Periodic Reporting for period 4 - HiChemSynPro (High-throughput combinatorial chemical protein synthesis as a novel research technology platform for chemical and synthetic biology)
Reporting period: 2021-09-01 to 2023-02-28
Chemical synthesis of proteins by methods of organic chemistry represents a versatile approach that allows for assembling proteins that contain non-canonical functionalities. The common strategy is to prepare short peptide segments by solid-phase peptide synthesis and then use chemoselective chemical ligation to join the segments together. Such methodology complements biological production of proteins by recombinant expression, moreover, the advantage of chemical approach is the possibility to incorporate residues that are not part of the genetic code at any site in the sequence. For instance, mirror-image proteins that are composed entirely of D-amino acids can be assembled in this way. While synthesis of individual proteins up to 300 amino acid residues long is a reality, preparation of large number of protein variants represents a challenge. Since proteins are combinatorial objects (their primary structure consists of a linear chain of amino acid building blocks), preparation of libraries should facilitate finding of the sequences with desired functions for various applications, for example, novel protein therapeutics, catalysts, enzymes, new biomaterials. Therefore, efficient combinatorial chemical synthesis of protein libraries can become a transformative technology. Two major directions were pursued in the HiChemSynPro project to develop such new methodologies: novel catalytic proteins to facilitate ligation of short peptide segments and the development of new microfluidic tools for chemical ligation of peptide segments. Intrinsically disordered proteins (IDPs) were used as targets to test these ideas.
We have designed and synthesized a soluble 24-kDa “covalently-tethered” amyloid analogue by a combination of solid-phase peptide synthesis and chemoselective ligation chemistries [Chem. Sci. 2018, 9, 5594]. It is one of a kind synthesis, where a large polypeptide construct with a non-linear backbone topology was prepared in a homogeneous form. N-methylation of the selected residues permitted to keep the structure soluble and to characterize its properties by biophysical methods such as solution-phase NMR, which is otherwise impossible for insoluble amyloid fibers. In the future, the synthesis of combinatorial libraries of such constructs will permit to identify optimized N-methylation patterns for most potent inhibition of selected amyloid polymorphs.
The tumour suppressor protein p53 is a transcription factor that controls the expression of a large number of genes in response to various stress conditions and regulates cell-cycle, senescence and programmed cell death. Transactivation domain of p53 (TAD p53) is a well-known ‘hub’ involved in many protein-protein interactions related to the function of p53. The post-translational modifications such as phosphorylation regulate the TAD p53 conformational and binding properties. There are 9 possible phosphorylation sites resulting in 512 possible combinations of phospho-variants. We have established chemical synthesis of wild type TAD p53 based on ligation of three peptide segments [Tetrahedron 2019, 75, 703-708]. Selected phospho-variants as well as “proteomimetics” containing triazole backbone surrogates were synthesized to study binding properties of these variants to various known protein partners interacting with p53 [two manuscripts are in preparation].
Previously, our team introduced an approach to interfere with the complex formation formed by intrinsically disordered protein domain by "conformational editing" of the domain itself through the incorporation of conformationally constrained non-canonical alpha-methylated amino acids [Chem. Commun. 2017, 53, 7369]. By combinatorial chemical synthesis we have prepared more than 50 alpha-methylated variants of intrinsically disordered activation domain from activator for thyroid and retinoid receptors (ACTR from p160 protein family) [Chem. Sci. 2021, 12, 1080]. A modified protein variant was discovered that possesses 10-fold stronger affinity to the cancer-related transcriptional co-activator CREB-binding protein (CBP) which can be used to interfere with the formation of wild type protein complex of CBP with co-activator ACTR. Moreover, this approach resulted in the first X-ray structure of the corresponding IDP complex. Furthermore, by using so-called “racemic crystallography“, i.e. by co-crystallization of mirror image forms of the corresponding complexes we obtained several crystal structures of modified ACTR domains in complex with nuclear coactivator binding domain (NCBD) of CBP at a resolution 1.2 Å [manuscript in preparation]. This enabled us to obtain structures of multiple conformers of ACTR domain/NCBD complex and to directly visualize different conformational states with unprecedented detail. This is a remarkable result given the difficulties to obtain crystal structures of dynamic “fuzzy” complexes of IDPs. Such result also highlights the advantages of using non-canonical amino acids to study and to modulate the properties of IDPs.
To achieve the synthesis of larger protein libraries several distinct approaches for combinatorial synthesis of proteins were considered. In the project, we have demonstrated the feasibility of chemical ligation of several protein segments using new engineered soluble protein supports (in contrast to solid-phase supports used previously) which facilitates handling and purification after each ligation step [manuscript in preparation]. For the development of new catalytic proteins (artificial ligases) we have explored a de novo designed three-helix bundle scaffold. By installing a catalytic cysteine residue at the selected site we have obtained an analogue possessing catalytic activity [J. Am. Chem. Soc. 2021, 143, 3330]. Further modifications based on the X-ray structure of this de novo protein were introduced resulting in an enhancement of the catalytic activity which in combination with immobilization of the protein variant onto the polymer beads enabled us to use it for the catalysis of peptide synthesis in a microfluidic flow reactor [manuscript in preparation]. In the future, we will apply these novel tools for the synthesis of protein libraries composed of hundreds of protein variants.
The results of the project led to 7 publications and several more manuscripts are currently in preparation. The key findings were presented at 15 conferences as oral and poster presentations. 4 PhD theses related to this project have been defended and 5 postdoctoral fellows received training that resulted in their further academic and industrial employment. Intellectual property protection will be obtained for results that have commercialization potential.
The tumour suppressor protein p53 is a transcription factor that controls the expression of a large number of genes in response to various stress conditions and regulates cell-cycle, senescence and programmed cell death. Transactivation domain of p53 (TAD p53) is a well-known ‘hub’ involved in many protein-protein interactions related to the function of p53. The post-translational modifications such as phosphorylation regulate the TAD p53 conformational and binding properties. There are 9 possible phosphorylation sites resulting in 512 possible combinations of phospho-variants. We have established chemical synthesis of wild type TAD p53 based on ligation of three peptide segments [Tetrahedron 2019, 75, 703-708]. Selected phospho-variants as well as “proteomimetics” containing triazole backbone surrogates were synthesized to study binding properties of these variants to various known protein partners interacting with p53 [two manuscripts are in preparation].
Previously, our team introduced an approach to interfere with the complex formation formed by intrinsically disordered protein domain by "conformational editing" of the domain itself through the incorporation of conformationally constrained non-canonical alpha-methylated amino acids [Chem. Commun. 2017, 53, 7369]. By combinatorial chemical synthesis we have prepared more than 50 alpha-methylated variants of intrinsically disordered activation domain from activator for thyroid and retinoid receptors (ACTR from p160 protein family) [Chem. Sci. 2021, 12, 1080]. A modified protein variant was discovered that possesses 10-fold stronger affinity to the cancer-related transcriptional co-activator CREB-binding protein (CBP) which can be used to interfere with the formation of wild type protein complex of CBP with co-activator ACTR. Moreover, this approach resulted in the first X-ray structure of the corresponding IDP complex. Furthermore, by using so-called “racemic crystallography“, i.e. by co-crystallization of mirror image forms of the corresponding complexes we obtained several crystal structures of modified ACTR domains in complex with nuclear coactivator binding domain (NCBD) of CBP at a resolution 1.2 Å [manuscript in preparation]. This enabled us to obtain structures of multiple conformers of ACTR domain/NCBD complex and to directly visualize different conformational states with unprecedented detail. This is a remarkable result given the difficulties to obtain crystal structures of dynamic “fuzzy” complexes of IDPs. Such result also highlights the advantages of using non-canonical amino acids to study and to modulate the properties of IDPs.
To achieve the synthesis of larger protein libraries several distinct approaches for combinatorial synthesis of proteins were considered. In the project, we have demonstrated the feasibility of chemical ligation of several protein segments using new engineered soluble protein supports (in contrast to solid-phase supports used previously) which facilitates handling and purification after each ligation step [manuscript in preparation]. For the development of new catalytic proteins (artificial ligases) we have explored a de novo designed three-helix bundle scaffold. By installing a catalytic cysteine residue at the selected site we have obtained an analogue possessing catalytic activity [J. Am. Chem. Soc. 2021, 143, 3330]. Further modifications based on the X-ray structure of this de novo protein were introduced resulting in an enhancement of the catalytic activity which in combination with immobilization of the protein variant onto the polymer beads enabled us to use it for the catalysis of peptide synthesis in a microfluidic flow reactor [manuscript in preparation]. In the future, we will apply these novel tools for the synthesis of protein libraries composed of hundreds of protein variants.
The results of the project led to 7 publications and several more manuscripts are currently in preparation. The key findings were presented at 15 conferences as oral and poster presentations. 4 PhD theses related to this project have been defended and 5 postdoctoral fellows received training that resulted in their further academic and industrial employment. Intellectual property protection will be obtained for results that have commercialization potential.
The key achievements of the project are: (1) chemical synthesis of protein library of more than 50 variants of activation domain of ACTR transcriptional co-activator, where each member of the library was obtained in highly purified form on several milligrams scale which enabled the detailed biophysical and biological characterization of each variant; (2) series of high-resolution X-ray structures obtained through quasi-racemic crystallization at very high resolution for a dynamic protein complex formed by intrinsically disordered proteins; (3) new protein catalyst able to catalyze amide bond formation and in conjunction with on-beads immobilization suitable for chemical synthesis of protein libraries.