I. Protein Structure and Hemoglobin

Lab Instructions

Jmol Visualization Tutorial - Self-paced Walkthrough

Key Words: quaternary structure, heme, allosteric cooperativity, ligand, protein conformation, zwitterion, functional groups, polarity

Directions:

• Open the tutorial: http://bioinformatics.org/jmol-tutorials/jtat/hemoglobin/contents/contents.htm (Chrome browser recommended)

• A colored molecule should automatically load in the JSmol window on the left and begin spinning (spheres representation).

• Note: the presentation works best if you click on the content pages and the grey buttons one at a time in the order they appear in the text. It will be helpful to avoid have too many browser windows open at once, and you may need to restart your browser periodically. Click the View button at the top of the page to reset the view if your molecule "blows up."

Did Jmol load properly? Try pressing your left mouse button over the spinning molecule and holding while dragging to view it from different angles.

The Hemoglobin tutorial covers material from Module 4: Proteins.

Introduction

1. Right-click on the protein and in the context menu click on Select> followed by All.

2. Right-click over the protein again and click Style> Scheme> Cartoon.

Page 1. Glycine representations. (Modules 2 & 3) (First two sections offline)

To switch to page 1, click the grey Next -> button. http://www.umass.edu/molvis/tutorials/hemoglobin/glycine.htm

A ball-and-stick representation of the simplest alpha amino acid should appear and begin spinning in view.

• Question 1a: Identify and name the molecule shown.

• Q1b: How can you tell what the charge is on the nitrogen (blue)? ..on the carboxyl group?

• Q1c: Consult your pka sheet. Would you expect to see this molecular species at pH 7 or some other pH? Explain.

Click on each of the four grey View buttons one at a time and rotate the view to familiarize yourself with the ball-and-stick, space-filling, and licorice representations. Note that for View 2, the white hydrogen atoms do not contribute much to the shape of the van der Waals (VDW) spheres. Hydrogens bonded to carbon are typically omitted in structural representations.

page 2: Peptides & Backbones (Modules 3 & 4)

To switch to the page, click the grey Next -> button. http://www.umass.edu/molvis/tutorials/hemoglobin/pepstruc.htm

Click the View 2 button and manually rotate the tripeptide backbone.

• Q2a: How many amide functional groups are present? How many atoms lie in the same plane of an amide?

• Q2b: Note that the structure is not symmetric. How do you know which end is the N-terminus and which is the C-terminus?

We number residues from the N-terminus and typically orient it on the lower left (compare slides 03-32 and 04-15). Click toggle spinning to turn off followed by View 3.

• Q3: Manually rotate one of your alanine (Ala) residues so that it aligns with the view in slide 03-08. Remembering that a wedge comes out of the page and a dash goes into the page, is this the correct chirality for L-alanine in proteins?

Click View 5 and then View 6, taking note of all the added chiral centers.

• Q4: Using your amino acid sheet, write the sequence for the tetrapeptide in 1-letter code.

Judging from the backbone amides being randomly exposed to solvent (not H-bonds in the helix axis or sheet plane), this peptide is in a disordered chain conformation (no regular secondary structure).

page 2: Hemoglobin & Heme (Module 4)

http://bioinformatics.org/jmol-tutorials/jtat/hemoglobin/2heme/chapter.htm

(To switch pages, click the grey Next -> button.)

First, press your left mouse button and hold while dragging over the molecule to view hemoglobin from different angles. You are looking at a space-filling, or van der Waals (VDW) spheres, representation of the protein surface. The four heme prosthetic groups are colored red.

• Q5: Each protein monomer, or chain, is a different color. How many monomers make up this protein oligomer?

The structure of hemoglobin has a large central cavity in between the monomers. Click the toggle spinning button and then manually rotate the view so you are looking down the central cavity. The cavity is large enough to accomodate many solvent water molecules.

• Q6: Do you expect the residues lining this cavity to have hydrophobic or hydrophilic sidechains? Does your answer make sense given that bisphosphoglycerate is known to bind at Lys 82?

Click View 2. Note how the alpha carbon backbone traces out the alpha helices surrounding each heme. Now click View 4.

• Q7: Locate the structure for heme (slide 04-34). How many carboxyl groups does the heme molecule contain? The oxygen atoms are colored red in VDW representation (view 4). Why do you suppose the carboxyl groups point out from the hemoglobin surface? This is most evident if you click on Contents to go back to the main page and view the VDW hemes within the space-filling tetramer.

Be sure to click on View 10. Note how the sp2 lone pair from a histidine sidechain coordinates iron, which together with the O₂ ligand, completes the hexavalent metal ion coordination complex (octahedral geometry). Because the heme is always bound, hemoglobin is a metalloprotein.

page 3: Hemoglobin - Secondary structure (Module 4)http://bioinformatics.org/jmol-tutorials/jtat/hemoglobin/3secstruc/chapter.htm

(To switch pages, click the grey Next -> button.)

Question 8:

• View 1- What color are the loop or turn regions connecting the alpha helixes?

• View 2- Are the helixes left-handed or right-handed? (It doesn't matter which end is the N-terminus. Compare to slide 04-16: the front parts of a right-handed helix always go up and to the right.)

• View 7- Describe the end of the backbone that corresponds to the N-terminus.

• View 8- The carbonyl oxygen of the second residue hydrogen bonds with the N-H of which residue?

• View 10- Determine the number of residues and write the 1-letter code amino acid sequence for this helix.

Note how polar and nonpolar sidechains alternate every 3.6 residues, so that they face opposite sides of the "amphipathic" helix. One side is exposed to water, while the other faces the hydrophobic protein core. A typical distribution of charged, polar, and hydrophobic amino acids throughout a protein is seen on Pages 5 and 6. Compare this information to your helical wheel in Activity 4.2.

page 5: Polarity (Module 4)

http://bioinformatics.org/jmol-tutorials/jtat/hemoglobin/5phob/chapter.htm

(To switch pages, click the grey Next -> button.)

At your leisure, compare the information on this page, about sickle aggregation, to slide 04-40, about amyloid fibrils/plaques.

Alternatively, check out this medicinal chemistry tutorial relevant to Module 5: Enzymes:

• www.proteopedia.org/wiki/index.php/Drug_Metabolism_by_CYP450_Enzymes