Identifying the subproteome of kinetically stable proteins via diagonal 2D SDS/PAGE

Abstract

Most proteins are in equilibrium with partially and globally unfolded conformations. In contrast, kinetically stable proteins (KSPs) are trapped by an energy barrier in a specific state, unable to transiently sample other conformations. Among many potential roles, it appears that kinetic stability (KS) is a feature used by nature to allow proteins to maintain activity under harsh conditions and to preserve the structure of proteins that are prone to misfolding. The biological and pathological significance of KS remains poorly understood because of the lack of simple experimental methods to identify this property and its infrequent occurrence in proteins. Based on our previous correlation between KS and a protein's resistance to the denaturing detergent SDS, we show here the application of a diagonal 2D (D2D) SDS/PAGE assay to identify KSPs in complex mixtures. We applied this method to the lysate of Escherichia coli and upon proteomics analysis have identified 50 nonredundant proteins that were SDS-resistant (i.e., kinetically stable). Structural and functional analyses of a subset (44) of these proteins with known 3D structure revealed some potential structural and functional biases toward and against KS. This simple D2D SDS/PAGE assay will allow the widespread investigation of KS, including the proteomics-level identification of KSPs in different systems, potentially leading to a better understanding of the biological and pathological significance of this intriguing property of proteins.electrophoresis ͉ kinetic trap ͉ proteomics ͉ sodium dodecyl sulfate ͉ stability K inetic stability (KS) refers to the presence of an unusually high energy barrier that traps proteins in their native conformations, thereby dramatically decreasing their unfolding rate under a variety of conditions (1). This barrier allows kinetically stable proteins (KSPs) to maintain their native fold and activity for longer periods of time than would be expected for typical proteins, even in harsh environments. This unique feature is likely to play important biological roles, including the regulation of protein turnover, the protection of some proteins from proteolysis (1), and the entrapment of aggregation-prone proteins in their native state (2). Perhaps the best examples to illustrate the importance of KS are the proteins ␣-lytic protease and transthyretin (TTR). ␣-Lytic protease is synthesized with a pro-region that is essential for folding (1). Upon folding, ␣-lytic protease becomes active and cleaves off the pro region, leaving itself kinetically trapped in an active metastable conformation that is less stable than the unfolded state (3). In the case of TTR, the high thermodynamic and kinetic stability (KS) of the native tetramer is often compromised by mutation, leading to the population of a monomeric conformation that is the precursor of the aggregated species linked to the disease familial amyloid polyneuropathy (4). The large difference in the thermodynamic stability of ␣-lytic protease and TTR shows that KS need not correlate to th...

Identifying kinetically stable proteins with capillary electrophoresis

“…Because of the known correlation between SDSresistance and KS, 5,6 the SDS-PAGE boil-unboil assay was carried out to confirm the KS or lack thereof of the eight control proteins chosen for this study. As expected, the non-KSPs a-chymotrypsin (CHT), glucose dehydrogenase (GD), concanavalin A (ConA), and myoglobin (MYO) were denatured by SDS even when the samples were not heated, resulting in identical migration on the gel as the respective samples that were boiled [ Fig.…”

“…5 Here, we show a simple method to qualitatively identify KSPs by capillary zone electrophoresis (CE). In contrast to SDS-PAGE and capillary gel electrophoresis, in which the mobility of proteins depends on both their charge-to-mass ratio and their size, in CE the separation of proteins is determined only by their charge-to-mass ratio.…”

Designed protein reveals structural determinants of extreme kinetic stability

“…Protease resistance of a classic extremely kinetically stable protein, α-lytic protease, has been proposed to originate from its large and highly cooperative unfolding energy barrier resulting in a rigid native conformation with limited local openings and consequently limited proteolytically susceptible regions (35). Also, challenge by a high concentration of SDS has been used extensively for direct evaluation of protein kinetic stability based on the ability of SDS to induce denaturation by trapping hydrophobic residues exposed during even transient unfolding events (47,48). Given its high barrier to unfolding, we tested ThreeFoil for resistance to protease and SDS.…”

“…Extremely slow unfolding has been associated with the capacity to maintain native form and function under harsh conditions (44), such as high concentrations of protease (35,45,46) and detergent (46,47). Protease resistance of a classic extremely kinetically stable protein, α-lytic protease, has been proposed to originate from its large and highly cooperative unfolding energy barrier resulting in a rigid native conformation with limited local openings and consequently limited proteolytically susceptible regions (35).…”

“…The identified proteins include new (SI Appendix, Fig. S10) and previously reported (45,47) results from whole proteome screening to identify kinetically stable proteins, as well as new (Fig. 5 A and B and SI Appendix, Fig.…”