A remarkable microscopic world
Proteins are highly complex molecules which underpin the vast majority of biological systems. If we were to observe the world at the size of the peptide hormone insulin (around 30 Å in diameter) and view both the intracellular and extracellular spaces, we would see that proteins are ubiquitous both spatially and in functionality. Proteins convey both the structural function of biochemical systems but also act as dynamic interconnected nanorobots facilitating practically every known metabolic pathway via enzymatic mediated catalysis and associated functions. Moreover, certain proteins and peptides, like insulin for example, act as molecular messengers interacting with yet other types of proteins where these function as dynamic locks and doorways on cell membranes. In doing so, these receptor an associated biomolecules allow or disallow the passage of molecules (and even ions) in and out of cells.
From our vantage point within a cell, we would observe a highly dynamic three-dimensional world - an orchestrated molecular city society, consisting of an unfathomable number of interconnected protein mediated processes. Indeed, it can appear to the observer that the primary purpose of such processes is the maintenance of cellular function and integrity (although the reality of this is far more complicated). Outside of the cell, we would observe a remarkable microscopic universe teeming with activity; molecules and ions interacting with each other and continually visiting cell membranes. These visitors often facilitate specific changes in cellular function, by initiating intracellular cascades of metabolic processes; both for the benefit but also for the detriment of the cell viability. For example, one of the key functions of the peptide hormone insulin is to interact with specific receptors, on the surface of cells, to allow the influx of glucose into cells, powering cellular respiration or stored for later usage.
The ubiquitous nature of proteins underpins much of our continual understanding of biochemical processes and the resultant emergence of biological systems. Indeed, garnering an understanding of the in vivo state, continues to pose huge challenges to scientists. However, the closer we get to creating accurate snapshots of biochemical pathways and their resultant impact on physiology, the better we’re able to understand the underlying mechanisms which lead to the cause and development of specific diseases such as diabetes.
Our increasing understanding affords us the opportunity to design more targeted approaches to not only manage symptoms but also, in some cases, reverse disease progression. One such early example of this is in the pioneering treatment of diabetes, which dates to the early 1920s. Fred Banting and colleagues at the university of Toronto, isolated and purified insulin from the pancreases of pigs and cows, to treat type 1
diabetic patients. The effects were remarkable, transforming what was once a lethal condition into one that could be managed and allow for patients to lead a near normal life1. However, at that time, the nature of insulin was unknown, and it would take over a decade before its molecular structure would begin to be elucidated.
Elucidating the structure of insulin and the importance of protein 3D structure
In 1935, scientists at John Hopkins University proved the hypothesis that insulin was a single protein, however, it wasn’t until the 1950s that English biochemist Frederick Sanger, successfully elucidated the primary amino acid sequence1. Nonetheless, this was not the complete story, as it transpired that the shape of the insulin molecule (as observed for all proteins), was key to its function and this could not be elucidated from its primary sequence.
After de novo protein synthesis, either through cellular translation or synthetic synthesis, peptides and proteins form secondary structures. These include alpha helices, beta sheets, beta turns and omega loops. Proteins often then go on to form tertiary states, creating complicated 3D structures and sometimes incorporating non-proteinaceous molecules such as porphyrins and metals in the process. These ligands can further act to dynamically change the tertiary structure based on for example, the oxidation state of the complexing agent such as a metal ion. Also related to this, proteins can undergo post translational modifications further extending their functional ubiquity. Finally, certain proteins will then form intermolecular associations with other tertiary subunits forming quaternary structures. An example of this is observed with haemoglobin molecule, which assembles to form a complex of two pairs of different protein subunits (α+β) each housing a single haem molecule.
The reason why the 3D shape of a protein is so important is that proteins work on shape selectivity such as ‘lock and key’ interactions with other proteins and non-proteinaceous molecules. Often, the functional centre of a protein is not found as a contiguous chain of amino acids within the protein sequence but instead, functional centres form when certain regions of the protein fold to form a configuration bringing key amino acids and groups into close proximity. An example of this is an active site of an enzyme or membrane receptor. Or in the case of insulin, the peptide hormone folds in such a way that it fits into specific receptors on membrane proteins and in doing so, allows a confirmational change in the receptor to encourage an influx of glucose into the cell2. Another key point here is that these are not static processes as proteins often undergo dynamic conformational changes when exposed to conditions such as ligand binding.
Understanding protein conformational changes, protein associations and ligand binding associations is essential to comprehending biological processes. For example, most drugs are used to treat diseases by interacting with proteinaceous receptors to either
inhibit or stimulate metabolic downstream processes. Understanding where and how ligands interact with receptors - whether they are innate molecular messengers, or synthetic drug molecules - will allow us to develop more efficacious treatments. Furthermore, it is important for us to understand the relevance of the impact of external influences (such as disease) on changes in protein structure. This is to allow us to get a clearer understanding of disease processes enabling the innovation of new more efficacious treatments
How do we measure 3D protein structures?
One of the key techniques in measuring the molecular three-dimensional arrangement of proteins is x-ray crystallography. A key pioneer of this technique was Professor Dorothy Crowfoot Hodgkin at Oxford university, who earned a Nobel prise in Chemistry in 1964. Her pioneering work elucidated the structure of penicillin, vitamin B12 and insulin. She achieved this by beaming x-ray light through crystals and mapping and interpreting resultant diffraction patterns on photographic paper3. After 35 years of researching the structure of insulin - in the same year Apollo 11 landed on the moon - Professor Hodgkin co published a paper in Nature detailing the structure of rhombohedral 2 zinc insulin crystals4. This seminal work was critical to further the understanding of insulin’s role in controlling blood sugar and diabetes.
For decades, X-ray crystallography has been key to elucidating the structure of proteins, and in 2022, it was reported that around 100,000 protein structures had been published in a protein data bank using the technique5. However, nowadays, x-ray crystallography is just one tool in an increasing toolbox. This is because, the technique does not suit all proteins and is also laborious. As a result, there are now other key methods which are also employed to elucidate protein structure, conformational changes and ligand interactions. These include NMR, cryo-electron microscopy and mass spectrometric (MS) related techniques. One of these MS techniques employs a method called hydrogen deuterium exchange or HDX.
Hydrogen Deuterium Exchange (HDX)
Pioneered in the mid-1960s, HDX exploits a phenomenon observed with proteins that when exposed to deuterated aqueous solvents, protons from the amide backbone (as well as some other areas of the protein) will be exchanged for deuterium. However, replace the solvent with a protonated variant then the deuterated protein reverts to its protonated state. This phenomenon can be exploited to elucidate protein structure, confirmational change and ligand interactions. This is because the rates of deuteration and protonation are directly related to their close environments. Shielding the local environment due to protein folding, ligand-binding or an antigen-antibody association, considerably slows down the exchange process . By coupling this technique with a means to identify and quantify proteins and protein fragments, we can not only elucidate
protein structure but also map dynamic folding and interactions6. Moreover, HDX LC-MS lends itself to full automation making sample preparation and analysis more routine allowing for a wide pallet of uses.
Summary
We are living in very exciting times; rapid improvement of technology is allowing us to gain more insight into biological systems. Over the years, techniques like x-ray crystallography and HDX coupled with LC-MS are allowing us to peer into the cellular and extracellular machinery in increasing levels of clarity. Over the next few months, we will delve deeper into HDX and how it is being used to better understand the nature of proteins and how these can be exploited to improve diagnosis, vaccination and medical treatments.
In 1994, Dorothy Crowfoot Hodgkin passed away aged 84, having helped to revolutionise the way we elucidate the three-dimensional structure of molecules. Her pioneering research has and will continue to save and improve countless lives worldwide. It would be amazing to show Professor Hodgkin the rapid progress we have seen in the last 30 years since her passing and how science has benefited from her brilliance and determination to make a difference.
1 https://www.acs.org/education/whatischemistry/landmarks/insulin.html
2 https://doi.org/10.1038/s12276-023-01101-1
3 https://www.nobelprize.org/womenwhochangedscience/stories/dorothy-hodgkin
4 https://www.nature.com/articles/224491a0
6 https://www.frontiersin.org/journals/analytical-science/articles/10.3389/frans.2023.1118749/full