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High-throughput combinatorial chemical protein synthesis as a novel research technology platform for chemical and synthetic biology

Periodic Reporting for period 2 - HiChemSynPro (High-throughput combinatorial chemical protein synthesis as a novel research technology platform for chemical and synthetic biology)

Reporting period: 2018-09-01 to 2020-02-29

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 fragments together. Such methodology complements biological production of proteins by recombinant expression, moreover, the advantage of chemical approach is the possibility to incorporate at any site in the sequence residues that are not part of the genetic code. 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. The objectives of the HiChemSynPro are to develop such new methodologies. Two major directions are envisaged in the project: development of new microfluidic tools for automation of chemical ligation of peptide fragments and novel catalytic proteins (enzymes) to facilitate ligation of short peptide segments. In the project, the plan is to validate new developed methods by using intrinsically disordered protein targets.
Several intrinsically disoredered proteins have been considered as targets.
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 - a large polypeptide construct with non-linear backbone topology was prepared in a homogeneous form. N-methylation of selected residues permitted to keep the structure soluble and to characterize its properties by solution-phase biophysical methods such as NMR, which is otherwise impossible for insoluble amyloid fibers. These studies provided insights for the development of selective binders to different structural polymorphs of amyloids. 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]. Next step is to synthesize combinatorial library of phospho-variants and to study binding properties of these variants to various known protein partners interacting with p53.
Recently, 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]. More recently, 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, also known as p160) [manuscript in preparation]. 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.
To achieve synthesis of larger protein libraries two distinct approaches for microfluidic synthesis of proteins are currently under study in our laboratory. For both strategies, we are close to reach the proof-of-concept stage. For the development of new catalytic proteins (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 modest catalytic activity. Currently, work is undergoing to enhance its catalytic activity.
Chemical synthesis of protein library of more than 50 variants of activation domain of ACTR transcriptional co-activator is the first major step towards synthesis of larger libraries of hundreds to thousands of variants. Furthermore, this strategy was used to perform systematic substitutions of residues for alpha-methylated amino acids (alpha-methylation scan). This is an innovative protein engineering approach particularly suitable to study intrinsically disordered proteins. Indeed, using this method we identified an ACTR variant possessing two alpha-methylations which has 10-fold enhanced affinity than wild type to its native binding partner - a domain in CREB-binding protein (CBP). In addition, we solved the first X-ray structure of this protein complex.
We expect to increase the size of libraries to several hundred members using microfluidic reactors and artificial peptide ligases. Thus, in addition to ACTR libraries we plan to synthesize hundreds of analogues (phosphorylated, N- and alpha-methylated) for different protein targets.