Lab 8 Computer_Models_Proteins

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Dec 6, 2023

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Molecular Modeling Handout 1 Introduction: In this exercise, we will be examining protein crystal structures to understand the relationships between primary, secondary, tertiary, and quaternary structure. We will examine how protein structures are stabilized by hydrogen bonding and the burial of hydrophobic sidechains. We will examine how proteins are able to bind cofactors and how binding of oxygen changes the quaternary structure of hemoglobin. Primary sequence: The primary sequence of a protein is an accounting of the amino acids that are present and is read from the N to C terminus. Proteins are often classified based on sequence homology , where similar patterns in the primary sequence are observed. When looking at the protein sequence, one can classify protein by either looking at the identity of each amino acid, or by looking for patterns in polarity, charge, and hydrophobicity. For example, let’s look at the first 30 amino acids in EF-1/EF-tu proteins in several species. Each letter represents the single-letter code for each amino acid in the sequence. While there are some differences in the amino acids in each protein sequence, you will notice a pattern of polar, charged, and hydrophobic amino acids. Hydrophobic = purple , polar = blue , charged = red . Species Sequence Halobacterium IG H V DH G K S T M VG R LL Y E T G S VP EH VI EQH Homo Sapiens IG H V D S G K S T TT G H LI Y K C G GI DKR T I EK F Escheria coli IG H V DH G K T T L T AA - - - - - - - - - - - - I TT V Secondary structure: Protein secondary structure is a description of the conformation of a particular segment of the protein chain. Certain conformations are favored over others, largely because they minimize steric clashes within the protein structure, allow for favorable hydrogen-bonding of backbone residues. In the folded structure, the amide backbone of a protein is shielded from water, so the amide backbone must form hydrogen bonds to be stable. Two common structures that are observed in proteins are  -helices and  -sheets. Both of these structures satisfy the necessary hydrogen bonding of the backbone and minimize steric clashes of atoms. In  -helices, amides form intra-strand hydrogen bonds with groups that are located on the next loop of the helix. In this lab, we will determine the hydrogen- bonding pattern in  -helices. In  -sheets, inter-strand hydrogen bonds are formed.

Molecular Modeling Handout 2 Tertiary structure Isolated  -helices and  -sheets are not very stable in solution and are in equilibrium with unfolded structures. The reason for this is simply that there is no driving-force for an unfolded peptide to form an isolated  -helix or  -sheet . In general, the driving force for protein folding is the removal of hydrophobic residues from water. The hydrophobic residues are alkanes or aromatic rings that lack charge or polarity, and hence are unable to hydrogen- bond with water. An isolated secondary structure does not have a way to bury hydrophobic residues upon folding. However, one can image that if two or more structures associate, they can bury hydrophobic amino acids. In proteins, it is often the case that different parts of the protein will adopt different secondary structures, where these parts are connected by loops to create a complex overall structure. This is called a tertiary structure. In this lab, we will observe how the burial of hydrophobic amino acids is able to direct these structures. We will also observe what happens to polar residues that are in the protein core. Quaternary Structure: The term quaternary structure is used to describe complexes containing two or more folded proteins. Each component is called a subunit or a domain. While each protein fold may be distinct, it is often the case that the proteins interact in interesting ways. For example, hemoglobin is a protein complex consisting of four subunits, each of which can bind a single molecule of oxygen. Upon oxygen binding to one subunit, the protein changes shape to increase the binding of oxygen to the other subunits. In this lab, we will examine this shape change and consider why the tertiary structure is important for hemoglobin function. Procedure: We will be using a program called Chimera to examine structures. Chimera can read *.pdb files as 3-D images that can be manipulated. Throughout the exercise you will be asked to Observe, and Reflect. Complete your observations (drawings, comments, etc.) and reflections on the report sheet at the end of the lab. You will also be asked to submit pictures from your work on the computer. 1. Using the program: 1a: computer commands • Commands from the pulldown menu bar will be given in bold . For example, File → Open is used to open a file. Titles for windows, buttons, and other items that appear on the screen will also be described in bold. • Keyboard commands will be given in “ italics” for example: press “ control ” while holding the mouse over an atom to select it. • Directories (or folders within folders) will be specified by “ \ .” For example, Desktop\Chem130\ data.pdb would refer to a file called data.pdb in a folder called Chem130 on the desktop. Notice that the file was named with underlined italics . 1b: downloading chimera and extracting files Go to the site: https://www.cgl.ucsf.edu/chimera/download.html to download and install Chimera to your computer. The program is free. All files are available in the lab information section on Blackboard. Save these files to your desktop.

Molecular Modeling Handout 3 1c: opening a file. To open a file go to File → Open in the pull down menu. Navigate to the location of your file. Use the scroll bar at the bottom of the window to scroll back to users/username/desktop. Once you have done that, your screen will look like this: *DO NOT MAXIMIZE THE WINDOWS TO FULL SCREEN* 2: Exploring a small secondary sequence. In this section, we will explore the basic hydrogen-bonding pattern in a short  -helix. Open 2zta_1heptad.pdb What you are seeing is 7 residues of an alpha helix, just the backbone (no sidechains), with red representing oxygen atoms, blue for nitrogen atoms, and grey for carbon atoms, white for hydrogen. The residues numbered 1 through 7, plus an identifier to indicate the type of amino acid. 2a: examining Hydrogen bonds Now, we are ready to examine the structure and look for hydrogen bonds. Observe: Find an amide bond and draw the Lewis structure that includes all atoms that are in the same plane. Remember that this program does not indicate if a bond is a single or double bond! The atoms in each amide bond are coplanar. This imparts rigidity in the protein structure. Find another amide that is engaged in a hydrogen bonding with your amide Draw the hydrogen bonding. Your drawing should include two full amide groups. How many hydrogen bonds are in the structure 2sta_1heptad? To help you find the hydrogen bonds, Tools → structure analysis → FindHBond Observe: verify that the hydrogen bond you drew corresponds with what was found by the program.

Molecular Modeling Handout 4 2b: understanding hydrogen bond angles and distances Next, we will calculate hydrogen bond angles and distances. We will look for generalizations that describe the angles and distances. Observe: Calculate the distance for two hydrogen bonds in the structure. Measure an N- H bond as well for comparison. Fill in the values in a table on your report sheet Reflect: How does the hydrogen bond distance compare to that of a N-H bond? We are done with this peptide fragment. Click on File → save image (or take a screen shot). Follow the prompts to save the image as a tiff file for your lab report. Click on File → Close session. DO NOT CLOSE THE WINDOW 3: Exploring a secondary sequence: In this section, we will explore the interactions between two short alpha helixes. 3a: exploring a single helix Open 2zta_1chain.pdb This is the structure for GCN4-p1, one of the peptides from your prelab assignment. Use • Actions → Atoms/Bonds → side chain/base → show At this point, the backbone is likely represented as a ribbon, with sidechains represented as wires. We will highlight the hydrophobic amino acids by first selecting them and then making them space filling. • Select → Residue → Leu , Then Go to Actions → Atoms/Bonds → sphere • Repeat for Val Use the mouse to rotate the peptide. Observe: Draw the structures of Leu and Val. Are they hydrophobic or hydrophilic? Explain. Observe: Draw a sketch of the peptide, including where you would expect to find hydrogen bonds and the location of the hydrophobic amino acids. Tools → structure analysis → Distances opens a window that facilitates the measurement of distances between atoms. To select an atom: ctrl-click To select multiple atoms: shift-ctrl-click To add an atom to those selected, it is necessary to hold down BOTH “ control” AND “shift” at the same time while clicking on the new atom. To determine a distance, it is necessary to have two atoms selected. Try selecting two atoms that are engaged in a hydrogen bond. To clear selection: Select → Clear Selection (or ctrl-click on blank space) When you do this, you will notice that descriptors for these atoms appear in the Structure Measurements window. The descriptor tells you the type of amino acid, its place in the sequence, and the type of atom (O, H, N). Click create. You should now see the distance of the hydrogen bond in the window.

Molecular Modeling Handout 5 Predict: Do you think this structure is stable in aqueous solution? Why or why not? Examine the model you made for the pre-lab to help formulate your thoughts. We are done with this peptide fragment. Click on File → save image (or take a screen shot). Follow the prompts to save the image as a tiff file for your lab report. Click on File → Close session. 3b: exploring a simple tertiary structure. In this part we will see an example of how two alpha helix secondary structures interact in a tertiary structure. Open 2zta.pdb We will recolor one of the chains before highlighting the Leu and Val side chains. • Select → Chain → A , then Actions → Color → Blue • Actions → Atoms/Bonds → side chain/base → show • Select → Residue → Leu , Then Go to Actions → Atoms/Bonds → sphere • Select → Residue → Val , Then Go to Actions → Atoms/Bonds → sphere Observe: Draw a sketch of the peptide, including where you would expect to find hydrogen bonds and the location of the hydrophobic amino acids. Reflect: How does primary sequence relate to the stability of the peptide’s folded structure? In other words, how does the pattern of hydrophobic residues effect protein stability? Briefly examine your prelab model and describe any changes you might make. We are done with this peptide fragment. Click on File → save image (or take a screen shot). Follow the prompts to save the image as a tiff file for your lab report. Click on File → Close session. 4: Exploring myoglobin, a more complex tertiary structure. 4a: Exploring the overall structure. Open 1MBO.pdb . This is the structure for myoglobin, a protein that carries and stores oxygen. Again, we want to determine the location of all the hydrophobic amino acids. We are focusing only on the most hydrophobic amino acids (Leu, Val, Ile, and Phe) to simplify viewing. • Select → Selection Mode → Append *allows selections to be added • Select → Residue → Leu , (repeat for Val, Ile, and Phe) • Actions → Atoms/Bonds → side chain/base → show • Actions → Atoms/Bonds → spheres Observe: What pattern do you observe with the placement of hydrophobic residues in the structure?

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