Abstract
This protocol describes how to measure protein-protein interactions by microscale thermophoresis (MST) using the MonolithTM NT.115 instrument (NanoTemper). We have used the protocol to determine the binding affinities between three different flavodoxin reductases (FNRs) and a flavodoxin-like protein, NrdI, from Bacillus cereus (Lofstad et al., 2016). NrdI is essential in the activation of the manganese-bound form of the class Ib ribonucleotide reductase (RNR) system. RNRs, in turn, are the only source of the de novo synthesis of deoxyribonucleotides required for DNA replication and repair in all living organisms.
Keywords: MST, Microscale thermophoresis, Protein-protein interaction, KD, Binding constant
Background
Protein-protein interactions are often characterised in terms of the associated dissociation constant, KD. The binding constant can be established using a variety of techniques, such as isothermal calorimetry (ITC), NMR spectroscopy, and surface plasmon resonance (SPR). An alternative method is based on thermophoresis, a phenomenon where distinct molecules (such as a protein-protein complex versus individual proteins) respond differently to a temperature gradient (Duhr and Braun, 2006; Seidel et al., 2013). This method is rapid, no sample immobilisation is needed and the sample requirement is low. Briefly, one of the proteins is labelled with a fluorescent dye and kept at a constant, low concentration. A dilution series is set up, where the other protein is diluted up to 16 times, creating a vast concentration range. The two proteins are subsequently mixed and loaded into capillaries, which are scanned in the MonolithTM NT.115 instrument, developed and sold exclusively by NanoTemper. The samples are subjected to a temperature gradient, and the movement of the fluorescently labelled molecule is tracked. The difference in the fluorescence of the molecule at the initial temperature and at the new temperature is used to generate a binding curve as a function of the concentration of the unlabelled protein.
Materials and Reagents
Equipment
Procedure
Data analysis
Notes
Recipes
Acknowledgments
This protocol is based on the user manual supplied with the MonolithTM NT.115 instrument (NanoTemper) and the FAQ: How do I determine the protein concentration after labeling and the degree of labeling (DOL)? (NanoTemper). This work is funded by the Norwegian Research Council (Projects 231669 and 214239). The MST instrument is operated with the financial support of the South-Eastern Norway Regional Health Authority (Grant 2015095; Regional Core Facility for Structural Biology). A brief description of this protocol has previously been published in Lofstad et al. (2016).
References
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