1-Chemical Synthesis of Proteins

For the past three decades, scientists have obtained proteins to conduct their studies by recombinant DNA-based expression in genetically engineered cells. This mature technology allows the production of large amounts of proteins of well-defined molecular compositions for studies at the molecular level. Unarguably, DNA-based expression has dramatically influenced our knowledge and understanding of the molecular basis of protein function and will remain the core for the study of biological systems. Yet, the method has some limitations, such as difficulties in expressing large multidomain proteins and over expression of proteins that are toxic to the cells (proteases). Moreover, studies in most cases are inherently limited to the 20 natural encoded amino acids, despite some successes of using cell-free synthesis to expand the repertoire of ribosomal synthesis to incorporate unnatural amino acids in proteins.

Chemists have offered an attractive and complementary approach to biological systems for protein production. Chemical synthesis of proteins allows for variations of the covalent protein structure, virtually in an unlimited manner. Although Solid-Phase Peptide Synthesis (SPPS) is reliable up to ~50 amino acids, protein synthesis is complicated due to the accumulation of side products during chain assembly and deprotection. Consequently, protein synthesis is dominated by approaches that depend on the assembly of smaller peptide fragments. Most Notably, the Native Chemical Ligation (NCL) utilizes a highly chemoselective coupling of two unprotected peptides, one of which bears an N-terminal cysteine residue, while the other contains a C-terminal thioester group. In continuation to these efforts, our lab is exploring new ligation methods to construct large polypeptides that correspond to folded proteins.

The ligation methods will be used in the context of the following fields:

A) Manipulating Proteins with Chemistry: Manipulating proteins with chemistry to study their biological function and to increase their stability has attracted synthetic chemists to contribute to these studies in ways that are difficult to achieve by using traditional biochemical approaches. The increasing momentum of the field is supported by the emerging chemical and biochemical approaches that allow the incorporation of unnatural functionalities into the protein of interest. In addition, there are an increasing number of organic reactions that can be carried out in aqueous media, which permit their use to modify proteins in physiological conditions. The introduction of unnatural entities into proteins at a specific position allows a subsequent chemistry to be carried out in a selective manner, contrary to previous methods where selectivity is sacrificed. We are investigating several chemistries that are compatible to physiological conditions in order to introduce the peptidomimetic motifs into protein scaffold. The effect of such modifications on the protein structure and activity will also be examined.

B) Posttranslationally Modified Proteins: Posttranslational modifications play an important role in regulating protein structure and function in health and disease. Ubiquitylation is one example for such a modification wherein both the extent (polyubiquitylation vs mono-ubiquitylation) and the sequence position of this modification dictates the function and fate of the ubiquitylated protein.  In the ubiquitylation process three distinct enzymes, known as the E1-E3 system, collaborate to achieve a site-specific tagging of the lysine residue(s) in target protein. This condensation step generates an isopeptide linkage between the -NH₂ of the lysine residue and the activated C-terminal glycine of ubiquitin (Ub). Chemical synthesis of proteins offers exceptional opportunities to prepare homogeneous posttranslationally modified protein with high purity and large quantities for functional and structural analysis. We have recently reported a new method for peptide ubiquitylation employing mercaptolysine residue to mediate thioesterification followed by amino acyl transfer to form the isopeptide linkage between ubiquitin and specific lysine residue of the tagged protein. we are currently working to apply  this approach to study the effect of ubiquitylation and SSMoylation on the function of a wide-range of proteins. 

2-Developing novel chemical approaches to control protein self-assembly in health and disease

Protein misfolding and aggregation, more specifically amyloid formation, play a central role in the pathogenesis of several incurable systemic and neurodegenerative diseases that affect the population today.   Therefore, detailed mechanistic understanding of these processes in and outside the cell is of critical importance to elucidating the fundamental roles governing protein folding, understanding disease mechanisms and developing therapeutic strategies to prevent, treat and/or reverse these devastating diseases.  This joint interdisciplinary collaborative project (with Prof. Hilal A. Lashuel) aims to bring together expertise in organic chemistry, proteins biochemistry, biophysics and molecular/cellular biology to develop innovative chemical approaches and novel tools to control and characterize protein misfolding and self-assembly of peptides and proteins in and outside of living cells.  Our efforts will focus on developing new chemical switch elements to facilitate mechanistic studies aimed at elucidating the mechanisms of protein misfolding and aggregation and their role in the pathogenesis of amyloid diseases.  More specifically, we will seek to  develop new chemical tools to enhance the solubility and allow controlled disruption of folding and self-assembly in amyloid formation. We will then apply these tools to elucidate the molecular mechanisms underlying the misfolding, self-assembly and amyloid formation of various systems of increasing complexity. Our work represents the first attempt to extend the concept of molecular switches based on secondary structure disruption elements and acyl migration to control the folding, self-assembly, and aggregation properties of proteins.  The work will result in the development of new chemical tools that allow greater spatial and temporal control over protein structure and function without altering the native sequence of the protein. These tools will contribute significantly to addressing many of the technical and experimental limitations to study protein folding and self-assembly. The knowledge to be gained from the proposed studies will have significant impact on our understanding of the molecular basis of amyloid diseases, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and Type II diabetes, and contribute to advances in biotechnology, where protein aggregation remains a major challenge in the development of protein therapeutics.

Towards these goals,  we have recently shown a new transformation based on the Staudinger reaction and demonstrate its application in the design of a novel switch element to control the folding of the NPY peptide from random coil to α-helix conformation. The azido functionality in the depsipeptide unit is activated rapidly in water using TCEP via Staudinger reaction. Our findings expand the repertoire of uses of the Staudinger reaction in chemical biology and the number of available triggers for use in switch peptides. Current efforts in our laboratories are focused on applying the azide switch, with other known switches, in the design and characterization of switch proteins,  and self-assembling systems.