Characterization of Protein-Protein Interactions

The interaction between two or more proteins (PPI) is the core of most biological processes. These interactions have been proven to be very diverse in thermodynamics, dynamics, structure, and regulation. Nuclear magnetic resonance (NMR) is a powerful technique for studying protein-protein interaction in solution. NMR spectroscopy has many valuable uses as a tool for studying PPI.

As an expert in the field of nuclear magnetic resonance, Creative Biostructure provides characterization services of protein interactions for our customers. We can provide customers with structural, kinetic, and thermodynamic information from NMR measurements of protein-protein interactions. The characterization of these interactions not only contributes to our understanding of biology, but also increasingly helps to design molecules that regulate protein-protein interaction (PPI).

Why Use NMR Spectroscopy

Nuclear magnetic resonance can,

• Provide specific information about the physical properties of a single functional group, such as ionization state, pKa, and hydrogen bond.
• Provide specific position information of dynamic motion of skeleton and side chain in solution.
• It is used to determine the contact between a single atom of a protein and its binding partner, and to study the dynamics of ligand binding.

Our Methods

Chemical shift perturbations (CSPs) analysis

Nuclear Overhauser Effects (NOEs)

Residual Dipolar Couplings (RDCs)

Paramagnetic Restraint

Solid-state NMR

Low-abundance species analysis

Relying on the above technologies. We can help our customers to deepen their understanding of the mechanism of protein-protein interactions.

Binding Site Analysis Services of PPI

Due to the changes in the physical and chemical environment of the protein interface, the formation of a protein complex (or any other chemical exchange process) will cause chemical shift perturbations (CSP), so chemical shift mapping is a sensitive indicator of protein binding sites. We provide a variety of analysis services.

Analysis service based on ¹⁵N-HQSC

We can use ¹⁵N-HSQC technology to complete PPI detection experiments for customers. From the ¹⁵N-HSQC titration experiment, a large amount of information about binding affinity and kinetics can be collected, which will form the basis for further analysis of the complex. The features of ¹⁵N-HSQC technology include,

• High sensitivity and good signal dispersion.
• Simplicity and economy of 15N marking.
• Good sampling of the interaction surface.

Analysis of PPIs involving large protein complexes

For large protein complexes, we use site-specific ¹³C-labeling of metal-bearing and aromatic side chains to label protein interaction sites. For large single peptides and large multi-subunit complexes, we use the "divide-and-conquer" strategy to allocate smaller units through traditional methods and then transfer the allocation to larger combinations. Then use ¹H/¹³C CSPs to draw the interaction diagram with the partner/substrate protein.

Analysis of complex PPIs

We can also reveal more complex protein-protein interactions through CSP analysis, such as

• There are two or more combining points in fast exchange.
• The collection of specific complex and low-affinity non-specific complex in exchange.
• A compound exchanged between two conformational states in a rapid exchange.

Structural and Kinetic Analysis Service of PPI

Nuclear Overhauser effects analysis service

One of the most valuable sources of structural information used to define the binding interface is the nuclear Overhauser effect. We can provide "Straight" NOE and Transferred NOEs (trNOE) analysis services.

Figure 1. Isotope selecting and editing schemes for NOE /cross-saturation experiments (Gell et al., 2018)

Residual dipolar couplings analysis service

For protein-protein complexes, RDCs can produce the relative orientation of a single subunit. This long-range information provides a valuable supplement to the local distance information provided by NOEs and CSPs.

Paramagnetic restraints analysis service

Figure 2. Schematic representation of RDC, PRE, and PCS experiments (Gell et al., 2018)

Provide various long-distance (up to 35 Å) and directional constraints. Here, we focus on the structural analysis of protein complexes using paramagnetic relaxation enhancement (PRE) and pseudo-contact displacement (PCS).

Creative Biostructure is committed to providing high-quality NMR analysis services to advance the life sciences fields. If you have any questions or needs, please contact us and our customer service staff will help you the first time.

Ordering Process

Reference

1. Gell D A, Mackay J P. NMR spectroscopy in the analysis of protein-protein interactions. Modern Magnetic Resonance. 2006: 1339-1346.