title: Protein folding on biosensor tips: folding of maltodextrin glucosidase monitored by its interactions with GroEL.
abstract: Protein folding has been extensively studied for the past six decades by employing solution-based methods such as solubility, enzymatic activity, secondary structure analysis, and analytical methods like FRET, NMR, and HD exchange. However, for rapid analysis of the folding process, solution-based approaches are often plagued with aggregation side reactions resulting in poor yields. In this work, we demonstrate that a bio-layer interferometry (BLI) chaperonin detection system can identify superior refolding conditions for denatured proteins. The degree of immobilized protein folding as a function of time can be detected by monitoring the binding of the high-affinity nucleotide-free form of the chaperonin GroEL. GroEL preferentially interacts with proteins that have hydrophobic surfaces exposed in their unfolded or partially folded form, so a decrease in GroEL binding can be correlated with burial of hydrophobic surfaces as folding progresses. The magnitude of GroEL binding to the protein immobilized on bio-layer interferometry biosensor inversely reflects the extent of protein folding and hydrophobic residue burial. We demonstrate conditions where accelerated folding can be observed for the aggregation-prone protein maltodextrin glucosidase (MalZ). Superior immobilized folding conditions identified on the bio-layer interferometry biosensor surface were reproduced on Ni-NTA sepharose bead surfaces and resulted in significant improvement in folding yields of released MalZ (measured by enzymatic activity) compared to bulk refolding conditions in solution.
authors.affiliation: Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, India.
authors.forename: Ashutosh
authors.identifier: 0000-0002-9866-6753
authors.initials: A
authors.lastname: Pastor
authors.affiliation: Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, India.
authors.forename: Amit K
authors.initials: AK
authors.lastname: Singh
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Kansas Medical Centre, KS, USA.
authors.forename: Mark T
authors.initials: MT
authors.lastname: Fisher
authors.affiliation: Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, India.
authors.forename: Tapan K
authors.initials: TK
authors.lastname: Chaudhuri
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D018834:Chaperonin 60 / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D007368:Interferometry / Q000379:methods*; D011487:Protein Conformation; D058849:Protein Refolding; D013050:Spectrometry, Fluorescence; D013696:Temperature
chemical_list: D018834:Chaperonin 60; D055767:Immobilized Proteins; D018698:Glutamic Acid; D006026:Glycoside Hydrolases
keywords: Bio-layer interferometry; GroEL binding to monitor protein folding; immobilized protein folding; protein folding on biosensor tips