The HDX field is ever growing in terms of the number of applications. It is used in the field of protein structure elucidation where it can be used to complement other techniques such as NMR and x-ray crystallography. One area where HDX is heavily used is in drug development research, as well as biopharmaceutical manufacturing. In its purest form, biologists can use HDX to help elucidate protein structure such as, understanding the three dimensionality and dynamic nature of disordered structures. >
HDX applications are also as useful for understanding the dynamics of proteins such as those found in membrane bound receptor assemblies. Furthermore, HDX is also useful in examining proteinaceous or non-proteinaceous covalent bonding. Examples of this are, mapping out locations of disulfide bridges of cysteine residues and also revealing sites of post translational modifications such as glycosylation events.
A very exciting field of HDX is understanding the associations of proteins of interest with various proteinaceous and non-proteinaceous ligands. For example, as discussed in the first blog in this series, the hormone insulin initiates signal transduction by binding to its receptor in doing so causes specific intracellular molecular cascades. These include activating machinery for the glycogen pathway, as well as signalling changes in conformation of both GLUT4 and GLUT1 transporters - allowing glucose to enter the cell . Just in this one interaction alone, there are several fascinating opportunities for HDX analysis. However, one key opportunity is understanding where insulin binds on the receptor. As many drug molecules act like hormones by interacting with receptors, understanding binding sites interactions, affords us the opportunity to design more efficacious drugs to target diseases such as diabetes.
It is not just the ligand protein interactions of small molecules (such as hormones and drugs) which are interesting targets for HDX analysis but HDX is ideal for large molecule interactions too These include for example, formation of histone and nucleosome complexes [ii]. Moreover, a key application for HDX is epitope mapping which has applications both in vaccine research but also in the design of effective monoclonal antibody therapies. Knowing where antibodies bind to proteins is critical to understanding their function. For example, during the Covid-19 pandemic, effort was made to understand the role of neutralising antibodies. Such antibodies targeted the receptor binding domain of the SAS-CoV-2 spike protein preventing the virus attaching to the angiotensin-converting enzyme 2 (ACE2) receptor on various cells including those of the lungs iii.
Below are a list of the growing number of applications for HDX and a short description for each. Over the coming HDX blogs, we will dive deeper into a number of these applications and how they are contributing not only to protein science but also medical research.
HDX is used to observe the activity of a target ligand in the presence and absence of a candidate drug. Furthermore HDX allows us to Identify allosteric effects and pathways through which different candidates work and targets respond. In drug discovery, the use of HDX can improve the efficiency with which drug candidates are selected. Another example is identification of drug chaperones for crossing the blood-brain barrier.
Identifying how two or more proteins interact in the biological state is an excellent use of HDX. It is valuable for investigating the mechanisms of protein misfolding and aggregation that cause diseases such as Cystic Fibrosis, Gaucher, Diabetes and amyotrophic lateral sclerosis (ALS). HDX can also be used to further understand processes in the body that are regulated by the presence or absence of functionally active proteins.
HDX is used to identifying the binding site, or "epitope", of an antibody on its target antigen, or the paratope of the antibody. This for example is used in vaccine development and also valuable in the production of data to support IP protection in the form of a recognized sequence, mapped binding sites and defined interaction pathways.
Many proteins a ‘well behaved’ in solution with respect to the fact that stable 3D structures can be successfully mapped by a number of techniques including NMR, X-ray crystallography and HDX. However, there are a class of proteins called Intrinsically disordered proteins (IDPs) which are highly dynamic in solution. As a result, this can make analysis by NMR and X-ray crystallography problematic and sometimes impossible. The reason is that the relatively high concentration needed for effective measurement for NMR affects the stability of the IDPs. Furthermore, the dynamic nature of these molecules makes protein crystallisation a challenge. HDX on the other hand is not impacted in the same way and due to the fact that HDX can operate temporally at as low as 100s of milliseconds, mapping these molecules is possible.
Membrane bound proteins are a critical class of proteins to understand, not only are they involve with ligand interactions but they also undergo conformational changes which are useful to elucidate. However, these proteins are challenging to work with due to their hydrophobic nature which means they tend to aggregate in high aqueous environments. To overcome this, solutions such as the use of detergents, formation of micelles, liposomes or nanodiscs are employed but this makes NMR and X-ray crystallography highly challenging if not impossible. However by employing trap columns to remove lipid components, HDX is a successful technique for analysis of these most challenging of molecules.
Understanding post translational modifications (PTMs) is a critical technique for understanding protein function. Proteins with identical amino acid sequences but different PTMs can have vastly different functions. For example Interferon-γ (IFN-γ) can be glycosylated or acetylated. If glycosylated, then the impact of the PTM alters the proteins interaction with receptors but instead, if acetylated then the protein is involved with inflammatory responsesviii. Although challenging, HDX can be used to look at PMTs and a big focus is elucidation of the heterogeneity of glycosylation events using the technique.
Screening out non-epitope residues of an antigen as binding ineligible during computational docking. This approach can assist in prior identification of Antigen/Epitope structure and reduces the time needed for computational identification and designation of binding sites.
HDX is a complimentary technique to provide alternative constructs for hard to crystalize proteins & complexes. This same information can also be used for computational modelling and docking studies, expanding available data sets and decreasing analytical time.
HDX is used in conjunction with Cryo-Electron microscopy to provide structural and functional data for a wide range of proteins with complex states. HDX can also be used to study proteins whose flexibility otherwise restricts the use of Cryo-EM and is suitable for heterogeneous samples.
Biopharma companies have a need to demonstrate and prove equivalence of the candidate biological drug to the parent drug in order to obtain regulatory approval. HDX provides a way to short-circuit this process considerably by demonstrating the active sites are structurally equivalent and similar in action, resulting in huge savings in time and cost.
HDX can be deployed in detection of small amounts of misfolded species within a population of native state biomolecules, during manufacture and postproduction, in storage. This allows for optimization of formulations to improve stability and storage conditions to increase shelf-life.
The applications of HDX continues to grow, shedding increasing light on how proteins function in biological systems. Like all techniques, there are many challenges to overcome such as, lipid removal for measuring membrane proteins, or enzymatic removal of glycosylation sites, to allow for effective protein digestion. Another challenge is speed. Many HDX methods run over ‘minutes to hours’ time scales but for certain experiments such as, examining certain confirmational changes, < 1 second HDX experiments are needed. These present practical challenges that need to be overcome. Also processing HDX data can be time-consuming sometimes leading to many days of manual data processing and interpretation.
Trajan CHRONECT Workstation HDX systems can be configured to reliably run automated HDX experiments from as little as <1 second duration through to many hours AND remarkably all in the same HDX run. In terms of processing data, Trajan has cutting edge software solutions automate and accelerate HDX data interpretation from many days to just hours. As a result, Trajan HDX solutions have transformed, what was once a very laborious niche application, into a routine method for the expanding field of protein science.
To find out more about how Trajan can optimise your HDX methods or if you want to get started in using HDX for your structural elucidation work, please contact Trajan and one of our subject matter experts will be happy to assist you.
2 https://pubmed.ncbi.nlm.nih.gov/30293810/
3 https://pmc.ncbi.nlm.nih.gov/articles/PMC7299284/
4 https://pubmed.ncbi.nlm.nih.gov/20660185/
5 https://pmc.ncbi.nlm.nih.gov/articles/PMC2242592/
6 https://pubmed.ncbi.nlm.nih.gov/19170039/
7 https://www.mdpi.com/2227-9059/8/7/224
8 DOI: 10.1042/bj3030831
9 https://pubmed.ncbi.nlm.nih.gov/22407975/
10 https://pmc.ncbi.nlm.nih.gov/articles/PMC4832280/
11 https://pubmed.ncbi.nlm.nih.gov/32487353/
12 https://pubmed.ncbi.nlm.nih.gov/31841688/
13 https://pubmed.ncbi.nlm.nih.gov/33278412/