title: Chaperonin-Based Biolayer Interferometry To Assess the Kinetic Stability of Metastable, Aggregation-Prone Proteins.
abstract: 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.
authors: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center , Kansas City, Kansas 66160, United States.
authors: Wendy A
authors: WA
authors: Lea
authors: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center , Kansas City, Kansas 66160, United States.
authors: Pierce T
authors: PT
authors: O'Neil
authors: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center , Kansas City, Kansas 66160, United States.
authors: Alexandra J
authors: AJ
authors: Machen
authors: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center , Kansas City, Kansas 66160, United States.
authors: Subhashchandra
authors: S
authors: Naik
authors: Indian Institute of Technology , Delhi, India.
authors: Tapan
authors: T
authors: Chaudhri
authors: fortéBIO (a division of Pall Life Sciences) , Menlo Park, California 94025, United States.
authors: Wesley
authors: W
authors: McGinn-Straub
authors: Division of Hematology, Department of Internal Medicine, Mayo Clinic , Rochester, Minnesota 55902, United States.
authors: Alexander
authors: A
authors: Tischer
authors: Division of Hematology, Department of Internal Medicine, Mayo Clinic , Rochester, Minnesota 55902, United States.
authors: Matthew T
authors: MT
authors: Auton
authors: Department of Molecular Microbiology and Immunology, University of Missouri , Columbia, Missouri 65212, United States.
authors: Joshua R
authors: JR
authors: Burns
authors: Department of Molecular Microbiology and Immunology, University of Missouri , Columbia, Missouri 65212, United States.
authors: Michael R
authors: MR
authors: Baldwin
authors: Center for Computational Biology and Department of Molecular Biosciences, University of Kansas , Lawrence, Kansas 66045, United States.
authors: Karen R
authors: KR
authors: Khar
authors: Center for Computational Biology and Department of Molecular Biosciences, University of Kansas , Lawrence, Kansas 66045, United States.
authors: John
authors: J
authors: Karanicolas
authors: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center , Kansas City, Kansas 66160, United States.
authors: Mark T
authors: MT
authors: Fisher
mesh_terms: D015374:Biosensing Techniques; D018834:Chaperonin 60 / Q000737:chemistry*; D007700:Kinetics; D008024:Ligands; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D018834:Chaperonin 60; D008024:Ligands; D011506:Proteins