CHAPTER 2 - PROTEIN STRUCTURE

D:  PROTEIN FOLDING AND STABILITY

BIOCHEMISTRY - DR. JAKUBOWSKI

Last Update:  3/1/16

D7.  Redox Chemistry and Protein Folding  

In general we envision the interior of a cell to be in a reducing environment.  Cells have sufficient concentrations of "b-mercaptoethanol"-like molecules (used to reduce disulfide bonds in proteins in vitro) such as glutathione (g-Glu-Cys-Gly) and reduced thioredoxin (with an active site Cys) to prevent disulfide bond formation in cytoplasmic proteins.  For disulfide bonds to occur in a protein, a free sulfhydryl reacts with another one on a protein to form the more oxidized disulfide bond.  This reaction occurs more readily if one of the Cys side chains had a lowered pKa (due to its immediate environment) making it a better nucleophile in the reaction.  Most cytoplasmic proteins contain Cys with side chain pKa > 8, which would minimize disulfide bond formation as the Cys are predominantly protonated at that pH. 

Disulfide bonds in proteins are typically found in extracellular proteins, where they serve to keep multisubunit proteins together as they become diluted in the extracellular milieu.  These proteins destined for secretion are cotranslationally inserted into the endoplasmic reticulum (see below) which presents an oxidizing environment to the folding protein and where sugars are covalently attached to the folding protein and disulfide bonds are formed (see Chapter 3D:  Glycoproteins - Biosynthesis and Function).   Protein enzymes involved in disulfide bond formation contain free Cys which form mixed disulfides with their target substrate proteins.   The enzymes (thiol-disulfide oxidoreductases, protein disulfide isomerases) have a Cys-XY-Cys motif and can promote disulfide bond formation or their reduction to free sulfhydryls.  They are especially redox sensitive since their Cys side chains must cycle between and free disulfide forms.

 Intracellular disulfide bonds are found in protein in the periplasm of prokaryotes and in the endoplasmic reticulum (ER) and mitochondrial intermembrane space (IMS) of eukaryotes.  For these proteins, the beginning stage of protein synthesis (in the cytoplasm) is separated temporally and spatially from the site of disulfide bond formation and final folding.  Disulfide bonds can be generated in a target protein by concomitant reduction of a disulfide in a protein catalyst, leaving the net number of disulfides constant (unless the enzyme is reoxidized  by an independent process). Alternatively, a disulfide can be formed by transfer of electrons to oxidizing agents such as dioxygen.

In the ER, disulfide bond formation is catalyzed by proteins in the disulfide isomerase family (PDI).  To function as catalysts in this process, the PDIs must be in an oxidized state capable of accepting electrons from the protein target for disulfide bond formation.  A flavoprotein, Ero1, recycles PDI back to an oxidized state, and the reduced Ero1 is regenerated on passing electrons to dioxygen to form hydrogen peroxide.  In summary, on formation of disulfides in the ER, electrons flow from the nascent protein to PDIs to the flavin protein Ero1 to dioxgen (i.e. to better and better electron acceptors).  The first step is really a disulfide shuffle, which, when coupled to the subsequent steps, leads to de novo disulfide bond formation.

 In the mitochondria, disulfide bond formation occurs in the intermembrane space (IMS) and is guided by the �mitochondria disulfide relay system.�   This system requires two important proteins: Mia40 and Erv1.  Mia40 contains a redox active disulfide bond cys-pro-cys and oxidizes cys residues in polypeptide chains.  Erv1 can then reoxidize Mia40 which can in turn get reoxized itself by the heme in cytochrome c.  Reduced cytochrome C is oxidized by cytochrome C oxidase of electron transport  through passage of electrons to dioxygen to form water.  The importance of IMS protein oxidation is less understood, but it is believe that the oxidative stress caused by a dysfunction could lead to neurodegenerative diseases.

A recent review by Riemer et al  compares the ER and mitochondrial processes for disulfide bond formation:

• Many more and diverse proteins form disulfides in the ER compared to the IMS.  Most in the IMS have low molecular mass and have two disulfide bonds between helix-turn-helix motifs.  These protein substrates include chaperones that facilitate localization of proteins in the inner membrane, and in proteins involved in electron transport in the inner membrane.

• There are many PDIs in the ER, probably reflecting the structural diversity of protein substrates in the ER.  However Mia40 appears to be the only PDI in the IMS.

• "De novo" disulfide bond formation is initiated by Ero1 in the ER and Erv1 in the IMS.   Convergent evolution led to the similar structures for both - a 4-helix bundle that binds FAD with two proximal Cys.

• The mitochondria pathway lead to water formation on reduction of dioxygen, not hydrogen peroxide, minimizing the formation of reactive oxygen species in the mitochondria.  The peroxide formed in the ER is presumably convert to an inert form.

• The IMS is in more intimate contact with the cytoplasm through outer membrane proteins called porins which would allow some glutathione access.  The IMS presents a more oxidizing environment than the cytoplasm (with more glutathione).   The ER, without a porin analog, would be more oxidizing.

• Reversible formation of disulfides in the ER regulates protein activity. 

Disulfide bond regulation in the Periplasmic Space of Bacteria

The redox sensitivity of the Cys side chain found in disulfide bonds is important in regulating protein activity.  In particular, the thiol group of the amino acid Cys, an important nucleophile often found in active site,  can be modified to control  protein activity.  The formation of a disulfide bond or the oxidation of free thiols to sulfenic acid or further to sulfinic or sulfonic acid can block protein activity.  The E. Coli periplasmic proteins DsbA (disulfide bond A) converts adjacent free thiols into disulfide-linked Cystine, in the process becoming reduced.  DsbB reoxidized DsbA back to its catalytyically active form.   What about periplasmic protein like YbiS with an active site Cys?   Since the environment of the periplasm is oxidizing, YbiS mist be protected from oxidative conversion of the free Cys to either sulfinic or sulfonic acids causing the protein to become inactive.  The mechanism involves two periplasmic proteins known as DsbG and DsbC which are similar to thioredoxin.  These two proteins are able to donate electrons to the unprotected thiol preventing it from becoming oxidized, which allows YbiS to remain active in the periplasm.   To maintain activity, DsbG and DsbC are reduced by another periplasmic protein, DsbD. 

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