Elements of Protein Structure

Discussion questions; Module 2

The questions posed below are to be answered through the "Discussion" link that is in the same folder as this page. Your responses are Due by 11:55 (EST) pm on 21 March. You may use the threaded discussions to discuss with your classmates as you like prior to answering. The quiz itself is not timed; you may take as long as you like. Only your first answer to each question is accepted as your response. You may find it helpful to formulate your responses in an external editor then copy and paste your responses to the "quiz".

RNase is a digestive protein excreted into the small intestine. Its function is to break up the RNA molecules that you eat into monomers so that they can be absorbed.

Urea is a chemical destroys the native 3 dimensional structure of proteins. Effectively unraveling into a truly random structure (meaning each molecule does not have the same fold but rather a rapidly changing randomly occurring structure) This process is called denaturing the protein. It does not have any effect on disulfide bonds, however. The Cysteines involved in a disulfide remain covalently linked with treatment by urea only.

  1. A classic experiment that demonstrated the importance of the forces in protein folding was performed with RNase many years ago. RNase Has 8 Cysteines all of them involved in disulfide bonds (there are 4 disulfides). In the native structure (just as you would expect) amongst the possible arrangements of Cysteines into disulfides only one is observed. RNase can be denatured with Urea and dithiothreitol (DTT). What is the function of DTT in this experiment? Why is DTT required to completely denature RNase? Would it be required for most cellular proteins as well?
  2. Upon the removal of the Urea (to refold the protein) and then the DTT (in this order) how many different arrangements of disulfides might you expect? why?
  3. In the reverse experiment One can oxidize the Cysteines (remove the DTT) and then refold the protein (remove the Urea). How would (if at all ) this change the results from the previous experiment? why?
  4. Fibrin, is the protein that actually forms the blood clot. It goes from a soluble single subunit protein "drifting" in the serum to a network containing many, many fibrin molecules (acquires quaternary structure). This requires modification of the structure to at least a small degree. Thinking ONLY of the noncovalent forces and generalizations about water soluble proteins... How do you think the overall structure might have changed?
  5. The noncovalent fibrin network is later covalently crosslinked by the active form of Factor XIIIa. (crosslinking means that the SIDECHAIN of an amino acid in one protein molecule is covalently bonded to the SIDECHAIN of an amino acid in a differnt protein molecule...)
    Thinking of amino acid side chain structures, and the types of bonds we have made so far propose two different types of covalent linkages that might be present between fibrin molecules.