Vaccines – and their use in immunization programs around the world – are considered one the most important medical developments of the last century. They are very effective medical interventions that prevent infections, save millions of lives, and provide significant global economic benefits every year.
Despite their benefits, not everyone has good access to vaccines. Logistical challenges around so-called ‘cold-chains’ are a major hurdle to their wide-spread use. The requirements for refrigerated storage and distribution add significant costs to immunization programs and make the delivery of vaccinations to some communities, particularly those in rural areas in low and middle-income countries, challenging. In addition, many vaccine doses are lost every year due to cold-chain failures.
Vaccination programs would benefit from a simple, cheap platform that keeps the proteins in vaccines stable without refrigeration. Currently when left unrefrigerated for too long these proteins unfold, aggregate and become irreversibly damaged such that they no longer work as intended. One proposed technique is to encapsulate them inside a porous molecular framework.
The idea is that a rigid framework, known as a metal-organic framework (MOF), holds the protein in tight confinement that prevents it from unfolding. As protein folding is associated with protein stability, the theory is that if a protein retains its shape and structure, it will retain its stability and function. This technique could also have applications in other fields. For instance, encapsulating enzymes in rigid frameworks could also increase their thermostability, so that they can be used at higher temperatures.
MOFs are self-assembling structures composed of metal centres – the nodes of the framework – connected by organic ligands. Previous research has shown that proteins released from MOFs retain their original structure and stability, even after being exposed to high temperatures.
It is not clear, however, what happens to a protein inside a MOF while exposed to temperatures that would normally cause it to unfold. To explore this, chemical and biomolecular engineers at the Georgia Institute of Technology and the University of Chicago, used a National Science Foundation-funded beamline (15-ID-D of NSF’s ChemMatCARS) at the Advanced Photon Source (APS), a U.S. Department of Energy (DOE) Office of Science user facility at DOE’s Argonne National Laboratory.
The researchers performed small-angle X-ray scattering (SAXS) to probe the structure of bovine serum albumin (BSA), a commonly used model protein, when embedded and not embedded in MOFs. The team used two variations of a class of MOFs known as zeolitic imidazolate frameworks (ZIFs). Both the free proteins and the proteins embedded in the MOFs were heated to 70°C and held at that temperature for three hours while being analysed.
As the researchers were trying to analyse the protein, they had to do some analytical work on the X-ray data of the BSA embedded in the MOFs to subtract the signal of the MOFs, to reveal the structure of the protein only.
The X-ray analyses showed that when embedded in MOFs the proteins retained their structure while being heated to and kept at 70°C. In comparison, unencapsulated BSA unfolded as it was heated, and its shape and structure changed.
Importantly, the work demonstrated that the encapsulated protein retained the same structure throughout the heating process. This discounts the possibility that the protein changes in some way during heating, but then reverts to its original shape, structure, and folding when released from the MOF. This reduces the chance that there has been some fundamental change in the protein that could damage it and impact its functionality.
As well as demonstrating that MOFs can keep proteins stable by keeping them physically trapped, the researchers also established a new technique for analysing encapsulated proteins. Their method of subtracting the signal of the MOF from the BSA could be used to probe other encapsulated systems to advance fundamental research and explore real-world applications, such as vaccine storage. – Michael Allen
See: R. Murty1, M. K. Bera2, I. M. Walton1, C. Whetzel1, M. R. Prausnitz1, K. S. Walton1, “Interrogating Encapsulated Protein Structure Within Metal-Organic Frameworks at Elevated Temperature,” J. Am. Chem. Soc. 145, 13, 7323-7330 (2023)
Author affiliations: 1Georgia Institute of Technology; 2University of Chicago.
We thank Donna Bondy for administrative support. NSF’s ChemMatCARS Sector 15 at Argonne National Laboratories is supported by the National Science Foundation under grant number NSF/CHE-1834750. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science user facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. This work benefited from the use of the SasView application, originally developed under NSF award DMR-0520547. SasView contains the code developed with funding from the European Union’s Horizon 2020 research and innovation program under the SINE2020 project, grant agreement no. 654000. This work was also supported in part by the Bill and Melinda Gates Foundation under grant no. INV-003561. XRD and SEM experiments were performed at the Georgia Tech Institute for Electronics and Nanotechnology, a member of the National Nanotechnology Coordinated Infrastructure (NNCI), which is supported by the National Science Foundation (ECCS-2025462).
The U.S. Department of Energy's APS at Argonne National Laboratory is one of the world’s most productive x-ray light source facilities. Each year, the APS provides high-brightness x-ray beams to a diverse community of more than 5,000 researchers in materials science, chemistry, condensed matter physics, the life and environmental sciences, and applied research. Researchers using the APS produce over 2,000 publications each year detailing impactful discoveries and solve more vital biological protein structures than users of any other x-ray light source research facility. APS x-rays are ideally suited for explorations of materials and biological structures; elemental distribution; chemical, magnetic, electronic states; and a wide range of technologically important engineering systems from batteries to fuel injector sprays, all of which are the foundations of our nation’s economic, technological, and physical well-being.
Argonne National Laboratory seeks solutions to pressing national problems in science and technology. The nation's first national laboratory, Argonne conducts leading-edge basic and applied scientific research in virtually every scientific discipline. Argonne researchers work closely with researchers from hundreds of companies, universities, and federal, state and municipal agencies to help them solve their specific problems, advance America's scientific leadership and prepare the nation for a better future. With employees from more than 60 nations, Argonne is managed by UChicago Argonne, LLC, for the U.S. DOE Office of Science.
The U.S. Department of Energy's Office of Science is the single largest supporter of basic research in the physical sciences in the United States and is working to address some of the most pressing challenges of our time. For more information, visit the Office of Science website.