The ability to systematically modify proteins through site directed mutagenesis has transformed protein science, enabling structure-function studies to be performed in a routine manner. However, despite the utility of these methods, there is a growing demand for proteins that are modified in a manner incompatible with normal cellular ribosomal expression. Chemical protein synthesis is attractive for these studies since there are very few limitations on the structural perturbations that can be introduced. As a result, proteins have been synthesized incorporating unnatural amino acid side chains, backbone elements, site-specific post- translational modifications and biophysical probes and have provided numerous unique insights into the molecular basis of protein function. We have addressed the challenge of synthesizing these large organic molecules through the development of chemical ligation strategies that facilitate the conjugation of unprotected peptides derived from solid phase peptide synthesis. However, despite the demonstrated utility of these methods, synthetic protein chemistry remains a challenging endeavor that is practiced by a relatively few number of laboratories. In this proposal, we describe a comprehensive strategy to refine the existing tools of synthetic protein chemistry and introduce novel synthetic approaches with the aim of developing efficient and high yielding syntheses of proteins. Specifically, we propose the development of novel selenium containing amino acids and N-terminal auxiliary groups to facilitate the chemoselective linking of unprotected polypeptides in aqueous solution. To complement these methods, we further develop an approach for the electrophilic thioester peptides using Fmoc based solid phase peptide synthesis approaches. Together, these methodological advances promise to significantly extend the use of chemistry to address questions in protein science. We will test the utility of these methods in the synthesis of several proteins including dihydrofolate reductase, single chain antibodies and alpha synuclein. Overall, we aim to develop and refine innovative synthetic protein chemistry methods with the goal of making protein synthesis a robust and general methodology for the introduction of non-coded probes into biologically interesting and synthetically challenging protein systems.
Synthetic protein chemistry combines the tools of solid phase peptide synthesis and chemoselective ligation reactions to enable the total synthesis of proteins. Using these techniques, proteins can be generated that are free of the biological constraints normally place on protein structure. In this proposal we aim to develop new chemical methods for the synthesis of proteins and to apply them to challenging protein targets to interrogate the utility of the chemistry and to facilitate new investigations into the molecular basis of protein function.
|Getz, Ted; Qin, Jingdong; Medintz, Igor L et al. (2016) Quantum dot-mediated delivery of siRNA to inhibit sphingomyelinase activities in brain-derived cells. J Neurochem 139:872-885|
|Reichart, Timothy M; Baksh, Michael M; Rhee, Jin-Kyu et al. (2016) Trimerization of the HIV Transmembrane Domain in Lipid Bilayers Modulates Broadly Neutralizing Antibody Binding. Angew Chem Int Ed Engl 55:2688-92|
|Walters, Ryan; Medintz, Igor L; Delehanty, James B et al. (2015) The Role of Negative Charge in the Delivery of Quantum Dots to Neurons. ASN Neuro 7:|
|Blanco-Canosa, Juan B; Nardone, Brunello; Albericio, Fernando et al. (2015) Chemical Protein Synthesis Using a Second-Generation N-Acylurea Linker for the Preparation of Peptide-Thioester Precursors. J Am Chem Soc 137:7197-209|
|Agarwal, Rishabh; Domowicz, Miriam S; Schwartz, Nancy B et al. (2015) Delivery and tracking of quantum dot peptide bioconjugates in an intact developing avian brain. ACS Chem Neurosci 6:494-504|
|Muttenthaler, Markus; Albericio, Fernando; Dawson, Philip E (2015) Methods, setup and safe handling for anhydrous hydrogen fluoride cleavage in Boc solid-phase peptide synthesis. Nat Protoc 10:1067-83|
|Assem, Naila; Ferreira, David J; Wolan, Dennis W et al. (2015) Acetone-Linked Peptides: A Convergent Approach for Peptide Macrocyclization and Labeling. Angew Chem Int Ed Engl 54:8665-8|
|Lamberto, Ilaria; Lechtenberg, Bernhard C; Olson, Erika J et al. (2014) Development and structural analysis of a nanomolar cyclic peptide antagonist for the EphA4 receptor. ACS Chem Biol 9:2787-95|
|Thompson, Darren A; Evans, Eric G B; Kasza, Tomas et al. (2014) Adapter reagents for protein site specific dye labeling. Biopolymers 102:273-9|
|Walters, Ryan; Kraig, Richard P; Medintz, Igor et al. (2012) Nanoparticle targeting to neurons in a rat hippocampal slice culture model. ASN Neuro 4:383-92|