OXIDATIVE FOLDING OF SELENO-HIRUDIN ANALOGS

Hirudin, originally isolated from the leech Hirudo medicinalis, is a 65-residue protein that is stabilized by three paired cysteines [6−14, 16−28, and 22−39] (Figure 1a).
The oxidative folding mechanism of hirudin represents a model to many proteins. Folding is initiated by a near-random packing, followed by the reorganization and fine adjustment of partially compact intermediates to attain the native state of hirudin. The process of packing is observed as the unfolded hirudin flows sequentially via three groups of equilibrated intermediates, namely one-disulfide, two-disulfide, and three-disulfide (scrambled species) isomers (Figure 1b). The rate-determining step is the rearrangement of the scrambled isomers into the native state by means of thiol-disulfide interchange reaction.

The greater polarizability of selenium in comparison to sulfur and the lower pK_a of selenol compared to thiol makes selenium a better nucleophile and electrophile in thiol-disulfide-like interchange reactions. This is the reason behind the enhancement of oxidative protein folding observed with selenocysteine substitution in polypeptides and proteins.

Here we aim to study the effect of selenocysteine substitutions on the oxidative folding of hirudin. Wildtype hirudin and its seleno-analogs containing one or two selenocysteine substitutions at different positions that could form native or nonnative diselenide bridges will be prepared using solid-phase peptide synthesis (SPPS) and native chemical ligation (NCL).