Quantifying the Kinetic Stability of Hyperstable Proteins via Time-Dependent SDS Trapping

Abstract

Globular proteins are usually in equilibrium with unfolded conformations, whereas kinetically stable proteins (KSPs) are conformationally trapped by their high unfolding transition state energy. Kinetic stability (KS) could allow proteins to maintain their activity under harsh conditions, increase a protein's half-life, or protect against misfolding-aggregation. Here we show the development of a simple method for quantifying a protein's KS that involves incubating a protein in SDS at high temperature as a function of time, running the unheated samples on SDS-PAGE, and quantifying the bands to determine the time-dependent loss of a protein's SDS resistance. Six diverse proteins, including two monomer, two dimers, and two tetramers, were studied by this method, and the kinetics of the loss of SDS resistance correlated linearly with their unfolding rate determined by circular dichroism. These results imply that the mechanism by which SDS denatures proteins involves conformational trapping, with a trapping rate that is determined and limited by the rate of protein unfolding. We applied the SDS trapping of proteins (S-TraP) method to superoxide dismutase (SOD) and transthyretin (TTR), which are highly KSPs with native unfolding rates that are difficult to measure by conventional spectroscopic methods. A combination of S-TraP experiments between 75 and 90 °C combined with Eyring plot analysis yielded an unfolding half-life of 70 ± 37 and 18 ± 6 days at 37 °C for SOD and TTR, respectively. The S-TraP method shown here is extremely accessible, sample-efficient, cost-effective, compatible with impure or complex samples, and will be useful for exploring the biological and pathological roles of kinetic stability.

Transthyretin as both a Sensor and a Scavenger of β-Amyloid Oligomers

“…This method prevents re-association or aggregation of dissociated monomer. Dissociation rate constants k d for wt and L82A were indistinguishable (Table 1), and the rate constant for wt is consistent with published data (19). Thus, the lack of tetramer dissociation for L82A incubated with Aβ is not due to an increase in L82A stability relative to wt.…”

“…We previously showed that L110A and L82A mutations did not cause a loss of secondary, tertiary, or quaternary structure in TTR (13). We tested whether these mutations caused a more subtle effect on tetramer stability by measuring dissociation at high temperature using the S-Trap method (19). This method prevents re-association or aggregation of dissociated monomer.…”

Proteins in a brave new surfactant world

“…This is also illustrated very well in a more recent study by the Colón group 13 . Hyperstable proteins such as superoxide dismutase or transthyretin are incubated with SDS at elevated temperatures such as 70 o C, and samples can then be removed and monitored by SDS-PAGE which can also quantify the amount of folded and unfolded protein.…”

Increased levels of hyper-stable protein aggregates in plasma of older adults

“…We have shown that hyper-stable (i.e., kinetically stable) proteins are resistant to SDS (Manning and Colon 2004;Xia et al 2012) and have developed a diagonal two-dimensional (D2D) SDS-PAGE method to identify such proteins at a proteomics level (Xia et al 2007). In this study, we used human plasma as a model system and carried out D2D SDS-PAGE to determine whether the plasma of older age adults contains higher amounts of SDS-resistant aggregates (SRA) than the plasma of younger adults.…”