## 11. Comparing Proteins: Hemoglobins

For the following exercises, obtain the file 1HHO.pdb from the PDB. You will also need the file 3HHB, which you used in section 10.

### A. Comparing Conformations: Deoxyhemoglobin versus Oxyhemoglobin

In this section, you will compare two different conformations of the same protein, the deoxy and oxy forms of hemoglobin. DeepView allows you to superimpose all or parts of different models, and to color the models to emphasize conformational differences.

Load 1HHO.pdb, the alpha and beta subunits of oxyhemoglobin (HbO2). Then load 3HHB.pdb, the alpha and beta subunits of deoxyhemoglobin (Hb). First, you will carry out a simple superposition of the hemes in the beta subunits, in order to examine conformational changes that accompany the binding of O2 to hemoglobin.

Press help or = to center the two models. Before you compare them, get to know some features for handling multiple models.

Wind: Layer Infos
The Layer Information window provides convenient control of multiple models. Place the window below the graphics window, and adjust the size of the graphics window so that you can always see Layer Infos.

Color: Layer
DeepView colors the two models yellow (first model loaded) and blue (second model). If more models are present, DeepView assigns different colors to all.

Click on the Control Panel, then click and hold on the name 1HHO at the top. A menu appears, showing the names of all loaded models or layers. Select 3HHB. Examine the effects of the visible and can move buttons on 3HHB. Restore movement and visibility for 3HHB. You can also control selection, activity, visibility, and movement of layers by using checkmarks in the Layer Infos window.

<tab>
<tab>
Notice the effect on the file-list menu at the top of the Control Panel. Each time you press tab, DeepView changes the active layer. If more than two models are loaded, DeepView cycles through them when you press tab repeatedly.

<ctrl> -<tab>, <ctrl> -<tab>, <ctrl> -<tab> (hold down ctrl and press tab repeatedly)
Notice the effect of this "blinking" on the Control Panel and the Layer Infos window. If you hold down the ctrl key and press tab repeatedly, you make successive layers active and visible. End with 1HHO active and visible.

Color: Secondary Structure
Blink to confirm that this command affects only the active layer. Unless you specify otherwise, most menu commands affect only the active layer.

<shift> - Color: B-Factor
Hold down the shift key while pulling down the menu. Blink to confirm that both models are colored according to their B-factors. With menu commands, the shift extends actions to all layers. For any key to have its effect on a menu command, you must press the key before pulling down the menu.

Color: Layer
This action restores different colors to the two models.

Blink to make 3HHB active. Scroll to the bottom of the Control Panel list and select HEM1 of chain B. Press return. Then switch to the 1HHO layer, select HEM1 of chain B, press return and press help or =. You should now see the two beta-chain heme groups, colored yellow and blue. The long bent line from the center of the yellow heme represents the O2 molecule of HbO2. Rotate the hemes so that you are looking at their faces. Switch off movement of 1HHO (remove the checkmark from the mov column of the Layer Infos window, or from the can move box of the Control Panel), and translate the blue heme to the left of the yellow heme. When they are side by side, restore movement to the blue heme.

Click the superimpose button, the third button from the right on the graphics window. This button is labeled with three red dots, three blue dots, and a curved arrow. The function controlled by this button allows you to select three corresponding atoms on both models, and superimpose them as well as possible. Read the next paragraph in its entirety, and then carry out its instructions.

Following the instructions at the top of the graphics window, pick an atom of the yellow heme, then the corresponding atom of the blue heme. Then pick a second yellow atom and the corresponding blue atom. Repeat for the third atoms. Good choices of atoms on each heme are ring atoms that form a large triangle around the periphery of the heme. When you pick the third pair, DeepView superimposes the first pair of atoms exactly, and the second and third ones as well as possible. Try it.

Now the two hemes should be superimposed.

Select: Neighbors of Selected aa...
Use the dialog to add to the view all groups within 4 angstroms of the selected hemes in both layers. Make 3HHB the active layer.

<shift>Select: Visible Groups
Color: RMS
Make 1HHO the active layer. Color it CPK.

Now zoom in and study your handiwork. In CPK colors is the heme region of HbO2, including the O2 molecule bound to the heme iron. In shades of blue and green is the corresponding region of Hb. The hemes are superimposed, so differences in positions of other groups reflect changes in their positions relative to heme upon binding O2. Groups in Hb are colored according to how far they lie from corresponding groups in HbO2. If they superimpose exactly, they are dark blue, with colors farther up the visible spectrum assigned for greater distances from corresponding atoms in the other layer. This is called RMS coloring because DeepView calculates the root-mean-square distances between corresponding backbone atoms to arrive at the color assignment for a group.

With this view, you can see that, when O2 binds to Hb, the proximal histidine residue (HIS92 -- label it) moves closer to the iron atom at the center of the heme. Binding of O2 to the heme iron reduces its diameter and allows it to move into the plane of the heme (notice that the iron is out of plane and the heme is cup-shaped in Hb, but flat in HbO2). The distal histidine (HIS63 -- label it also) also moves noticeably, moving aside to accomodate O2 and form an H-bond with it. Compute H-bonds in the 1HHO layer to see this interaction.

Hold down control and press tab. Each time you press tab, one layer becomes invisible, and the other visible. So you can switch back and forth (called blinking) between views of Hb and HbO2. It's like watching hemoglobin breathe.

Take another look at the Layer Infos window. It allows you to control layer displays without making the Control Panel active. By clicking to add or delete checkmarks, you can control -- from left to right -- visibility, movement, display of coordinate axes, alpha carbons (display of full backbone or alpha carbons only), backbone O atoms, hydrogens (if present in model), H-bonds, H-bond distances, side chains, and water (if present). The cyc column controls which models are displayed during blinking. When you hold down control and press tab repeatedly, DeepView cycles through the layers that are checked in the cyc column. Finally, the Sel column shows you how many groups (residues or hetero groups) are currently selected in each layer.

The conformational changes that occur at this site are transmitted throughout the Hb tetramer to alter O2 affinity at the other heme sites. Display the full beta chain of 3HHB in order to see the full extent of atom movements relative to the heme positions.

Take time to PLAY with the tools introduced in this section.

Close all files before proceeding to the next section.

### B. Comparing Homologous Proteins: Alpha and Beta Chains of Hemoglobin

In Section A, you compared two models of the same protein in different conformations. In this section, you will compare two homologous protein chains, the alpha and beta chains of hemoglobin. These two chains are very similar in overall conformation, but differ substantially in amino-acid sequence. With DeepView, you can superimpose the chains and quickly locate amino acids that are identical, similar, and different in the two chains. In addition, you can print out an alignment of the sequences.

Load the file 3HHB.pdb, deoxyhemoglobin. Select chain A only.

Save Selected Residues...
Name the file 3HHBAlpha.pdb, direct it to a convenient folder, and click Save. Next, select chain B only. Save it as before, naming it 3HHBBeta.pdb. Close 3HHB and open your two new files, 3HHBAlpha.pdb first (remember that the File menu lists recently used files for easy opening).

Wind: Sequences Alignment
The Align window appears at the bottom left with the sequences of the two chains in one-letter abbreviations. The name of the currently active layer is shown in red, with the reference layer at the top. You can click a layer name to make it active, and you can also select residues and switch layers from this window, in much the same way as you use the Layer Infos window or the Control Panel.

Color: Layer
Fit: Magic Fit (Quick Test)...
Look at the options on the dialog that appears, but do not change anything. The program will attempt to superimpose the CA atoms of the beta chain onto those of the alpha chain. Notice that the alpha chain is listed first, which means that it will remain fixed while the other model is fitted onto it by a least-squares procedure. In a superposition, the model that remains fixed is called the reference. By default, the first model loaded is the reference (but you can change that with the menus in this dialog if needed). Click OK. DeepView superimposes the two models. Make 3HHBBeta the active layer.

Fit: Calculate RMS Deviation...
Accept the default settings in the dialog box by clicking OK. DeepView calculates the root-mean-square distance between all corresponding CA atoms in the alpha and beta chains, and reports the number of atoms used (138) and the RMS distance (1.57 angstroms). You can make this calculation only with the non-reference layer active.

Fit: Fit Molecules (from selection)
Again accept the default settings in the dialog and click OK. This time, DeepView first carries out an alignment of the sequences that is guided by the Magic Fit superposition. Then it carries out the superposition using only the residues that are structurally aligned (these are automatically selected during Magic Fit). Calculate the RMS deviation again and you will find it improved: a lower RMS deviation, based on a smaller number of better-corresponding residues.

Scroll across the sequences in the Alignment window. Notice that certain pairs of aligned residues are highlighted. These are the residues that were aligned with each other by the fitting operations you conducted. Place the pointer on (but do not click) a residue of the reference layer. Its identity appears at the left end of the window, and the residue blinks in the graphic window (you may have to look carefully to find a blinking residue). Move the pointer to a residue in the lower sequence. Now you learn its identity and its distance from the corresponding residue in the reference layer.

Select: aa Identical to ref.
In both layers, DeepView selects and highlights the aligned pairs that are identical. Scroll through the sequence alignment. Approximately what percentage of residues are identical in the beta chain to those in the alpha chain? The alpha and beta chains probably evolved by divergence from a common ancestral globin gene. The highlighted residues are those that have been conserved during this evolution. Conserved residues are likely to be essential to protein function.

Select: aa Similar to ref.
Now additional pairs are selected. These pairs are either identical (the residues selected before) or they similar in chemical properties. Find similar pairs in which both are positively charged, negatively charged, small, polar, or nonpolar. Mutations that replace one residue with one of similar properties, such as lysine replaced by arginine, are called conservative mutations.

Select: Inverse Selection
In the beta-chain sequence, the highlighted residues are those that are neither identical nor similar to those in the alpha chain. These residues may serve different functions in the two subunits, or their specific side chains may not be essential to the function of the chain. Color all these residues green, and hide side chains. Are most of the differences that distinguish alpha from beta buried or on the surface of this subunit?

Display the Layer Infos window and notice the number of residues selected in the 3HHBBeta layer (Sel column). This number, 47, is the number of amino-acid differences between the alpha and beta chains of human hemoglobin. Use the Select menu and this Layer Infos window to determine the numbers of identical and similar residues in the two chains.

At the left end of the Align window, click on the small text icon. The text page that appears summarizes the current structural alignment. Below aligned pairs are asterisks (*) if the pairs are identical, dots if the pairs are similar, and no mark if they are neither. You can save this text file with File: Save: Sequence Alignment, and then open and print it with any word processor.

#### Other Alignment Options

Examine the commands under the Fit menu. Notice that you can also align models using only selected residues. This allows you to choose specific regions or chains of models to align, with unselected residues following the selected ones, but not affecting the alignment. For example, you might want to see how the beta chains of hemoglobin move with respect to the alpha chains in an alpha-beta dimer. You could accomplish this by selecting the alphas only and carrying out Fit: Fit Selected Residues. DeepView would superimpose on the basis of the the alphas, and comparison of the models would highlight differences between the positions and orientations of the betas. The only restriction on fitting selected residues is that you must select exactly the same number of residues in each layer.

### Review

- Wind:Sequences Alignment provides a window showing how the residues of all displayed models are alignment with each other. Before any alignment operations, sequences are aligned simply by number, 1 with 1, and so forth. After alignment commands, the display shows the current alignment, including gaps.
- DeepView provides several way to superimpose models by structure, and then to make structure-based sequence alignments. These structural alignments are more likely to be correct than alignments based solely on sequence. Here are brief descriptions of some of the alignment tools:
  1. Fit:Magic Fit is the simplest fit tool. It superimposes two models on their geometric centers, then rotates one (the target) for best fit on the other (the reference), giving a useful starting fit. It also selects the residues in each model that align well, giving the same number of residues selected in each model. This prepares the models for more rigorous fit tools (see 3).
  2. Fit:Interative Magic Fit might improve this intial alignment.
  3. Fit:Fit Molecules (from selection) superimposes the model for best fit of selected residues. The number of residues selected in each model must be the same, but this is simple if Magic Fit is done first (see 1).
  4. Fit: Calculate RMS... displays on the Tool Bar the RMS deviations between corresponding (aligned) residues in the two models. Well-aligned and similar models give RMS deviations of less than 1.0 anstroms. Higher RMS values signal that at least part of the alignment is poor.
- Several selection tools allow you to select and explore the nature of aligned residues:
  1. Select:AA Identical to Ref selects residues in the current model that are indentical to their aligned residues in the Fit reference model. This amounts to selecting conserved residues if the two models are evolutionary relatives.
  2. Select:AA Similar to Ref selects residues in the current model whose side chains are chemically similar (or identical) to that of their aligned residues in the Fit reference model. This amounts to selecting for conservative mutations in the Fit target, if the two models are evolutionary relatives.
  3. To select residues that a neither identical nor similar, use Select:AA Similar to Ref followed by Select:Inverse Selection.
- You can save sequence alignments from Fit operations using one of the options under File:Save Sequence.

Take time to PLAY with the tools introduced in this section.

Reminder: See Summary: Action of Key Modifiers for more information on the effects of the shift, option, control and command keys.

##

End of Basic Tutorial

If you have worked through all of Sections 1-11, you have used many of the functions of DeepView. The program has additional advanced features, for which the program's creator, Nicolas Guex, provides tutorials. Section 12 and USM Asignment 2 includes introductions to the advanced tutorials and projects on which you can hone your skills. The advanced tutorials and projects teach you more about

- analyzing binding sites,
- using Ramachandran Plots,
- building protein models to specifications,
- comparing proteins,
- viewing and interpreting electron-density maps,
- computing surfaces and electrostatic potentials, and
- building homology models by threading the sequences of unknown proteins onto the known structure of homologous proteins.

You can carry out these assignments and master these skills in any order you choose. Just click on the next section heading to begin.

####

To Next Section: 12. Making Images for Publication. Also see ADVANCED TUTORIALS from DeepView Developers (Contents frame).

## Now You're On Your Own!

You are now an experienced user of one of the world's most modern tools for viewing and analyzing the structure of macromolecules on personal computers. If you have assimilated these lessons well, it is likely that you know more about molecular graphics and modeling than many practicing biochemists and molecular biologists, many of whom are now scrambling to learn these powerful new tools.

DeepView will continue to grow and develop in step with new tools for exploring structure. You can grow with it by continuing to use it your studies, and by updating your copy when new versions appear. Keep abreast of updates of all ExPASy tools by subscribing to Swiss-Flash e-mail.

If you know how to use one modeling program, you will find it easy to learn others. (or as Ronald Reagan put it, "If you've see one redwood, you've seen 'em all.") Although there are many different modeling programs, all of them contain basic tools for manipulating, selecting, displaying, and measuring. Because you now know what to look for in any modeling program, you are better equipped to learn new ones, and to recognize specific or unique features that make a particular program the right tool for your needs.

Happy Modeling!

To The Molecular Level