Nuclear Magnetic Resonance to Study Atomic Level Protein-Protein Interactions

Abstract

Source: Winkelaar, G., et al. Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST). J. Vis. Exp. (2018).

This video describes the nuclear magnetic resonance spectroscopy technique to study protein-protein interactions between ¹⁵N-labeled wild-type and mutant envoplakin proteins and the unlabeled vimentin protein. The successful interaction between wild-type envoplakin and vimentin leads to extensive line broadening and peak disappearance in the NMR spectra, whereas the absence of an interaction between the mutated envoplakin and vimentin results in well-resolved peaks in the NMR spectra.

Protocol

1. NMR Methods NMR Sample Preparation Purify 15N-labeled wild-type or R1914E E-PRD protein using 20 mM Tris-HCl, 1 mM DTT, pH 7. Protein stock solutions usually range from 0.3 to 1 mM with volumes of about 1 mL. NOTE: Protein can be concentrated to >100 µM using an MWCO 3 kDa, 5 mL centrifugal ultrafiltration device to bring the concentration into a suitable range for sample preparation. Purify a sample of unlabeled VimRod pr…

Representative Results

Figure 1: Screen Capture of the Setup of the NMR Experiment. The window shown is used to set up a standard experiment to collect an HSQC dataset. Experiment parameters are read adjacent to Experiment. The ZGPR experiment shown is chosen as an initial experiment to load the standard and solvent-dependent proton parameters. The Title window is used to input experimental details for record-keeping p…

Materials

Ammonium chloride (15N, 99%)	Cambridge Isotope Laboratories	NLM-467	Isotope labelling for NMR
D2O, Deuterium Oxide (D, 99.8%)	Cambridge Isotope Laboratories	DLM-2259	NMR sample preparation
DSS, Sodium 2,2-dimethyl-2- silapentane-5-sulfonate-D6 (D,98%)	Cambridge Isotope Laboratories	DLM-8206	Reference for NMR
Precision 5 mm NMR Tubes, 7” long	SJM/Deuterotubes	BOROECO-5-7	NMR tubes
NMR spectrometer (14.1 Tesla)	Bruker		Acquisition of NMR data
TCI 5mm z-PFG cryogenic probe	Bruker		Acquisition of NMR data
Software
Bruker TopSpin 4.0.1	Bruker		Processing of NMR data