Name: Nuclear Magnetic Resonance to Study Atomic Level Protein-Protein Interactions
Uploaded: 2023-05-31T11:07:20
Description: Source: Winkelaar, G., et al. Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale ...

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1. NMR Methods
<ol>
	<li>NMR Sample Preparation
	<ol>
		<li>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 &#62;100 &#181;M using an MWCO 3 kDa, 5 mL centrifugal ultrafiltration device to bring the concentration into a suitable range for sample preparation.</li>
		<li>Purify a sample of unlabeled VimRod pr...

nuclear magnetic resonance to study atomic level protein-protein interactions

Source: Winkelaar, G., et al. <a href="https://app.jove.com/v/58537">Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST).</a> 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.

<img alt="Figure 1" src="/files/ftp_upload/21407/21407_Figure1.jpg" /> 
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...

Nuclear Magnetic Resonance to Study Atomic Level Protein-Protein Interactions

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

Nuclear magnetic resonance, or NMR, spectroscopy enables atomic-level protein-protein interaction detection.
To study interactions between globular and helical proteins, label purified wild-type or mutant globular proteins with 15N &#8212; a nitrogen isotope. In an NMR tube, mix the labeled globular and unlabeled helical proteins in a solvent containing an NMR standard. Insert the tube into the NMR spectrometer.
The spectrometer magnets generate a strong magnetic field, aligning the 15N-labeled globular protein nuclei parallel to the field. A radiofrequency, or RF, pulse excites the 15N-nuclei to align antiparallel to the field.
The pulse frequency required for the transition of 15N nuclei between the two states defines the resonance frequency. Normalizing the 15N-resonance frequency to the NMR standard measures the 15N-chemical shift.
The magnetic field is adjusted to align the 1H-nuclei &#8212; bonded to 15N &#8212; parallel to the field. The RF pulses are adjusted, and the 1H-chemical shift is recorded.
During protein interactions, the chemical environment surrounding the interacting amino acid residues changes, altering the 15N-1H chemical bonds and corresponding chemical shifts. Plot the 15N-1H NMR spectra.
Without helical proteins, wild-type globular proteins exhibit well-resolved spectral peaks. When helical and wild-type globular proteins interact, the spectral peak is reduced. In contrast, incubating helical proteins with mutant globular proteins results in the spectral peaks reappearing, indicating no interaction between them.

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Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-protein interactions

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1H NMR is shown for the purine-specific enzyme, while 19F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...

JoVE Journal

Chemistry

Monitoring Protein-Ligand Interactions in Human Cells by Real-Time Quantitative In-Cell NMR using a High Cell Density Bioreactor

In-cell NMR is a unique approach to observe the structural and dynamic properties of biological macromolecules at atomic resolution directly in living cells. Protein folding, chemical modifications, and conformational changes induced by ligand binding can be observed. Therefore, this method has great potential in the context of drug development. However, the short lifetime of human cells confined in the NMR spectrometer limits the application range of in-cell NMR. To overcome this issue, NMR bioreactors are employed that can greatly improve the cell sample stability over time and, importantly, enable the real-time recording of in-cell NMR spectra. In this way, the evolution of processes such as ligand penetration and binding to the intracellular protein target can be monitored in real time. Bioreactors are often limited by low cell viability at high cell numbers, which results in a trade-off between the overall sensitivity of the experiment and cell viability. We recently reported an NMR bioreactor that maintains a high number of human cells metabolically active for extended periods of time, up to 72 h. This setup was applied to monitor protein-ligand interactions and protein chemical modification. We also introduced a workflow for quantitative analysis of the real-time NMR data, based on multivariate curve resolution. The method provides concentration profiles of the chemical species present in the cells as a function of time, which can be further analyzed to obtain relevant kinetic parameters. Here we provide a detailed description of the NMR bioreactor setup and its application to monitoring protein-ligand interactions in human cells.

This protocol describes the setup of an NMR bioreactor to keep encapsulated human cells viable for up to 72 h, followed by time-resolved in-cell NMR data acquisition and analysis. The methodology is applied to monitor intracellular protein-ligand interactions in real time.

In-cell NMR is a unique approach to observe the structural and dynamic properties of biological macromolecules at atomic resolution directly in living ...

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic resonance (MR) microscopy data. While it is possible to perform MR collections on living, excised mammalian tissue, such experiments have traditionally been constrained by resolution limits and are thus incapable of visualizing tissue microstructure. Conversely, MR protocols that did achieve microscopic image resolution required the use of fixed samples to accommodate the need for static, unchanging conditions over lengthy scan times. The current protocol describes the first available MR technique that enables imaging of living, mammalian tissue samples at microscopic resolutions. Such data is of great importance to the understanding of how pathology-based contrast changes occurring at the microscopic level influence the content of macroscopic MR scans such as those used in the clinic. Once such an understanding is realized, diagnostic methods with greater sensitivity and accuracy can be developed, which will translate directly to earlier disease treatment, more accurate therapy monitoring and improved patient outcomes.
While the described methodology focuses on brain slice preparations, the protocol is adaptable to any excised tissue slice given that changes are made to the gas and perfusate preparations to accommodate the tissue&#39;s specific metabolic needs. Successful execution of the protocol should result in living, acute slice preparations that exhibit MR diffusion signal stability for periods up to 15.5 h. The primary advantages of the current system over other MR compatible perfusion apparatuses are its compatibility with the MR microscopy hardware required to attain higher resolution images and ability to provide constant, uninterrupted flow with carefully regulated perfusate conditions. Reduced sample throughput is a consideration with this design as only one tissue slice may be imaged at a time.

The current protocol describes a method by which users can maintain viability of acute hippocampal and cortical slice preparations during the collection of magnetic resonance microscopy data.

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic ...

Nuclear Magnetic Resonance to Study Atomic Level Protein-Protein Interactions

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