record the energy in your notebook. Save this file on your U: drive. (You should print a copy of your structure in both the sticks and in the spheres renderings before you leave the lab. You should paste these pictures into your notebook.) --Beta Strand: Select New under File Menu, and do not "Save current changes: (untitled)." Your -helix will disappear. Open the Databases window and select Amino Acid. Select beta sheet, begin with Ace, then six repeats of your tripeptide, culminating with Nme. Under display menu select Recompute H-bonds. What do you see? Comment. Again measure the distance from the N-terminus to the C-terminus by choosing the Select Tool and clicking on the N-terminal nitrogen and the C-terminal capping carboxamide nitrogen atom. Note the distance in your notebook. What is the distance per amino acid in a -strand? How does this value compare with that for an amino acid in an -helix? Discuss briefly. Rotate the image of your strand and note the position of the side chains. Can you see the pleats? --No you can’t! So, let’s make a pleated -strand. The appropriate dihedrals are phi = -139o and psi = +135o (for a strand that will participate in a typical anti-parallel -sheet). Now you should be able to see the pleats. Note that the side chains are perpendicular to the planes of the peptide bonds and that they alternately point in opposite directions-perfect for forming silk! Again, you will minimize the energy of your strand to eliminate any particularly unfavorable interactions. Open the Compute Menu and select Geometry Optimization. Select Steepest Descent algorithm on the left part of the dialogue window. Set the termination conditions for the calculation to be an RMS gradient of 0.1 kcal/o mol or 10 maximum cycles, and Screen refresh on the bottom to 1 cycle. Click on OK. The computer will start the calculation and show the energy in kcal/mole, gradient in kcal/o mol, and whether or not the gradient converges on the bottom of the screen. Again, the calculation won't converge. Record the energy in your notebook. Why is this energy so much higher than that calculated for your helix? Save your file on your U: drive; again you should print a copy of your structure 21 in both the sticks and in the spheres renderings before you leave the lab. PART IV: DOGFISH LACTATE DEHYDROGENASE In this section, you will look at aspects of the structure of a protein, namely the enzyme lactate dehydrogenase (LDH). This enzyme catalyzes the reaction: --Exit from HyperChem. To look at this protein, we are going to use a program which reads pdb files containing coordinates of the atoms from a model for the structure determined by X-ray crystallographic analysis. The program translates these coordinates into a visual image of the location of the atoms which can be easily manipulated. There are several such programs available for free on the internet, including DeepView (seen in class) developed by the Swiss Protein Database group, Cn3D developed by the NCBI, and Protein Explorer developed by Eric Martz at UMass using the visualization engine known as RasMol. Of these, Protein Explorer is the easiest to use, and, for many purposes, gives the most useful images. It is a steadily evolving program, and changes without warning – if you discover that some detail in the instructions provided here does not seem to correspond to the screen that you see, congratulate yourself on your modernity, and carry on. 22 To open Protein Explorer, open the Firefox web browser. A recent modification claims to work in any browser, so pick your poison according to your preferences; instructions here were written while using Mozilla. In the URL window, type www.proteinexplorer.org and hit enter. Notice that you end up somewhere else, namely at a Umass webset, looking at a webpage styled the "FrontDoor" to Protein Explorer. There are a world of treasures accessible from this web page, virtually none of which will be investigated here – that's why you have spare time. To gain real fluency with the program, you should try out the Tutorial, or even the QuickTour. Links to both can be found in the right hand column just below the yellow banner. For this laboratory, however, we will jump right into it, by calling up a structure file by name. We will start with the structur of a single subunit of dogfish lactate dehydrogenase, stored in the protein databases as the file 6ldh.pdb. To open and view this protein, enter "6ldh" (no quotes) in the box provided) in the center column just below the yellow banner) and click the "Go" box. The visualization program will load, fetch the pdb file from a (different) web server, and calculate a visualization. When it's done, the program will present you with three windows, two stacked on the left side of the screen, and one on the right containing the visualized structure. This structure will appear spinning in the field of view, and obscured by red balls which are actually the oxygen atoms of water molecules present (and resolved) in the crystal. Notice the lower window on the left side of the screen – a few basic characteristics of the structure are noted there. Most of the work we will do will be done using the upper window on the left. In particular, you will see two button in this window, labeled "Toggle spinning" and "Hide/Show water". Click these, and observe the result in the image. Notice that when you click them, a sequence of commands appears in the lower window on the left. These are RasMol commands, and there is a line provided to allow you to enter such commands yourself. Place the mouse on any empty space in the right window, click, and drag. You will see the molecule move in response to your mouse movements. The movement makes it easier to see how the molecule is distributed in three dimensions, and allows you to look on all sides of the structure. SECONDARY STRUCTURE Let's examine the structure in more detail. Find the link to "QuickViews" in the upper left hand window, and click on it. This will divide the upper left hand window into two windows. The top contains tools, the middle contains information and options. In the upper window, find the buttons for spin and water, and turn off both if you haven't already. Now look at the structure in the right hand window. This "worm" is the path of the protein backbone. Pull down the "Color" menu by clicking on the little blue box on its right, and select N>C rainbow. Identify the N-terminus. Now let's investigate a few aspects of the secondary structure in this protein. Pull down the "Color" menu again and select "Structure". Pull down the "Display" menu and select "Cartoon". Just admire this image for a moment and chat approvingly with your 23 lab partners. Note the sheets and helices. By now, you and your partners should have noticed that the core of the structure is constructed from three -sheet domains. Describe each (number of strands, whether parallel or antiparallel, relationship between order in the sequence and order in the sheet domain). Are there any instances of parallel strands which are not linked by the sheet-helix-sheet motif? Are there helices that do not function as simple connectors for sheet strands? We learned in lecture about specific effects that stabilize or destabilize these secondary structures. Let's have a look at a couple. Pull down the "Display" menu, and select "HBonds". Then go to the central window and select the first box. Note the caveat – not all Hbonds will be visualized. Now examine the structure, where H bonds appear as thin bars. Confirm that the hydrogen bonding properties advertised in theory for sheets and helices are observed in this real structure. In doing so, note the differences in the hydrogen bond pattern between strands at the junction between two -sheet domains, and between strands in the large sheet domain. In addition, note the backbone hydrogen bonding patterns in the loops connecting helices or sheet strands. Draw three different arrangements in your notebook. Note that one substantial loop seems to largely lack hydrogen bonds. Discuss with your partners and provide an explanation for this situation in your notebook. Hide the Hbonds by selecting "Hide" under the Display menu, and then checking the Hbond box in the central window on the left. You remember from earlier in the laboratory that proline is incapable of fitting neatly into an -helix. To see this principle in action, go to the little box at the top of the lowest window, erase the text saying that you can enter commands there, and type in the following "select pro and helix" (not the quotes, of course) and hit enter. This command will find any prolines which the crystallographers assigned to helix regions. The window below the command line should tell you that 21 atoms have been selected (the number of nonhydrogen atoms in 3 proline residues). To see where they are, we only need to make them look different. Go to the Display menu, and select "Spacefill", and then the Color menu to select "Green". Note the significance of the location of these residues in your notebook. TERTIARY STRUCTURE Of course, a protein does not look like a collection of curlicues and curvy arrows. To see a more reasonable representation, go to the Select menu, and choose Protein, then to the Display menu and choose Spacefill. Note that the protein now appears to be a relatively compact collection of atoms. By rotating the molecule, you can get a good idea of the surface topology of the protein. Now go to the Color menu and select Polarity2. This coloring scheme colors all the atoms of each amino acid either white, if the amino acid is hydrophobic, or purple if the amino acid is polar. Note the distribution of polar and nonpolar amino acids on the surface. In your notebook, speculate on the reason for the presence of hydrophobic patches. 24 In this view, the protein interior is invisible. To see into the interior, find the "Slab" button in the QuickViews window at the top of the left side of the screen. Click on this button, and observe the effect on the image of the molecule. Essentially the molecule has been sliced in half vertically, and you are looking at the cut surface, i.e., the protein interior. You can move the plane of slicing forward and back by holding down the Control (Ctrl) key while dragging the mouse up and down in the viewing window. By doing this, you can have a look at all parts of the protein interior. Note the distribution of hydrophilic and hydrophobic amino acids in the cell interior, as well as the degree of close-packing which you find there. EVERYBODY HAS ONE If you check, you'll find that the window containing the Front Door to Protein Explorer is still open. Go to that window and load a new structure by entering the code "1LLD" in the little window in the center column. This file contains the structure of an LDH molecule isolated from a bacterium. While we and dogfish are rather close cousins, it's been a long time (2.5-3 billion years or so) since there was an organism which could have counted both us and any bacterium among its descendants. What could we have inherited in common from that old geezer? To investigate one possibility, examine the organization of secondary structure in this bacterial LDH molecule (display as cartoon, colored by structure). To do that, you'll need to look at only one of the two subunits. You can do that by choosing "Chain A" from the Select Menu, followed by "Only" from the Display menu. If you follow this immediately by clicking on the "Center" button, and then hit Cancel, this subunit will be centered in the viewing window for your convenience. By switching between windows. compare this structure with the structure of the dogfish LDH. Comment on the similarities and differences in your notebook. 25