Protein Investigator

The Protein Investigator Screen
Entering and Editing Protein Sequences
Folding Your Proteins
Using the History List
Using Game Mode
Printing Your Structures
Assumptions, approximations, etc.

(1) The Protein Investigator Screen

The Protein Invesitgator Screen is shown below with key parts numbered.

The Amino Acid Table. This is a reference for the 20 amino acids found in proteins. Both the one-letter and three-letter codes for each amino acid are shown.
- The shades of the amino acids indicate their relative hydrophobicities: the shades blend from white (hydrophilic) through gray (intermediate) to black (hydrophobic).
- Amino acids with positively-charged side chains have a + in their symbol.
- Amino acids with negatively-charged side chains have a - in their symbol.
- Amino acids whose side chains can make hydrogen bonds have a * in their symbol.
Upper Folding Window: Amino acid Sequence Box. Enter the amino acid sequence you want to fold here. See under Entering for instructions on how to enter and edit sequences. See under Folding for how to fold sequences.
Upper Folding Window: Folded Protein Pane. The structure of the folded protein will appear here.
Lower Folding Window. You can enter and fold another protein in this window. You can then compare the two protein sequences and structures.
History List. Each time you fold a protein, it is saved in the History List. Sequences in the history list can be loaded into either folding window for further analysis.

(2) Entering and Editing Protein Sequences

You can enter a protein sequence by clicking in the Amino Acid Sequence Box and typing the single letter code for the desired amino acids. The program will automatically fill in the three-letter code for easier reading.

You can edit the amino acid sequence as you would any line of text: you can click on a place to insert an amino acid (type the single-letter code); you can select amino acids and delete them with the delete or backspace key (the program will automatically delete complete amino acids).

Note that once you change the amino acid sequence, the border of the Folded Protein Pane changes to pink - this indicates that the amino acid sequence in the Sequence Box no longer matches that of the Folded Protein. The "Fold" button is then activated so you can fold your new protein.

(3) Folding Your Proteins

To fold your protein, click on the "Fold" button in the appropriate Folding Window or click the "Return" key on the keyboard. In a few seconds, depending on the length of your protein and the speed of your computer, the folded protein will appear in the Folded Protein pane. The border of the Folded Protein Pane will then turn gray to indicate that your protein is folded and the "Fold" button will be deactivated until you change the Amino acid sequence. The backbone of your protein will be shown in magenta.

By selecting either "Disulfide Bonds On" or "Disulfide Bonds Off", you can fold the protein under conditions where disulfide bonds can form (oxidizing - typically outside of a cell) or not (reducing - typically inside a cell), respectively. When enabled, disulfide bonds can form between pairs of cysteines; they are stronger than ionic bonds and are shown by yellow lines.

(4) Using the History List

The History List shows all the proteins you have folded during this session. All proteins are lost once you quit the program.

You can move a folded protein from the History List to one of the Folding Windows by double-clicking on the desired protein in the History List; its border will change to green indicating that it has been selected. Then choose either the "Send to Upper Panel" or "Send to Lower Panel" items from the menu that pops up to load that protein into the upper or lower folding window, respectively. You can then compare the two proteins in the different folding windows as well as edit and re-fold either sequence. You can also add a note to the item in the history list that will appear when you leave the cursor over the item for a few seconds.

If you leave the cursor over a protein in the History List, a small window will pop up that shows that protein's amino acid sequence and/or any notes you have added.

You can save the proteins in your History List to a file by choosing "Save As..." from the "File" menu. Once you have designated a file to save into, choosing "Save" from the "History List" menu will automatically save to that file. History List files must end with the extension .histlist; the program will add it if you don't.

You can load in a History List that you have saved in a .histlist file by choosing "Load" from the "History List" menu and selecting the desired .histlist file. The current History List will be replaced by the one in the file.

You can delete an protein from the History List by clicking on the protein in the History List that you want to delete and choosing "Delete Selected" from the "History List" menu. This action cannot be un-done.

You can clear the History List by selecting "Clear" from the "History List" menu. This action cannot be un-done.

You can save all the proteins in the History List as a web page by selecting "Save as Web Page..." from the "History List" menu. You will be asked to supply the name of a folder in which all the relevant files will be stored. You can view the web page by double-clicking the file named "index.html" in the directory you have named. The web page cannot be loaded into Protein Investigator; only a .histlist file can do this.

(5) Using Game Mode

In Game Mode, the object is to design a protein with the same shape as a target molecule you have chosen. Protein Investigator will then determine if the shape of your protein matches the target. Using Game Mode is a good way to practice your protein engineering skills.

In Game Mode, there are two levels of shape matching which can be selected by choosing the Strict Matching Mode from the Game menu:

Strict Mode although the amino acid sequences of your protein need not match that of the target, both the shape of the protein and the direction of the backbone must be the same as the target. Also, the folded protein must be in the same orientation as the target on order to register a match. This is a much more challenging level of play than Non Strict Mode.
Non-strict Mode matches your protein based on shape only and will try matching your protein with the target rotated into different orientations.

You can switch between these two modes by choosing the Strict Matching Mode from the Game menu; in Strict Mode, a check will appear to the left of the Strict Matching Mode in the menu.

You use game mode as follows:

Choose Choose a Target Shape... from the Game menu.
Double-click on one of the target shapes in the list that appears.
A window will pop up showing the target shape you chose. In Strict Mode the backbone will be shown and the amino acids will be numbered to show the proper direction of the protein chain. In Non Strict Mode, only the shape of the target protein will be shown. In either case, no specific amino acids will be shown since your task is to match the shape of the target, not necessarily the sequence.
Go back to one of the Folding Windows and design a protein to match the shape of the target you have chosen.
To see if your shape is a match to the target, click the Check Upper/Lower Protein for Matching Shape button as appropriate. A window will pop up telling you if you were successful or not.
When you are done with this target, click the Cancel button.

(6) Printing Your Structures

You can print the amino acid sequences and folded structures of the proteins in both Folding Windows by choosing "Print" from the "File" menu.

(7) Assumptions, approximations, etc.

This is a highly-simplified model of protein folding. It is not intended to predict the correct structures of any proteins; it is designed to illustrate the major principles involved in that process. Our model of protein folding includes the following factors:

The hydrophobic effect: Proteins are folded so as to minimize the number of hydrophobic amino acids exposed to the water surrounding the protein. Different amino acids have different hydrophobicities that span a wide range of values. This is the weakest force controlling the shape of the proteins.
Hydrogen Bonds: Proteins are folded so as to maximize contacts between amino acids that can form hydrogen bonds. Amino acids are either able to make hydrogen bonds or not; there are no intermediate levels of hydrogen bonding ability. Hydrogen bonds have an intermediate strength between the other two interactions.
Ionic Bonds: Proteins are folded so as to maximize contacts between side chains with unlike charges and to minimize contacts between side chains with like charges. Amino acids are (+), (-), or neutral; no intermediate levels of charge are allowed. Ionic bonds are the strongest of the interactions.

Even though these provide some important insights into protein folding, you should always keep in mind that this is an approximation. The most important "gotcha's" to be aware of are:

This program folds proteins in 2-dimensions only; real proteins fold in 3-dimensions.
This program treats all amino acids as equal-sized circles; real amino acids have different sizes and shapes.
This program folds the protein strictly based on the interactions between the side chains; in real proteins, side chains can interact with the backbone and proteins are often folded with the help of chaperones.
This program does not model secondary or quaternary structure; real proteins often have these.
This program assumes that all side chains with hydrogen bonding capability can bond with each other; in real proteins this is not always possible.