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MIT 7 014 - Biochemistry—Proteins

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Recitation Section 3 Answer Key February 15-16, 2006 Biochemistry—Proteins A. CBS proteins, 3D representation The protein we will consider today and periodically throughout the term is the yeast cystathionine beta synthase (CBS) protein. Today we will see gels visualizing wild-type and mutant versions of the yeast CBS protein. Yeast that lack CBS protein can not synthesize the amino acid cysteine. Mutant versions of the human CBS protein can lead to a very serious disease called homocystinuria. Some symptoms include mental retardation, early strokes and heart disease. First consider a crystallographic model of a form of CBS protein. Open the CBS structure html file in Internet Explorer. Rotate the molecule and get an idea of the three dimensional structure of CBS. The true 3-dimensional structure of CBS can be observed using the “Show Spacefill” button, and a trace of the peptide backbone can be observed using the “Show Ribbon” button. 1. What is the primary structure of a protein? What information does the modeling program provide us about the primary structure of CBS? The primary structure of a protein is the linear sequence of amino acids. It is impossible to determine this for CBS with the representation that has been provided. We could look up the sequence in the Protein Data Bank and it would read something like (…Trp-Ile-Arg-Pro-Asp-Ala-Pro-Ser…) through all 430 amino acids. 2. What is the secondary structure of a protein? What information does the modeling program provide us about the secondary structure of CBS? The secondary structure is described as α-helical or β-sheet. Remember that secondary structure is controlled by backbone hydrogen bonds between amino acids. CBS contains both types of secondary structure. The majority of the secondary structure elements are α-helices. There are 4 β-sheets. 3. What is the tertiary structure of a protein? What information does the modeling program provide us about the tertiary structure of CBS? The tertiary structure is determined by the side-chain interactions between amino acids in adjacent secondary structures. Although it is hard to resolve the desired interactions from this view, it is clear that amino acids in both the α-helices and β-sheets are interacting with amino acids in other secondary structure elements to contribute to the overall tertiary structure. 4. What is the quaternary structure of a protein? What information does the modeling program provide us about the quaternary structure of CBS? (Hint: Use the “Color Ribbon by Protein Subunit” button) The quaternary structure of a protein is created by a number of distinct interacting amino acid chains. If we color CBS by chain we find that there are two amino acid strands indicating that the protein is a homodimer.B. Protein gels 1. Yeast cells produce enough CBS to fulfill their needs depending on the conditions in which they are growing. In order to procure large quantities of the enzyme, we created special bacterial cells that mostly produce CBS. After we grew up a large number of these bacterial cells, what did we have to do to end up with pure CBS for loading on the gel? We had to grow up a lot of cells, break them all open, and then go through a gradual process of removing all parts of the cell we didn’t need. We removed membranes, nucleic acids, carbohydrates, and small molecules. We then removed the majority of other proteins that were in the cell at the time of harvest, leaving us with mostly pure CBS. Some technical issues: 2. What is a gel? A gel is a jelly-like matrix. Bigger objects have more trouble fitting through the matrix, and, therefore, travel slower through the gel. 3. How does gel electrophoresis work? Our sample preparations and the buffer used in the gel set-up ensure that unfolded proteins have negative charge overall. When the electric current is applied, they move toward the anode. 4. If you run two proteins on the same gel, and one travels farther than the other, what can you say about the relative sizes of these proteins? The one that travels farther has lower molecular weight. 5. With gel electrophoresis, two different conditions are often used. a. Denaturing gel: detergent is added to the gel and a reducing agent is added to the protein sample. Reducing agent converts the S-S disulfide bonds to SH and SH. b. Native gel: Physiological pH conditions where no detergent or reducing agent is added. Compare the 1°, 2°, 3°, and 4° structure of CBS for each of these conditions: Denaturing gel Native gel 1° Primary structure is not disrupted. Same as that for CBS proteins present in a cell and for those run on native gels. Primary structure is not disrupted. Same as that for CBS proteins present in a cell and for those run on denaturing gels. 2° Secondary structure lost due to presence of detergent and reducing agent. Secondary structure is not disrupted. Same as that for CBS proteins present in a cell. 3° Tertiary structure lost due to presence of detergent and reducing agent. Tertiary structure is not disrupted. Same as that for CBS proteins present in a cell. 4° Unable to form quarternary structures due to presence of detergent and reducing agent. Quaternary structure is not disrupted. Same as that for CBS proteins present in a cell. 6. Information about what level(s) of protein structure do you expect to gain from a denaturing gel? Adding detergent and a reducing agent ensures that the protein will lose its secondary and tertiary structure, so the information we get from running such a denaturing gel is the size of each subunit of the protein complex.7. Information about what level(s) of protein structure do you expect to gain from a native gel? Not adding detergent and a reducing agent allows the protein to enter the gel as a 3D structure, so we would be able to gain information about its apparent 3D size. 8. What could we learn from comparing the native and the denaturing gels? If we know the molecular size of a single subunit of the protein from the denaturing gel, the native gel would provide information on the number of subunits involved in the protein complex. Let’s now talk about the proteins themselves. In the 3D representation of the CBS protein, consider a single subunit of CBS. Now consider the whole CBS protein again. 9. Which of these forms would we be visualizing with each kind of gel? A native gel would give us an


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MIT 7 014 - Biochemistry—Proteins

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