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Chemical methods have enabled the total synthesis of protein molecules of ever-increasing size and complexity. However, methods to engineer synthetic proteins comprising noncanonical amino acids have not kept pace, even though this capability would be a distinct advantage of the total synthesis approach to protein science. In this work, we report a platform for protein engineering based on the screening of synthetic one-bead one-compound protein libraries. Screening throughput approaching that of cell surface display was achieved by a combination of magnetic bead enrichment, flow cytometry analysis of on-bead screens, and high-throughput MS/MS-based sequencing of identified active compounds. Direct screening of a synthetic protein library by these methods resulted in the de novo discovery of mirror-image miniprotein-based binders to a ∼150-kDa protein target, a task that would be difficult or impossible by other means. Keywords: xenoprotein; mirror-image miniprotein; D-protein; protein engineering; flow cytometry, United States. Defense Advanced Research Projects Agency (Award 023504-001)

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2017., Cataloged from PDF version of thesis. Page 186 blank., Includes bibliographical references., Natural proteins are comprised primarily of (L)-amino acids. (D)-amino acids are rare in protein structures. Solid-phase peptide synthesis (SPPS) and native chemical ligation enable the total chemical synthesis of proteins. Using these techniques, it is possible to design and study polypeptides foreign to Nature. Herein, I describe the investigation and application of proteins simultaneously comprised of (L)- and (D)-amino acids. SPPS has traditionally been a time intensive endeavor. Recently, the Pentelute laboratory described a flow-based system that reduces the time required to synthesize a polypeptide by over an order of magnitude. We have systematically studied variables that influence peptide quality with this system. From these efforts, we established protocols used in the synthesis of heterochiral polypeptides. We proceeded to study two different heterochiral systems. In the first system, we examined folding of (L)-proteins containing loops of (D)-amino acids, and vice-versa. Protein loops are important structural features that mediate protein-protein interactions. Using Ecballium elaterium trypsin inhibitor II (EETI-ll), we discovered that strategic incorporation of linkers such as P-alanine or glycine can facilitate efficient folding of heterochiral proteins. We used NMR spectroscopy and molecular dynamic simulations to interrogate the structure and folding pathway of one such protein that possesses a (D)-amino acid core and a loop with 5 (L)-amino acids, 2 P-alanine, and 1 glycine. We also determined that our heterochiral proteins were more resistant to proteolysis than natural proteins. In the second system, we examined heterochiral antibody-drug conjugates (ADCs) for the treatment of P. aeruginosa infection. New therapeutic modalities are needed to address bacterial resistance to conventional antibiotics. We developed a biologically expressed antibody that targets P. aeruginosa and bears antimicrobial peptides chemically synthesized from (D)-amino acids, by Surin Khai Mong., Ph. D.

We report the convergent total synthesis of two proteins: DARPin pE59 and Bacillus amyloliquefaciens RNase (Barnase). Leveraging our recently developed fast-flow peptide-synthesis platform, we rapidly explored numerous conditions for the assembly of long polypeptides, and were able to mitigate common side reactions, including deletion and aspartimide products. We report general strategies for improving the synthetic quality of difficult peptide sequences with our system. High-quality protein fragments produced under optimal synthetic conditions were subjected to convergent native chemical ligation, which afforded native full-length proteins after a final desulfurization step. Both DARPin and Barnase were folded and found to be as active as their recombinant analogues., MIT Faculty Start-up Fund, Massachusetts Institute of Technology (Charles E. Reed Faculty Initiative Fund), Deshpande Center for Technological Innovation, Damon Runyon-Rachleff (Innovation Award), Sontag Foundation (Distinguished Scientist Award), AstraZeneca (Firm) (Distinguished Graduate Student Fellowship), Daniel S. Kemp Summer Fellowship, National Institutes of Health (U.S.). Biotechnology Training Program (5T32GM008334-25)

A flow-based solid-phase peptide synthesis methodology that enables the incorporation of an amino acid residue every 1.8 min under automatic control or every 3 min under manual control is described. This is accomplished by passing a stream of reagent through a heat exchanger into a low volume, low backpressure reaction vessel, and through a UV detector. These features enable continuous delivery of heated solvents and reagents to the solid support at high flow rate, thereby maintaining maximal concentration of reagents in the reaction vessel, quickly exchanging reagents, and eliminating the need to rapidly heat reagents after they have been added to the vessel. The UV detector enables continuous monitoring of the process. To demonstrate the broad applicability and reliability of this method, it was employed in the total synthesis of a small protein, as well as dozens of peptides. The quality of the material obtained with this method is comparable to that for traditional batch methods, and, in all cases, the desired material was readily purifiable by RP-HPLC. The application of this method to the synthesis of the 113-residue Bacillus amyloliquefaciens RNase and the 130-residue DARPin pE59 is described in the accompanying manuscript., MIT Faculty Start-up Fund, Massachusetts Institute of Technology (Charles E. Reed Faculty Initiative Fund), Deshpande Center for Technological Innovation, Damon Runyon-Rachleff (Innovation Award), Sontag Foundation (Distinguished Scientist Award), C. P. Chu and Y. Lai Fellowship, Daniel S. Kemp Summer Fellowship, National Institute of General Medical Sciences (U.S.). Biotechnology Training Program (Grant 5T32GM008334-25), National Institutes of Health (U.S.) (Fellowship F32GM101762)