The new cell-free protein crystallization method (CFPC), developed by Tokyo Tech, involves direct protein crystallization and is an important advance in structural biology. This technique will allow the analysis of unstable proteins that could not be studied by conventional methods. Analyzing these will increase our knowledge of cellular processes and functions.
While we are familiar with certain crystals such as salt and sugar that we use in our daily lives, there is another set of crystals, hidden from the naked eye, that is crucial to our biology. Microscopic protein crystals are found in living cells and help maintain processes such as immune system activation, protein storage and protection.
To better understand the relationship between the structure and function of protein crystals, scientists have developed the in-cell protein crystallization (ICPC) method, which can directly observe protein crystals in living cells, ensuring high-quality crystals without the need for purification processes or complex screening. methods. However, despite the many advantages, very few structures were reported because the crystals formed in living cells were not of the size and quality necessary for analysis. So a team of researchers from Japan, led by Prof. Takafumi Ueno of Tokyo Tech, wanted to develop a better method. And recently they made a breakthrough.
In their article published in Scientific Reports, the team reported developing a technique that would make protein crystallization and analysis more efficient and effective. This technique – a cell-free protein crystallization method (CFPC) – was a hybrid between in vitro protein crystallization and ICPC and allowed rapid and direct formation of protein crystals without the need for complicated crystallization and purification methods.
“ICPC is expected to become an important tool in crystal structure analysis, but we need a method to obtain protein crystal structures with better resolution. So we focused on achieving high-quality protein crystallization using CFPC with small-scale and fast reactions” , says Prof. Ueno, who also heads Tokyo Tech’s Ueno lab. Members of this laboratory study naturally occurring protein compositions, their structure and functions, with the aim of applying this knowledge to develop innovative biotechnology and energy solutions.
Returning to the team conducting the current study (some of whom are also members of the Ueno lab), they used a wheat germ protein synthesis kit, a tool for the synthesis of polyhedrin monomer, a viral protein produced in insect cells by cypovirus infection. This protein was then crystallized using the novel CFPC method, leading to the formation of nano-sized polyedra crystals (PhCs). The team was able to efficiently complete this process within six hours, using just 20 microliters of the reaction mixture.
Scanning electron microscopy images indicated that the PhCs had excellent purity, allowing their structure to be determined with a resolution as high as 1.95 Å (or 1.95 angstroms). To further explore the capabilities of their new system, the team performed the structural analysis of crystalline inclusion protein A (CipA). Its structure was determined at a high resolution of 2.11 , something that had never been reported before for this study.
This work represents a major leap forward in the field of structural biology, as the method it proposes will allow for the analysis of low-yield unstable proteins that cannot be studied by conventional methods. This technology is also intended to aid in the development of advanced techniques for small-scale and rapid protein crystallization and analysis. “The high-quality protein crystals produced by our method will broaden the horizons of structural determination and provide us with useful and unprecedented insights into the complex environment of living cells,” says Prof. Ueno.
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Satoshi Abe et al, Cell-free protein crystallization for nanocrystal determination, Scientific Reports (2022). DOI: 10.1038/s41598-022-19681-9
Quote: Novel Cell-Free Protein Crystallization Method to Promote Structural Biology (2022, October 3), retrieved October 3, 2022 from https://phys.org/news/2022-10-cell-free-protein-crystallization-method-advance.html
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