Graph Representations of Oxidative Folding Pathways - Essays in Bioinformatics - page 211

Figure 2. A. Thiol-disulfide exchange mechanism: in the pH range above 8,
cysteine thiols are readily converted to thiolate anions (RS - ), which are potent
nucleophiles. RS - anions attack a disulfide bond, displacing one sulfur atom and
forming a new bond with the other sulfur atom (nucleophilic substitution). The rate-
determining step of this concerted process is the formation of a transition state with
a partial transfer of the negative charge (G - ) over the three sulfur atoms. B. The
formation of a disulfide bond on the polypeptide chain (solid curve) with the help of
a small molecule reagent (thiol form: RSH, disulfide form: RSSR). The two steps
both proceed via a thiol-disulfide exchange reaction. The first step shown is
intermolecular and the second intramolecular. The rate of the intramolecular step is
relevant to protein folding, since it also involves conformational changes.
The particular kind of folding that this article is concerned with is oxidative folding,
which is the fusion of native disulfide bond formation with conformational folding. This
complex process is guided by two types of interactions: first, non-covalent interactions
giving rise to secondary and tertiary protein structure, and second, covalent interactions
between cysteine residues, which transform into native disulfide bridges. The process of
disulfide formation is a simple chemical reaction in which two SH groups join to form a
disulfide link (Figure 2A). If the SH groups are on a polypeptide chain, the in vitro
reaction can be promoted by an external redox system such as a mixture of oxidized and
reduced glutathione, or cysteine and cystine, respectively. In vivo , the oxidative power
comes from specific agents such as the molecular chaperones protein disulfide isomerases.
The underlying mechanism is disulfide interchange (Figure 2B). There are two kinds of
reactions: in a redox reaction a protein disulfide bond is created (or abolished), i.e. the
oxidative state of the polypeptide is changed. This is the case when one of the participants
of the reaction (say RSH) is not part of the protein. In a shuffling reaction both participants
of the disulfide interchange are protein-bound, so the oxidative state of the polypeptide
does not change. In view of these possibilities it becomes obvious that there are a great
many ways in which disulfide bridges can form and rearrange during the folding process.
Today it is generally accepted that non-covalent interactions guide the process of folding
and formation of disulfide bridges will lock the protein into the right conformation. The
advantage of oxidative folding as opposed to general protein folding is that disulfide
intermediates can be chemically isolated and studied using such techniques as acid trapping
of the intermediates and analysis of the disulfide bridges using a combination of enzymatic
cleavage and mass spectrometry. There is a body of literature in describing the pathways of
oxidative folding in terms of disulfide intermediates [8-10], and our goal is show how
graph theory can be used for this purpose.