Chemistry 532: Key to Homework 4

FK506 Case Study and Protein Conjugation

  1. Does FK506 block calcium signaling during T-cell activation? Justify your answer.

    2. Yes and no. The initiation of intracellular calcium fluxes is triggered by a step that occurs upstream of the action of FK506 so, it does not block the increases in calcium. It does, however, block calcineurin activation , which is a calcium dependent process.

  2. Explain why it has been suggested that the immunoplillin proteins have a functional reactivity that is intermediate between chaperone proteins like BiP and heat shock proteins like Hsp70.

    3. Proteins like BiP use ATP to facilitate the refolding of improperly folded proteins while heat shock proteins like Hsp70 simply bind improperly folded proteins to prevent aggregation. The immunophilins apparently bind improperly folded proteins like Hsp70 and they catalyze folding, like BiP but, the latter is achieved without the use of ATP.

  3. One criticism of the suggestion that inhibition of proline isomerase activity is related to the immunosuppresant activity of FK506 is that the concentration of FK506 needed to block IL2 production by T cells is considerably lower than the concentration needed to inhibit isomerase activity. Given that FK506 still binds to the isomerase site and the complex then interferes with calmodulin mediated calcineurin activation, suggest how it achieves this block of calcineurin activation at a concentration that is lower than what is needed to fully inhibit the isomerase activity.

    4. Two things have to be true in order for this to work. One is that the affinity of the FK506-FKBP complex must have an affinity for the calcineurin-calmodulin complex that is very low, comparable to the concentration of FK506 used to block IL2 secretion. The second is that the concentration of the calcineurin-calmodulin complex has to very low and comparable to the small amount of FKBP that is bound by FK506 at the concentrations used to block IL2 secretion.

  4. One problem with using a probe protein conjugated to beads as an affinity matrix to pull out ligands from cell lysates is that many of the proteins eluted from these affinity columns turn out to have no affinity for the probe protein. Describe at least two procedures you would use to minimize the isolation of non-specifically bound proteins.

    5. The bead itself and any linkers used could be a source of non-specific binding. One can "pre-clear" the cell lysate by incubating it with beads that have not been conjugated with the probe molecule and to remove species that have an affinity for the bead matrix. If one has access to another molecule that is structurally similar to the probe but lacking the desired specificity (i.e., an isotype antibody), it would be better to pre-clear with beads conjugated to the other molecule first. Also, before eluting the affinity column one can elute with some other protein (or a protein cocktail) to elute species that are non-specifically bound.

  5. One functional group used to conjugate to protein lysine residues is an epoxide, which reacts to give an amino alcohol linkage. The problem is that the epoxides react slowly. Another functional group is a chloro-imidate, which reacts to an N-alkyl guanidinium linkage. The problem is that the imidate is highly reactive and, thus, much of the linker hydrolyzes in water. Despite these problems, both the epoxides and chloro-imidates have a significant advantage over activated acyl functional groups. What is the advantage and why is this important?

It is important not to neutralize too much of the protein’s surface charge because this can lead to aggregation and/or a decrease in activity. The chloro-imidate and epoxide linkers result in an amine or guanidinium ion, respectively, which are both positively charged. Thus, these linkers do not alter the protein’s surface charge.