Ribosomes can produce proteins in minutes and are largely constrained to proteinogenic amino acids. Here, we report highly efficient chemistry matched with an automated fast-flow instrument for the direct manufacturing of peptide chains up to 164 amino acids long over 327 consecutive reactions. The machine is rapid: Peptide chain elongation is complete in hours. We demonstrate the utility of this approach by the chemical synthesis of nine different protein chains that represent enzymes, structural units, and regulatory factors. After purification and folding, the synthetic materials display biophysical and enzymatic properties comparable to the biologically expressed proteins. High-fidelity automated flow chemistry is an alternative for producing single-domain proteins without the ribosome.

Chemical protein synthesis and racemic protein crystallization were used to determine the X-ray structure of the snow flea antifreeze protein (sfAFP). Crystal formation from a racemic solution containing equal amounts of the chemically synthesized proteins d-sfAFP and l-sfAFP occurred much more readily than for l-sfAFP alone. More facile crystal formation also occurred from a quasi-racemic mixture of d-sfAFP and l-Se-sfAFP, a chemical protein analogue that contains an additional -SeCH2- moiety at one residue and thus differs slightly from the true enantiomer. Multiple wavelength anomalous dispersion (MAD) phasing from quasi-racemate crystals was then used to determine the X-ray structure of the sfAFP protein molecule. The resulting model was used to solve by molecular replacement the X-ray structure of l-sfAFP to a resolution of 0.98 A. The l-sfAFP molecule is made up of six antiparallel left-handed PPII helixes, stacked in two sets of three, to form a compact brick-like structure with one hydrophilic face and one hydrophobic face. This is a novel experimental protein structure and closely resembles a structural model proposed for sfAFP. These results illustrate the utility of total chemical synthesis combined with racemic crystallization and X-ray crystallography for determining the unknown structure of a protein.

High-diversity genetically-encoded combinatorial libraries (10 8 −10 13 members) are a rich source of peptide-based binding molecules, identified by affinity selection. Synthetic libraries can access broader chemical space, but typically examine only~10 6 compounds by screening. Here we show that in-solution affinity selection can be interfaced with nano-liquid chromatography-tandem mass spectrometry peptide sequencing to identify binders from fully randomized synthetic libraries of 10 8 members-a 100-fold gain in diversity over standard practice. To validate this approach, we show that binders to a monoclonal antibody are identified in proportion to library diversity, as diversity is increased from 10 6-10 8. These results are then applied to the discovery of p53-like binders to MDM2, and to a family of 3-19 nM-affinity, α/β-peptide-based binders to 14-3-3. An X-ray structure of one of these binders in complex with 14-3-3σ is determined, illustrating the role of β-amino acids in facilitating a key binding contact.

The use of competitive inhibitors to disrupt protein–protein interactions (PPIs) holds great promise for the treatment of disease. However, the discovery of high-affinity inhibitors can be a challenge. Here we report a platform for improving the affinity of peptide-based PPI inhibitors using non-canonical amino acids. The platform utilizes size exclusion-based enrichment from pools of synthetic peptides (1.5–4kDa) and liquid chromatography-tandem mass spectrometry-based peptide sequencing to identify high-affinity binders to protein targets, without the need for ‘reporter’ or ‘encoding’ tags. Using this approach—which is inherently selective for high-affinity binders—we realized gains in affinity of up to ~100- or ~30-fold for binders to the oncogenic ubiquitin ligase MDM2 or HIV capsid protein C-terminal domain, which inhibit MDM2-p53 interaction or HIV capsid protein C-terminal domain dimerization, respectively. Subsequent macrocyclization of select MDM2 inhibitors rendered them cell permeable and cytotoxic toward cancer cells, demonstrating the utility of the identified compounds as functional PPI inhibitors.

The burial of hydrophobic side chains in a protein core generally is thought to be the major ingredient for stable, cooperative folding. Here, we show that, for the snow flea antifreeze protein (sfAFP), stability and cooperativity can occur without a hydrophobic core, and without α-helices or β-sheets. sfAFP has low sequence complexity with 46% glycine and an interior filled only with backbone H-bonds between six polyproline 2 (PP2) helices. However, the protein folds in a kinetically two-state manner and is moderately stable at room temperature. We believe that a major part of the stability arises from the unusual match between residue-level PP2 dihedral angle bias in the unfolded state and PP2 helical structure in the native state. Additional stabilizing factors that compensate for the dearth of hydrophobic burial include shorter and stronger H-bonds, and increased entropy in the folded state. These results extend our understanding of the origins of cooperativity and stability in protein folding, including the balance between solvent and polypeptide chain entropies.protein folding | cooperativity | kinetics | PP2 | hydrogen bonding T he basis of protein-folding stability and cooperativity remains a topic of great interest (1, 2). Most studies have focused on proteins with hydrophobic cores containing α-helices and β-sheets. Here, we study the folding of snow flea antifreeze protein (sfAFP), a globular protein that lacks these features. The 81-residue protein has a novel fold that is distinct from other proteins (3-6), containing only polyproline 2 (PP2) helices and turns with a core filled with H-bonds and no hydrophobic groups (Fig. 1).The H-bond network between the PP2 helices (6), PP2 1-6 , requires close packing, which would be precluded by the presence of a C β atom. Thus, the PP2 helices are defined by glycine (Gly) repeats (GXX or GGX, where X is any other amino acid), which enable the hydrogen-bond network. As a consequence, the sfAFP molecule has a low-complexity glycine-rich sequence [37/81 Gly residues, or 46%; typical is 6, or 7% (7)]. The sfAFP core is well packed with essentially no interior void volumes, even when probed using a 0.5-Å radius sphere (8). sfAFP also contains two disulfide bonds, C1-C28 and C13-C43.Notably, no side chains are buried in sfAFP's core (2). All 12 hydrophobic side chains (V 4 , K 2 , and P 6 ) are surface exposed. Upon folding, sfAFP buries about 20 Å 2 ·res −1 of hydrophobic surface area compared with an average of 50 Å 2 ·res −1 for a set of 34 proteins of similar size (Fig. 2A). The difference equates to a decrease in stability of ∼1 or 1.4 kcal·mol −1 ·res −1 , assuming a surface tension coefficient of γ ∼ 34 or 47 cal·mol −1 ·Å −2 based on classical (9) or Flory-Huggins theory (10), respectively. Even the lower bound of 1 kcal·mol −1 ·res −1 represents a significant stability loss, and it is not obvious which energetic terms could compensate for the reduction in hydrophobic burial.Because hydrophobic burial is generally regarded as the basis of protein stability a...

We report a site-selective cysteine-cyclooctyne conjugation reaction between a seven-residue peptide tag (DBCO-tag, Leu-Cys-Tyr-Pro-Trp-Val-Tyr), at the N or C-terminus of a peptide or protein, and various aza-dibenzocyclooctyne (DBCO) reagents. Compared to a cysteine peptide control, the DBCO-tag increases the rate of the thiol-yne reaction by 220-fold, enabling selective conjugation of DBCO-tag to DBCO-linked fluorescent probes, affinity tags, and cytotoxic drug molecules. Fusion of DBCO-tag with the protein of interest enables regioselective cysteine modification on proteins that contain multiple endogenous cysteines; these examples include green fluorescent protein and trastuzumab antibody. This study demonstrates short peptide tags could aid in accelerating bond forming reactions that are often slow to non-existent in water.

A methodology to achieve high-throughput de novo sequencing of synthetic peptide mixtures is reported. The approach leverages shotgun nanoliquid chromatography coupled with tandem mass spectrometry-based de novo sequencing of library mixtures (up to 2000 peptides) as well as automated data analysis protocols to filter away incorrect assignments, noise, and synthetic side-products. For increasing the confidence in the sequencing results, mass spectrometry-friendly library designs were developed that enabled unambiguous decoding of up to 600 peptide sequences per hour while maintaining greater than 85% sequence identification rates in most cases. The reliability of the reported decoding strategy was additionally confirmed by matching fragmentation spectra for select authentic peptides identified from library sequencing samples. The methods reported here are directly applicable to screening techniques that yield mixtures of active compounds, including particle sorting of one-bead one-compound libraries and affinity enrichment of synthetic library mixtures performed in solution.

Efficient total synthesis of insulin is important to enable the application of medicinal chemistry to the optimization of the properties of this important protein molecule. Recently we described ‘ester insulin’ – a novel form of insulin in which the function of the 35 residue C-peptide of proinsulin is replaced by a single covalent bond – as a key intermediate for the efficient total synthesis of insulin. Here we describe a fully convergent synthetic route to the ester insulin molecule from three unprotected peptide segments of approximately equal size. The synthetic ester insulin polypeptide chain folded much more rapidly than proinsulin, and at physiological pH. Both the D-protein and L-protein enantiomers of monomeric DKP ester insulin (i.e. [AspB10, LysB28, ProB29]ester insulin) were prepared by total chemical synthesis. The atomic structure of the synthetic ester insulin molecule was determined by racemic protein X-ray crystallography to a resolution of 1.6 Å. Diffraction quality crystals were readily obtained from the racemic mixture of {D-DKP ester insulin + L-DKP ester insulin}, whereas crystals were not obtained from the L-ester insulin alone even after extensive trials. Both the D-protein and L-protein enantiomers of monomeric DKP ester insulin were assayed for receptor binding and in diabetic rats, before and after conversion by saponification to the corresponding DKP insulin enantiomers. L-DKP ester insulin bound weakly to the insulin receptor, while synthetic L-DKP insulin derived from the L-DKP ester insulin intermediate was fully active in binding to the insulin receptor. The D- and L-DKP ester insulins and D-DKP insulin were inactive in lowering blood glucose in diabetic rats, while synthetic L-DKP insulin was fully active in this biological assay. The structural basis of the lack of biological activity of ester insulin is discussed.