Assessing Bacterial Chaperone Activity via Thermal Unfolding of a Model Protein

Abstract

Source: Dahl, J. et al. Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo. J. Vis. Exp. (2016)

This video demonstrates a fluorescence spectrophotometer-based assay to evaluate chaperone activity on a substrate protein under acid and heat stress. It outlines the steps for monitoring substrate unfolding and aggregation through light scattering, comparing conditions with and without the chaperone.

Protocol

1. Chaperone Activity Assay Using Thermally Unfolding Malate Dehydrogenase (MDH)

NOTE: The influence of purified HdeB on the aggregation of thermally unfolding porcine mitochondrial malate dehydrogenase (MDH) at different pH values was monitored as described below. All listed protein concentrations refer to the monomer concentration.

1. To prepare MDH, dialyze MDH at 4 °C overnight against 4 L buffer C (50 mM potassium phosphate, 50 mM sodium chloride (NaCl), pH 7.5) and concentrate the protein to approximately 100 µM using centrifugal filter units with a molecular weight cut-off of 30 kDa.
   

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Malate dehydrogenase (MDH)	Roche	10127914001
Potassium phosphate (Monobasic)	Fisher Scientific	BP362-500
Potassium phosphate (Dibasic)	Fisher Scientific	BP363-1
F-4500 fluorescence spectrophotometer	Hitachi	FL25
Fluorescence Cell cuvette	Hellma Analytics	119004F-10-40
Hydrochloric Acid	Fisher Scientific	A144-212
Sodium Hydroxide	Fisher Scientific	BP359-500
Amicon Ultra Centrifugal Filter Units 30K	Millipore	UFC803024
Sodium chloride	Fisher Scientific	S271-10
Spectra/Por 1 Dialysis Membrane MWCO: 6 kDa	Spectrum Laboratories	132650
Varian Cary 50 spectrophotometer	Agilent Tech