Ann Bohannon-Stewart
Class: Biochemistry
Professor: Dr. Whalen
Summery report:
Enhancing the Activity of a Protein by Stereospecific Unfolding
Insulin is a protein hormone structure that can be changed by making a substitution of (Ala) where (PheB24) is in the side chain. In this article they claim that the substitution of protein D-Ala of insulin is destabilizing to insulin, but the (Ala) protein diastereomer enhanced some of the activity with segmental unfolding of the strand. The article goes on to explain photoactivable derivatives that have L- or D-para-azido-Phe are able to link to the insulin receptor with better D-specificefficiency, and give exposure of hydrophobic bonding in the analogs and make them able to mix with accelerated fibrillation, a form of aggregation-coupled misfolding that is associated with cellular toxicity. When you trade out PheB24 for Ala, you see a changed effect of both the D and L substitution. This article explains both effects of the Stereospecific unfolding of the protein.
The article explains how insulin works and that insulin is a globular protein hormone made up of 2 chains. The two chains are, A chain which has 21 residues and B chain which has 30 residues. Now inside of the B chain is the area where many conformational changes take place, and the area of interest or (PheB24) is in the B side chain.
If you look on page 2 of the article it will show you a picture of insulin in it’s 2 different chains which is label A and B. In side chain B it shows Phe B24 as an arm that holds in a folding shape pocket of the protein insulin, and it tries to show how L-Ala and D-Ala will position when Phe B24 is removed and replaced with Ala L and D. The pictures show that its storage form, requires engagement of a detachable arm at an extended receptor interface. Because this active conformation resembles an amyloidogenic intermediate, it says the protein envisage that induced fit and self-assembly represent complementary molecular adaptations to potential proteotoxicity.
In order to understand why this change in insulin is complementary to its function you need to understand how insulin works. Insulin is stored as a zinc crystal inside the islet cells in the pancreas, so it has an important function to preserve insulin function. The article explains on page 1 that in pancreatic cells, insulin is stored as Zn2 stabilized hexamers which is shown on page 2 in Fig. 1B. This will form microcrystalline arrays within specialized secretory granules (1). The hexamers will than dissociate upon secretion into the bodies circulation. This will enable the hormone to function as a zinc-free monomer. The monomer is will than undergo a change in conformation upon receptor binding to the cell. This article shows how they investigated a site of conformational change in the B-chain (PheB24) which they tried to show a picture of this folding change in Fig. 1A on page 2 of this article. You see in the normal folding of the crystal structures of insulin, the aromatic side chain anchors an antiparallel B-sheet at the dimer interface which they showed a picture of in Fig. 1C on page 2. Total chemical synthesis is tudied and tested for comparison of corresponding D- and L amino ala amino acid substitutions at this site.
They do something they call “chiral mutagenesis”. Now I didn’t know what that chiral mutagenesis was when I first read it, but I looked it up on the web, and found an article about it which is named “Chiral mutagenesis of insulin. Foldability and function are inversely regulated by a stereospecific switch in the B chain.” In this article it stated on the first line, “How insulin binds to its receptor is unknown despite decades of investigation. Here, we employ chiral mutagenesis-comparison of corresponding D and L amino acid substitutions in the hormone-to define a structural switch between folding-competent and active conformations.” So they are doing close to the same thing in article I am working on to summarize for you. You can link to this article on chiral mutagenesis of insulin at this link posted below:
http://www.find-health-articles.com/rec_pu...rsely-regulated.htm I believe it better explains the process they used to make the changes in the insulin, so they could test their hypothesis.
They use many ways to procedures to study this change in insulin. The consequences of this conformational change are investigated by photomapping of the receptor-binding surface, and you can see in the experimental procedures section of this article they explain how they tested their theory. They ran these tests by using, Synthesis of Insulin Analogs (where human insulin was given by donors), Receptor Binding Assays (where receptor binding was tested), Circular Dichroism—Far-UV CD spectra, 1H NMR Spectroscopy, and Photocross-linking Studies where used to determine the structure of the insulin. Than finally Insulin Fibrillation was used and these tests were performed multiple times.
The results showed analogs of DKP-insulin containing L-amino acid substitutions at B24 (“L-analogs”) exhibit reduced receptor-binding affinities (relative to the parent monomer), whereas D-analogs exhibit enhanced affinities. No disproportionate changes were observed in the extent of low affinity
