Chaperonin-Based Biolayer Interferometry To Assess the Kinetic Stability of Metastable, Aggregation-Prone Proteins
| Autor: | Alexandra J Machen, Mark T. Fisher, Wendy A. Lea, Karen R. Khar, Matthew Auton, Alexander Tischer, Pierce T. O’Neil, Subhashchandra Naik, Tapan Chaudhri, Wesley Mcginn-Straub, John Karanicolas, Joshua R. Burns, Michael R. Baldwin |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Protein Denaturation Protein Folding 030102 biochemistry & molecular biology Proteins Biosensing Techniques Chaperonin 60 Protein aggregation Ligands Ligand (biochemistry) Biochemistry GroEL Article Chaperonin Kinetics 03 medical and health sciences chemistry.chemical_compound Crystallography 030104 developmental biology Monomer chemistry Thermodynamics Denaturation (biochemistry) Protein folding Target protein |
| Zdroj: | Biochemistry. 55:4885-4908 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/acs.biochem.6b00293 |
| Popis: | Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy for decreasing disease pathologies caused by protein folding defects or deleterious kinetic transitions. Current methods of examining binding of a ligand to these marginally stable native states are limited because protein aggregation typically interferes with analysis. Here, we describe a rapid method for assessing the kinetic stability of folded proteins and monitoring the effects of ligand stabilization for both intrinsically stable proteins (monomers, oligomers, and multidomain proteins) and metastable proteins (e.g., low Tm) that uses a new GroEL chaperonin-based biolayer interferometry (BLI) denaturant pulse platform. A kinetically controlled denaturation isotherm is generated by exposing a target protein, immobilized on a BLI biosensor, to increasing denaturant concentrations (urea or GuHCl) in a pulsatile manner to induce partial or complete unfolding of the attached protein population. Following the rapid removal of the denaturant, the extent of hydrophobic unfolded/partially folded species that remains is detected by an increased level of GroEL binding. Because this kinetic denaturant pulse is brief, the amplitude of binding of GroEL to the immobilized protein depends on the duration of the exposure to the denaturant, the concentration of the denaturant, wash times, and the underlying protein unfolding-refolding kinetics; fixing all other parameters and plotting the GroEL binding amplitude versus denaturant pulse concentration result in a kinetically controlled denaturation isotherm. When folding osmolytes or stabilizing ligands are added to the immobilized target proteins before and during the denaturant pulse, the diminished population of unfolded/partially folded protein manifests as a decreased level of GroEL binding and/or a marked shift in these kinetically controlled denaturation profiles to higher denaturant concentrations. This particular platform approach can be used to identify small molecules and/or solution conditions that can stabilize or destabilize thermally stable proteins, multidomain proteins, oligomeric proteins, and, most importantly, aggregation-prone metastable proteins. |
| Databáze: | OpenAIRE |
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