Structural Proteomics
Protein structure determination by hydrogen deuterium exchange (HDX) and cross-linkers
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Hydrogen deuterium exchange mass spectrometry (HDX-MS) is used to measure the exchange rate of amide hydrogens with deuterated solvent. The exchange rate of amide hydrogens is exquisitely sensitive to changes in secondary structure and solvent accessibility, and this technique has been used extensively to characterize protein folding, as well as protein-protein, protein-lipid, and protein-small molecule binding sites. Building on our >20 year history of using this technique with >80 publications using HDX-MS to study almost all aspects of protein dynamics we are now providing this service to interested academic and industrial partners.
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HDX-MS experiments can be carried out with a variety of stimuli, including protein binding partners, ligands, and membrane surfaces. Importantly, HDX-MS provides information on allosteric conformational changes. Our laboratory has made significant progress in the use of HDX-MS to study membrane localized signalling complexes that have been challenging to study by other structural approaches. We provide epitope mapping of antibody binding sites, and mapping of protein-protein interactions. We also provide screening of oncogenic specific drug binding interfaces.
Validation of Protein Dynamics
When a protein binds to another protein, the interface between the two proteins becomes more rigid and stable. This is because the interaction between the two proteins reduces the flexibility of the interface region, making it less susceptible to exchange with deuterium.
By comparing the extent of deuterium exchange in the interface region of a protein bound to its partner protein with that of the unbound protein, we can identify the residues involved in the interaction, as well as regions which have allosteric changes upon binding, and can validate the protein-protein interface.
Alphafold, a deep learning algorithm developed by researchers at the University of Cambridge, has revolutionized the field of biochemistry by significantly improving our ability to predict the three-dimensional structure of proteins. Before Alphafold, predicting protein structure was a time-consuming and error-prone process, but Alphafold has shown remarkable accuracy and speed in predicting protein structure, making it a valuable tool for researchers in biochemistry and related fields.
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However, the accuracy of Alphafold's predictions is not perfect and it may still produce errors, which could lead to misinterpretations and incorrect conclusions if not carefully validated by experimental methods. We can validate Alphafold structural predictions using HDX. This technique is particularly useful at studying disordered region interfaces.
Epitope Mapping of Antibodies
When an antibody binds to an antigen, it interacts with specific regions on the surface of the antigen known as epitopes. The binding of the antibody to the antigen can cause changes in the conformation and dynamics of the epitope, which can be detected by differences in the extent of deuterium exchange.
By comparing the deuterium exchange patterns in the epitope region of the antigen in the unbound and bound states, we can can identify the residues involved in the antibody-antigen interaction and map the antibody epitopes. This technique allows for rapid screening of multiple antibodies.
Small Molecule and Substrate Binding
Hydrogen deuterium exchange (HDX) is a very useful technique for studying the interactions between small molecule inhibitors and their target proteins. By measuring the rate of exchange of hydrogen and deuterium atoms in the protein before and after exposure to the inhibitor, we can identify the specific regions of the protein that are affected by the inhibitor binding.
This information can be used to optimize the inhibitor design, by identifying the key amino acid residues that interact with the inhibitor and designing modifications to improve binding affinity and specificity. HDX can also provide insights into the mechanism of inhibition, by revealing how the inhibitor affects the dynamics and conformational changes of the protein.
HDX-MS can also provide insights into the structural changes and dynamic behavior of proteins upon interaction with lipid membrane vesicles. This information can be used to identify the specific regions of the protein that interact with the membrane, as well as identify allosteric changes which occur upon lipid binding.