Development and application of gel electrophoresis methods for assaying protein kinetic stability

Abstract

Aside from the functional roles of the hyperstable proteins in these studies, bioinformatic analysis was performed on the identified KSPs to further elucidate structural trends common to the class of proteins. KSPs showed a preference for high oligomeric states and domains with mixed α/β secondary structure. Particularly, the 3-layer αβα sandwich architecture Rossmann fold was favored, potentially conveying stability through a complex topology and increased number of long-range contacts. Continued method development, identification, and analysis of hyperstable proteins would not only advance the understanding of protein kinetic stability but may provide information applicable to science and engineering endeavors with medical and biotechnological relevance.

The gel electrophoresis method D2D SDS-PAGE further exploits SDS-resistance and is coupled with matrix assisted laser desorption/ionization – time of flight mass spectrometry (MALDI-TOF MS) to explore the identity of KSPs in biological lysates. D2D SDS-PAGE was applied to cytoplasmic lysates from the bacterial pathogen model systems Pseudomonas aeruginosa and Staphylococcus aureus. While many of the identified KSPs possess roles commonly associated with kinetic stability, including stress response and primary metabolic functions, additional KSPs identified from P. aeruginosa are involved in toxin production and in conveying antibiotic resistance. A separate study explored the KSP profiles of the Bacteroides species, B. fragilis and B. thetaiotaomicron, common nanoaerobic members of the human gut microbiota. Through D2D SDS-PAGE visualization and MALDI-TOF MS, these bacteria were found to express the KSP, superoxide dismutase, at extremely high concentrations and contained several other KSPs involved in maintaining cellular redox homeostasis. It appears that these nanoaerobic bacteria are constantly primed to combat oxidative stress to protect themselves from damage and possibly to shield less aerotolerant organisms in the microbiota.

SDS is a highly denaturing detergent and only allows for the stability analysis of extremely stable proteins. In attempts to broaden the range of stabilities that can be studied, SDS was replaced throughout the B/U SDS-PAGE assay with alternative detergents. It was found that the anionic surfactant, sarkosyl, acts as a milder denaturant than SDS allowing the evaluation of proteins that are not quite resistant to SDS but still possess elevated stabilities. For initial testing, sarkosyl B/U gels were run on a series of legume lysates, as legumes are known to possess highly expressed hyperstable proteins. While the dichotomous result of resistant or susceptible to sarkosyl provided significant insight into the stabilities of the proteins, PAGE gels were also run with varying ratios of sarkosyl and SDS to further evaluate proteins within the range of sarkosyl and SDS susceptibility. Analysis of the data resulted in unfolding transition profiles that semi-quantitatively categorized the stability of proteins in each lysate.

S-TraP provides a quantitative view of protein stability and has previously been performed with purified, commercial proteins. The work here shows application to complex samples proving the accessibility of S-TraP analysis and its ability to be performed on highly abundant proteins without purification. The KSP phaseolin is both highly stable and highly expressed in many legumes making it an ideal candidate. Lysates of navy bean, small red bean, black bean, and Lima bean were analyzed using S-TraP to quantify the stability of the phaseolin protein in each sample. Extrapolation to physiological temperatures show half-lives on the order of decades, with the highest stability belonging to Lima bean phaseolin with a half-life of 101 years at 24 °C. Such an elongated lifespan may contribute to the long shelf-lives of many legumes as well as issues involving dietary legume digestibility.

While most proteins are susceptible to denaturants, detergents, and proteases, hyperstable or kinetically stable proteins (KSPs) are highly resistant to unfolding and degradation. Of particular interest, KSPs are resistant to denaturation induced by the detergent, sodium dodecyl sulfate (SDS). The correlation between protein stability and SDS-resistance has led to the development of several gel electrophoretic methods for assaying protein kinetic stability by the Colón laboratory. Boiled/Unboiled (B/U) SDS-polyacrylamide gel electrophoresis (PAGE) provides a binary confirmation as to whether a protein possesses elevated kinetic stability, SDS-Trapping of Proteins (S-TraP) quantifies the stability of proteins, and Diagonal Two-Dimensional (D2D) SDS-PAGE enables proteome-scale isolation of KSPs from complex lysates. Presented here is the continued development and application of these methods using both plant and bacterial samples, as well as bioinformatic analysis of the systems examined.