## 11. Comparing Proteins: Lysozyme

UNDER CONSTRUCTION

For this tutorial, you will need three PDB files 1HEW (used in sections 1-9), 1HEL, and 1LZY. 1HEL is the same hen egg-white lysozyme as in 1HEW, but it lacks the trisaccharide inhibitor. 1LZY is egg-white lysozyme from turkey. In section A, you will compare the conformations of lysozyme with and without the inhibitor. In section B, you will compare homologous lysozymes from chicken and turkey.

### A. Comparing Conformations: Effect of Inhibitor Binding on Lysozyme Conformation

In this section, you will compare two different conformations of the same protein: lysozyme with and without an inhibitor. DeepView allows you to superimpose all or parts of different models, and to color the models to emphasize conformational differences.

Load 1HEL, chicken egg-white lysozyme. Then load 1HEW, the same enzyme with tri-N-acetylglucosamine, a competitive inhibitor of lysozyme.

Press help or = to center the two models. Before you compare them, let's review some features for handling multiple models.

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 1HEL at the top. A menu appears, showing the names of all loaded models or layers. Select 1HEW. Examine the effects of the visible and can move buttons on 1HEW. Restore movement and visibility for 1HEW.

<tab>
<tab>
Notice the effect on the file-list menu at the top of the Control Panel. With the Control Panel active, 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. End with 1HEL as the active layer.

Wind: Layer Infos
The Layer Information window appears. Move it to a convenient location if it is in the way. Notice that you can use this little window to control visibility and movement of multiple models, and that you can control many other features of the display as well. The check-marks work just like those in the Control Panel window.

Use the Layer Infos window to turn off movement of 1HEW and move 1HEL to the left, leaving it beside 1HEW.

Click on 1HEW.pdb in the Layer Infos window. Notice that the file name 1HEW.pdb becomes red, and the Control Panel heading changes to 1HEW.pdb. Without clicking on the Control Panel, you have made 1HEW the active layer.

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

<shift>Color: B-Factor
(Press and hold the shift key down before pulling down the menu.) Now both models are colored according to their B-factors. With menu commands, the shift key extends actions to all layers.

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

Fit: Magic Fit (Quick Test)...
On the resulting dialog, click OK. Now the two models are superimposed -- 1HEW moved onto 1HEL. SPdvB superimposed the two models by using the statistical method of least squares to fit the alpha-carbon coordinates of 1HEW onto those of 1HEL.

Prefs: General
Click to put a check-mark in the last box, marked "Scale RMS colors so that min = dark blue and max = red.

Color: RMS
This action affects 1HEW, the active layer. Now hold down the control key and press tab repeatedly. This action cycles through the current layers, showing only one at a time. In this manner, you can "blink" back and forth between two layers.

Stop blinking with 1HEW shown.

Next -- color NAGs CPK

Then explain color scheme (see old Hb stuff below).

Fit: Magic Fit
This time, look at the dialog.

Scroll to the bottom of the Control Panel list and select NAG210, NAG202, and NAG203 of 1HEW. Press return.

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

***

<shift>Select: Visible Groups
Color: RMS
Make 1HHO the active layer. Select (but do not display) the whole model, and 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.

Wind: Layer Infos
The Layer Infos table 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, 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 the last section, 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).

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 1HHBBeta the active layer.

Tools: 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.

Tools: Improve Fit (with Struct. Align.)
Again accept the default settings in the dialog and click OK. Calculate the RMS deviation again and you will find it improved: a lower RMS deviation, based on a smaller number of residues.

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 Control Panel.

Scroll across the sequences. Notice that certain pairs of aligned residues are highlighted (gray). 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 a larger number of 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.

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

You have used many of the functions of SwissPdbViewer. The program has additional advanced features, for which the program's creator, Nicolas Guex, provides tutorials. The following section includes introductions to the advanced tutorials and projects on which you can hone your skills. The 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, 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 USM Assignment 2

#### To Next Section: 12. Advanced Tutorials and Projects

## 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 Biochemistry Resources

HOME