Identifying kinetically stable proteins with capillary electrophoresis

Abstract

Unlike most proteins, which are in equilibrium with partially and globally unfolded conformations, kinetically stable proteins (KSPs) are trapped in their native conformations and are often resistant to harsh environment. Based on a previous correlation between kinetic stability (KS) and a protein's resistance to sodium dodecyl sulfate (SDS), we show here a simple method to identify KSPs by SDS-capillary electrophoresis (CE). Control non-KSPs were fully denatured by SDS and formed protein:SDS complexes that exhibited similar mobility in CE. In contrast, KSPs bound fewer SDS molecules, and showed a very different migration time and peak pattern in CE, thereby providing some insight about the structural heterogeneity of SDS:protein complexes and the relative KS of the various proteins.

Figure 1

SDS-PAGE of (A) non-KSP and (B) KSP samples that were unboiled (U) or boiled (B) in 1% SDS. The non-KSPs α-chymotrypsin (CHT), glucose dehydrogenase (GD), concanavalin A (ConA), and myoglobin (MYO) showed no migration difference before and after boiling. The unboiled KSPs glucose oxidase (GO), streptavidin (SVD), superoxide dismutase (SOD), and subtilisin carlsberg (SCA) migrated less than their boiled counterparts. None of the samples contained reducing agent to avoid disrupting the native disulfide bonds of GO and SOD, as this might compromise their KS. Therefore, boiled GO and SOD migrated as a dimer due to non-native intermolecular disulfide bonds.

Figure 2

Electropherograms showing the migration of unboiled and boiled samples of non-KSPs (A–D) and KSPs (E–H). Black solid lines and dash lines represent the data of samples incubated in SDS that were not boiled or boiled, respectively. Samples were incubated in 20 mM sodium phosphate buffer (pH 7.4) containing 1% (w/v) SDS for 10 min. The electropherograms of unboiled and boiled non-KSPs showed little difference, but unboiled KSPs had significantly faster migration.