Development and Application of a High Throughput Protein Unfolding Kinetic Assay

Wang, Qiang; Waterhouse, Nicklas; Feyijinmi, O.; Dominguez, Matthew J.; Martinez, Lisa M.; Sharp, Zoey L.; Bothe, Jameson R.; Stollar, Elliott J.

doi:10.1371/journal.pone.0146232

Cited by 4 publications

(2 citation statements)

References 28 publications

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“…All experiments were performed in a POLARstar Omega plate reader (BMG labtech, NC, USA) with a Hellma 96 well quartz plate at 30°C. The assays were performed as previously described (Wang et al, 2016 ) although modified to avoid injecting denaturants into the wells. For the equilibrium assay, in each well, 50 μL of protein started in high concentrations of urea (between 6 and 10.5 M) in 10 mM Tris, pH 8.1 (+salt) and were refolded by injection (dilution) using the same buffer, without urea.…”

Section: Methodsmentioning

confidence: 99%

How a highly acidic SH3 domain folds in the absence of its charged peptide target

et al. 2023

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Charged residues on the surface of proteins are critical for both protein stability and interactions. However, many proteins contain binding regions with a high net charge that may destabilize the protein but are useful for binding to oppositely charged targets. We hypothesized that these domains would be marginally stable, as electrostatic repulsion would compete with favorable hydrophobic collapse during folding. Furthermore, by increasing the salt concentration, we predict that these protein folds would be stabilized by mimicking some of the favorable electrostatic interactions that take place during target binding. We varied the salt and urea concentrations to probe the contributions of electrostatic and hydrophobic interactions for the folding of the yeast SH3 domain found in Abp1p. The SH3 domain was significantly stabilized with increased salt concentrations due to Debye–Huckel screening and a nonspecific territorial ion‐binding effect. Molecular dynamics and NMR show that sodium ions interact with all 15 acidic residues but do little to change backbone dynamics or overall structure. Folding kinetics experiments show that the addition of urea or salt primarily affects the folding rate, indicating that almost all the hydrophobic collapse and electrostatic repulsion occur in the transition state. After the transition state formation, modest yet favorable short‐range salt bridges are formed along with hydrogen bonds, as the native state fully folds. Thus, hydrophobic collapse offsets electrostatic repulsion to ensure this highly charged binding domain can still fold and be ready to bind to its charged peptide targets, a property that is likely evolutionarily conserved over 1 billion years.

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Section: Methodsmentioning

confidence: 99%

How a highly acidic SH3 domain folds in the absence of its charged peptide target

et al. 2023

Self Cite

View full text Add to dashboard Cite

show abstract

“…All experiments were performed in a POLARstar Omega plate reader (BMG labtech, NC, USA) with a Hellma 96 well quartz plate at 30 °C. The assays were performed as previously described(36) although modified to avoid injecting denaturants into the wells. For the equilibrium assay, in each well, 50 µL of protein started in high concentrations of urea (between 6 and 10.5 M) in 10 mM Tris, pH 8.1 (+ salt) and were refolded by injection (dilution) using the same buffer, without urea.…”

mentioning

confidence: 99%

How a highly acidic SH3 domain folds in the absence of its charged peptide target

Martinez

Dominguez

Bell

et al. 2023

Preprint

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Charged residues on the surface of proteins are critical for both protein stability and interactions. However, many proteins contain binding regions with a high net-charge that may destabilize the protein but are useful for binding to oppositely charged targets. We hypothesized that these domains would be marginally stable, as electrostatic repulsion would compete with favorable hydrophobic collapse during folding. Furthermore, by increasing the salt concentration we predict that these protein folds would be stabilized by mimicking some of the favorable electrostatic interactions that take place during target binding. We varied the salt and urea concentrations to probe the contributions of electrostatic and hydrophobic interactions for the folding of the 60-residue yeast SH3 domain found in Abp1p. The SH3 domain was significantly stabilized with increased salt concentrations according to the Debye-Huckel limiting law. Molecular dynamics and NMR show that sodium ions interact with all 15 acidic residues but do little to change backbone dynamics or overall structure. Folding kinetics experiments show that the addition of urea or salt primarily affects the folding rate, indicating that almost all the hydrophobic collapse and electrostatic repulsion occurs in the transition state. After the transition state formation, modest yet favorable short-range salt-bridges are formed along with hydrogen bonds, as the native state fully folds. Thus, hydrophobic collapse offsets electrostatic repulsion to ensure this highly charged binding domain can still fold and be ready to bind to its charged peptide targets, a property that is likely evolutionarily conserved over one billion years.

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A multi-column plate adapter provides an economical and versatile high-throughput protein purification system

Dominguez

Lantz

Rhode

et al. 2018

Protein Expression and Purification

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Protein purification is essential in the study of protein structure and function, and the development of novel therapeutics. Many studies require purifying multiple proteins at once, increasing the demand for improved purification methods. We hypothesized that multiple chromatography columns could be interfaced with a multi-well collection plate for rapid and convenient protein purification without the need of expensive instrumentation. As such, we developed a multi-column plate adapter (MCPA), which provides an economical yet versatile and time efficient, high-throughput protein purification system. The MCPA system simultaneously purified milligrams of different proteins under gravity or under vacuum for faster purification. The MCPA handles up to twenty-four 12 mL columns and multiple MCPA's in sequence allow milligram-scale purification of 96 different samples with relative ease. We also used the MCPA system for large scale affinity purification of four proteins, providing sufficient yields and purity for protein crystallization and biophysical characterization. The MCPA system is ideal for optimizing resin type and volume or any other purification parameter by customizing individual columns during the same purification. The high-throughput and versatile nature of this system should prove to be useful in obtaining adequate amounts of protein for subsequent analyses in any laboratory setting.

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Development and Application of a High Throughput Protein Unfolding Kinetic Assay

Cited by 4 publications

References 28 publications

How a highly acidic SH3 domain folds in the absence of its charged peptide target

How a highly acidic SH3 domain folds in the absence of its charged peptide target

How a highly acidic SH3 domain folds in the absence of its charged peptide target

A multi-column plate adapter provides an economical and versatile high-throughput protein purification system

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