The futur
of structural
biology
is bio.
Cell-Free fields of application
The functions of macromolecules within the cells are intimately associated and depend on their tridimensional structure (tertiary structure) which, in turn, depends on their basic composition (primary structure). Structural biology relates to the study of the structure of molecules and macromolecules, how they fold, and how structural modifications and misfolding can affect their cellular functions
Structural biology relates to the study of the structure of molecules and macromolecules and how they fold. It is also concerns the structural modifications that can affect their function. Proteins and nucleic acids adopt a specific three-dimensional structure (also called tertiary structure) that depends on their basic composition (primary structure). It is essential to study the structure of these molecules that lie at the heart of biological processes to better understand how they perform their functions. This is the primary purpose of structural biology.
Importance and applications
Structural biology has been used for many years. In particular, in 1953, it enabled the determination of the structure of DNA using X-ray diffraction. And more recently, the resolution of the structure of the Spike protein of the SARS-CoV2 virus allowed researchers to better understand the fusion mechanism of the virus and to study the interactions brought into play by neutralizing antibodies during infection or vaccination.
This constantly evolving field now allows us to solve more and more precise structures, to study larger and larger molecular complexes and to study processes that occur in less than a tenth of a trillionth of a second. Computational models, complementary to structure resolution methods, allow the design of new proteins not found in nature. These new proteins with useful functions may lead to potentially life-saving drugs.
The information gathered from structural biology finds applications in medicine, biotechnology and agriculture.
How it works?
Structural biology analysis are performed using imaging techniques such as cryo-electron microscopy (cryo-EM), X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and small angle X-ray scattering, as well as computer modelling, to determine the 3D structure of biological macromolecules at the atomic level. These techniques allow the observation of isolated molecules but also of larger structures such as molecular complexes (notably the association of proteins and/or nucleic acids forming a functional unit), viruses and organelles.
Progression in structural biology comprises the merging of all imaging techniques, enabling to create a more precise and clearer map of macromolecules’ shape and how they interact with others.
Trends and challenges
The global structural biology market is expanding rapidly worldwide (Figure 1), as structural biology plays a central role in understanding the special arrangement and the complex folding of proteins, nucleic acids, carbohydrates and lipid membranes. The drug discovery segment is expected to capture the maximum market share.
Figure 1 – Evolution of the structural biology market size from 2023 to 2034.
One of the major difficulties in structural biology is protein structure prediction or modelling. Computational approaches, in parallel with experimental structure determination techniques, have been developed to predict the 3D structure of macromolecules, their dynamics and interactions. Two main strategies have been employed to achieve this, namely template-based and neural network (deep learning)-based approaches [1,2]. In order to understand how macromolecules work in a native complex environment and generate an integrated multiscale whole-cell model, an integrative structural biology approach will be required, congregating improvements and expansion of structural training sets for enhanced modelling, and the integration of diverse data from complementary fields like proteomics (Figure 2).
Another growing trend in structural biology is the study of 4D structures of complex entities (enzymes and other spatially and temporally heterogeneous structural ensembles) throughout a functional cycle, in which the fourth dimension is time [1,2]. This will require new techniques and methodologies aimed at capturing and analysing the dynamic aspects of biomolecular structures when they respond to different environmental conditions or during biological processes.
Figure 2 – Integrated structural biology – the interplay between various technologies. Reproduced from [2].
Systèmes acellulaires et biologie structurale
Cell-free protein synthesis (CFPS) using Escherichia coli cell extracts has successfully been applied to protein sample preparation for structure determination by X-ray crystallization and NMR spectroscopy. Milligram quantities of proteins can be synthesized by the dialysis mode of the cell-free reaction in several hours, and a great variety of proteins can be produced including mammalian proteins, heteromultimeric protein complexes, and integral membrane proteins, with advantages over the recombinant protein expression methods with bacterial and eukaryotic host cells.
Cell-free synthesis enables to surpass one of the limitations in structural analysis of proteins, namely the production of high-yield and high-purity target proteins. A multiscale eukaryotic wheat germ cell-free protein expression pipeline was developed to generate functional proteins of different sizes from multiple host organism and DNA source origins [3]. The system was able to produce highly pure (> 98%) proteins compatible with all three major protein sample formats used for structural biology including single particle analysis with electron microscopy, and both two-dimensional and three-dimensional protein crystallography. Furthermore, by using an eukaryotic ribosome, the cell-free system would enable the synthesis of complex eukaryotic proteins including, for example, protein complexes and membrane proteins [4].
In cell protein crystallization is a technique that has been advantageously employed in structural biology studies since it does not require protein purification and a complicated crystallization process. Nonetheless, only a few protein structures have been determined so far since the crystals are incidentally formed in living cells and are insufficient in size and quality for structure analysis.
Recently, application of CFPS to in-cell protein crystallization was reported for the synthesis of the polyhedra crystal, one of the most highly studied in-cell protein crystals [5]. The so-called cell free protein crystallization (CFPC) method involved direct protein crystallization using a cell free system and was able to perform crystallization and structure determination of nano sized polyhedra crystal at high resolution. The CFPC focused on:
. establishing crystallization of proteins using CFPS with small scale and rapid reactions and
. manipulating the crystallization by adding chemical reagents. The advantage of using CFPC is that various reagents can be added to the reaction mixture without preventing protein synthesis. Thus, CFPC opens up the possibility of crystallizing unstable proteins and rapidly determining their structures.
Publications
References
To know more
Nature
Structures and distribution of SARS-CoV-2 spike proteins on intact virions
Alzheimer's association - Richard C. Mohs,Nigel H. Greig
Drug discovery and development: Role of basic biological research
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