I Experimental Procedures p. S2 II Supplementary Tables p. S9 III Supplementary Figures p. S14 IV Characterization of Reaction Products p. S28 V NMR Spectra p. S31 VI Nucleotide and Amino Acid Sequences of P411 Variants p. S39 VII Supplementary References p. S44 S2 I. Experimental Procedures General Unless otherwise noted, all chemicals and reagents were obtained from commercial suppliers (Sigma-Aldrich, VWR, Alfa Aesar) and used without further purification. Silica gel chromatography was carried out using AMD Silica Gel 60, 230-400 mesh. Synthetic reactions were monitored using thin layer chromatography (Merck 60 gel plates) using a UV-lamp for visualization. 1 H and 13 C NMR spectra were recorded on a Varian Inova 300 MHz or 500 MHz, or Bruker Prodigy 400 MHz instrument, in CDCl3 and are internally referenced to the residual solvent peak. Data for 1 H NMR are reported as follows: chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet, dd = doublet of doublets, dt = doublet of triplets, ddd = doublet of doublet of doublets), coupling constant (Hz), integration. High-resolution mass spectra were obtained at the California Institute of Technology Mass Spectrometry Facility. Chromatography. Analytical reversed-phase high-performance liquid chromatography (HPLC) was carried out using an Agilent 1200 series instrument with water and acetonitrile as mobile phases using an Agilent XDB-C18 column (4.6 x 150 mm, 5 µm). For quantitative HPLC analyses of reaction products, calibration curves using either 1,3,5-trimethoxybenzene or 1-phenyl-3propanol as internal standards were generated (Figures S11 to S20). Preparative-scale HPLC to purify enzymatic reaction products was performed using an Agilent XDB-C18 column (9.4 x 250 mm, 5 µm). The identity of enzymatic reaction products was confirmed by NMR analysis of products isolated from enzymatic reactions performed on preparative scale (vide infra). Cloning and site-saturation mutagenesis. Plasmid pET22b(+) (Novagen) with the pelB leader sequence removed was used as a cloning and expression vector for all constructs described in this study (see p. S39 for DNA sequences). Site-saturation libraries were generated using the 22c-trick method. 1 Primers were obtained from IDT (primer sequences are available upon request). PCR was performed using Phusion polymerase (NEB) and the resulting PCR products were digested with DpnI (NEB), gel purified (Zymo Research), repaired using the method of Gibson, 2 and used

While native scaffolds offer a large diversity of shapes and topologies for enzyme engineering, their often unpredictable behavior in response to sequence modification makes de novo generated scaffolds an exciting alternative. Here we explore the customization of the backbone and sequence of a de novo designed eight stranded β‐barrel protein to create catalysts for a retro‐aldolase model reaction. We show that active and specific catalysts can be designed in this fold and use directed evolution to further optimize activity and stereoselectivity. Our results support previous suggestions that different folds have different inherent amenability to evolution and this property could account, in part, for the distribution of natural enzymes among different folds.

The efficient and bioorthogonal chemical ligation reaction between potassium acyltrifluoroborates (KATs) and hydroxylamines (HAs) was used for the surface functionalizationof a self-assembled monolayer (SAM) with biomolecules. An alkane thioether molecule with one terminal KAT group (S-KAT) was synthesized and adsorbed onto a gold surface, placing a KATgroup on the top of the monolayer (KAT-SAM). As an initial test case, an aqueous solution of a hydroxylamine (HA) derivative of PEG (HA-PEG) was added to this KAT-SAM at room temperature to perform the surface KAT ligation. Quartz crystal microbalance with dissipation (QCM-D) monitoring confirmed the rapid attachment of the PEG moiety onto the SAM.The covalent conjugation of PEG by amide-bond formation was established by complementary surface characterization methods including contact angle, ellipsometry, and X-ray photoelectron spectroscopy (XPS). To test the applicability of this surface KAT ligation for the attachment of biomolecules to the surfaces, this KAT-SAM was subjected to the reaction with HA derivative of protein. A HA-derivatized green fluorescent protein (HA-GFP) was added in dilute concentrations to the KAT-SAM under aqueous conditions and rapid protein attachment was observed in real-time by QCM. Despite the fact that such biomolecules have a variety of unprotected functional groups within their structures, the surface KAT reaction proceeded efficiently in a selective manner. Our results clearly demonstrate the versatile applicability of the KAT ligation for the covalent attachment of a variety of biomolecules onto surfaces under dilute and biocompatible conditions to form stable, natural amide bonds.File list (2) download file view on ChemRxiv Surface_KAT_ACS_Mater_interface_main_text_25Mar20... (2.08 MiB) download file view on ChemRxiv Surface KAT_SI_25Mar2021_final.pdf (4.32 MiB)