Hallucination of closed repeat proteins containing central pockets.

In pseudocyclic proteins, such as TIM barrels, ß barrels, and some helical transmembrane channels, a single subunit is repeated in a cyclic pattern, giving rise to a central cavity that can serve as a pocket for ligand binding or enzymatic activity. Inspired by these proteins, we devised a deep-learning-based approach to broadly exploring the space of closed repeat proteins starting from only a specification of the repeat number and length. Biophysical data for 38 structurally diverse pseudocyclic designs produced in Escherichia coli are consistent with the design models, and the three crystal structures we were able to obtain are very close to the designed structures. Docking studies suggest the diversity of folds and central pockets provide effective starting points for designing small-molecule binders and enzymes.

De novo design of diverse small molecule binders and sensors using Shape Complementary Pseudocycles.

A general method for designing proteins to bind and sense any small molecule of interest would be widely useful. Due to the small number of atoms to interact with, binding to small molecules with high affinity requires highly shape complementary pockets, and transducing binding events into signals is challenging. Here we describe an integrated deep learning and energy based approach for designing high shape complementarity binders to small molecules that are poised for downstream sensing applications. We employ deep learning generated psuedocycles with repeating structural units surrounding central pockets; depending on the geometry of the structural unit and repeat number, these pockets span wide ranges of sizes and shapes. For a small molecule target of interest, we extensively sample high shape complementarity pseudocycles to generate large numbers of customized potential binding pockets; the ligand binding poses and the interacting interfaces are then optimized for high affinity binding. We computationally design binders to four diverse molecules, including for the first time polar flexible molecules such as methotrexate and thyroxine, which are expressed at high levels and have nanomolar affinities straight out of the computer. Co-crystal structures are nearly identical to the design models. Taking advantage of the modular repeating structure of pseudocycles and central location of the binding pockets, we constructed low noise nanopore sensors and chemically induced dimerization systems by splitting the binders into domains which assemble into the original pseudocycle pocket upon target molecule addition.

De Novo Protein Design Using the Blueprint Builder in Rosetta.

While native proteins cover diverse structural spaces and achieve various biological events, not many of them can directly serve human needs. One reason is that the native proteins usually contain idiosyncrasies evolved for their native functions but disfavoring engineering requirements. To overcome this issue, one strategy is to create de novo proteins which are designed to possess improved stability, high environmental tolerance, and enhanced engineering potential. Compared to other protein engineering strategies, in silico design of de novo proteins has significantly expanded the protein structural and sequence spaces, reduced wet lab workload, and incorporated engineered features in a guided and efficient manner. In the Baker laboratory we have been applying a design pipeline that uses the blueprint builder to design different folds of de novo proteins, and have successfully obtained libraries of de novo proteins with improved stability and engineering potential. In this article, we will use the design of de novo ß-barrel proteins as an example to describe the principles and basic procedures of the blueprint builder-based design pipeline. © 2020 Wiley Periodicals LLC. Basic Protocol 1: The construction of blueprints Alternate Protocol: Build blueprints based on existing protein .pdb files Basic Protocol 2: De novo protein design pipeline using the blueprint builder.

O-Methyltransferase-Mediated Incorporation of a ß-Amino Acid in Lanthipeptides.

Lanthipeptides represent a large class of cyclic natural products defined by the presence of lanthionine (Lan) and methyllanthionine (MeLan) cross-links. With the advances in DNA sequencing technologies and genome mining tools, new biosynthetic enzymes capable of installing unusual structural features are continuously being discovered. In this study, we investigated an O-methyltransferase that is a member of the most prominent auxiliary enzyme family associated with class I lanthipeptide biosynthetic gene clusters. Despite the prevalence of these enzymes, their function has not been established. Herein, we demonstrate that the O-methyltransferase OlvSA encoded in the olv gene cluster from Streptomyces olivaceus NRRL B-3009 catalyzes the rearrangement of a highly conserved aspartate residue to a ß-amino acid, isoaspartate, in the lanthipeptide OlvA(BCSA). We elucidated the NMR solution structure of the GluC-digested peptide, OlvA(BCSA)GluC, which revealed a unique ring topology comprising four interlocking rings and positions the isoaspartate residue in a solvent exposed loop that is stabilized by a MeLan ring. Gas chromatography-mass spectrometry analysis further indicated that OlvA(BCSA) contains two dl-MeLan rings and two Lan rings with an unusual ll-stereochemistry. Lastly, in vitro reconstitution of OlvSA activity showed that it is a leader peptide-independent and S-adenosyl methionine-dependent O-methyltransferase that mediates the conversion of a highly conserved aspartate residue in a cyclic substrate into a succinimide, which is hydrolyzed to generate an Asp or isoAsp containing peptide. This overall transformation converts an α-amino acid into a ß-amino acid in a ribosomally synthesized peptide, via an electrophilic intermediate that may be the intended product.

Substrate-assisted enzymatic formation of lysinoalanine in duramycin.

Duramycin is a heavily post-translationally modified peptide that binds phosphatidylethanolamine. It has been investigated as an antibiotic, an inhibitor of viral entry, a therapeutic for cystic fibrosis, and a tumor and vasculature imaging agent. Duramycin contains a ß-hydroxylated Asp (Hya) and four macrocycles, including an essential lysinoalanine (Lal) cross-link. The mechanism of Lal formation is not known. Here we show that Lal is installed stereospecifically by DurN via addition of Lys19 to a dehydroalanine. The structure of DurN reveals an unusual dimer with a new fold. Surprisingly, in the structure of duramycin bound to DurN, no residues of the enzyme are near the Lal cross-link. Instead, Hya15 of the substrate makes interactions with Lal, suggesting it acts as a base to deprotonate Lys19 during catalysis. Biochemical data suggest that DurN preorganizes the reactive conformation of the substrate, such that the Hya15 of the substrate can serve as the catalytic base for Lal formation.

Enzyme-Controlled Intracellular Self-Assembly of (18)F Nanoparticles for Enhanced MicroPET Imaging of Tumor.

Herein, we report the development of a new "smart" radioactive probe (i.e., 1) which can undergo furin-controlled condensation and self-assembly of radioactive nanoparticles (i.e., 1-NPs) in tumor cells and its application for enhanced microPET imaging of tumors in nude mice co-injected with its cold analog (i.e., 1-Cold). Furin-controlled condensation of 1-Cold and self-assembly of its nanoparticles (i.e., 1-Cold-NPs) in vitro were validated and characterized with HPLC, mass spectra, SEM, and TEM analyses. Cell uptake studies showed that both 1 and 1-Cold have good cell permeability. TEM images of 1-Cold-treated MDA-MB-468 cells directly uncovered that the intracellular 1-Cold-NPs were at/near the location of furin (i.e., Golgi bodies). MTT results indicated that 50 µM 1-Cold did not impose cytotoxicity to MDA-MB-468 cells up to 12 hours. MicroPET imaging of MDA-MB-468 tumor-bearing mice indicated that mice co-injected with 1 and 1-Cold showed higher uptake and longer attenuation of the radioactivity in tumors than those mice only injected with same dosage of 1. Tumor uptake ratios of 1 between these two groups of mice reached the maximum of 8.2 folds at 240 min post injection. Biodistribution study indicated that the uptake ratios of 1 in kidneys between these two groups continuously increased and reached 81.9 folds at 240 min post injection, suggesting the formation of radioactive NPs (i.e., 1-NPs) in MDA-MB-468 tumors of mice co-injected with 1 and 1-Cold. And the nanoparticles were slowly digested and secreted from the tumors, accumulating in the kidneys. Our ''smart'' probe (i.e., 1), together with the strategy of co-injection, might help researchers trace the biomarkers of interest within a longer time window.

Intracellular Self-Assembly of Taxol Nanoparticles for Overcoming Multidrug Resistance.

Multidrug resistance (MDR) remains the biggest challenge in treating cancers. Herein we propose the intracellular self-assembly of nanodrugs as a new strategy for overcoming MDR. By employing a biocompatible condensation reaction, we rationally designed a taxol derivative Ac-Arg-Val-Arg-Arg-Cys(StBu)-Lys(taxol)-2-cyanobenzothiazole (CBT-Taxol) which could be subjected to furin-controlled condensation and self-assembly of taxol nanoparticles (Taxol-NPs). In vitro and in vivo studies indicated that, compared with taxol, CBT-Taxol showed a 4.5-fold or 1.5-fold increase in anti-MDR effects, respectively, on taxol-resistant HCT 116 cancer cells or tumors without being toxic to the cells or the mice. Our results demonstrate that structuring protease-susceptible agents and assembling them intracellularly into nanodrugs could be a new optimal strategy for overcoming MDR.

Intracellular Disassembly of Self-Quenched Nanoparticles Turns NIR Fluorescence on for Sensing Furin Activity in Cells and in Tumors.

There has been no report on enzyme-controlled disassembly of self-quenched NIR fluorescent nanoparticles turning fluorescence on for specific detection/imaging of the enzyme's activity in vitro and in vivo. Herein, we reported the rational design of new NIR probe 1 whose fluorescence signal was self-quenched upon reduction-controlled condensation and subsequent assembly of its nanoparticles (i.e., 1-NPs). Then disassembly of 1-NPs by furin turned the fluorescence on. Employing this enzymatic strategy, we successfully applied 1-NPs for NIR detection of furin in vitro and NIR imaging furin activity in living cells. Moreover, we also applied 1-NPs for discriminative NIR imaging of MDA-MB-468 tumors in nude mice. This NIR probe 1 might be further developed for tumor-targeted imaging in routine preclinical studies or even in patients in the future.

Intracellular Self-Assembly and Disassembly of (19)F Nanoparticles Confer Respective "Off" and "On" (19)F NMR/MRI Signals for Legumain Activity Detection in Zebrafish.

(19)F MRI has higher selectivity but lower sensitivity than (1)H MRI for in vivo diagnosis. Therefore, to avoid using a high injection dose of the (19)F probe while, in the meantime, maintaining the high sensitivity of (19)F MRI has remained challenging. Local self-assembly and disassembly of (19)F nanoparticles could be one of the "smart" strategies to achieve this goal. Herein, we report a dual-functional probe 1 for glutathione (GSH)-controlled self-assembly and subsequent legumain (Lgmn)-controlled disassembly of its nanoparticles (i.e., 1-NPs). Self-assembly and disassembly of 1-NPs confer (19)F magnetic resonance (MR) signals "off" and "on", respectively. Employing this strategy, we successfully applied 1 for consecutive detections of GSH and Lgmn in vitro and in cells, imaging Lgmn activity in HEK 293T tumors in zebrafish at a low dosage under 14.1 T.

Controlled intracellular self-assembly and disassembly of 19F nanoparticles for MR imaging of caspase 3/7 in zebrafish.

Compared to (1)H MRI, (19)F MRI provides higher selectivity but lower sensitivity. Therefore, the need to inject high doses of the (19)F probe to improve its sensitivity for in vivo diagnosis remains a challenge. A "smart" strategy is needed that could locally concentrate a low-dose (19)F probe while avoiding the fast transverse relaxation of the probes. Locally self-assembling and disassembling (19)F nanoparticles may be an optimal measure to achieve this goal. Herein, we report a dual-functional probe 1 for glutathione (GSH)-controlled self-assembly and subsequent caspase 3/7 (Casp3/7)-controlled disassembly of formed nanoparticles (i.e., 1-NPs). Consecutive assembly and disassembly of 1-NPs translate to "off" and "on" (19)F magnetic resonance (MR) signal states, respectively. Employing this smart strategy, we successfully used 1 for the consecutive detection of GSH and Casp3/7 activity in vitro and in cells and imaging Casp3/7 activity in cells and in zebrafish at low doses with a 14.1 T magnetic field.

Multifunctional electroactive heteroatom-doped carbon aerogels.

The design and synthesis of highly active, durable, and cheap nanomaterials for various renewable energy storage and conversion applications is extremely desirable but remains challenging. Here, a green and efficient strategy to produce CoOx nanoparticles and surface N-co-doped carbon aerogels (Co-N-CAs) is reported by multicomponent surface self-assembly of commercially melamine sponge (CMS). In the methodology, the CMS simultaneously function as green N precursor for surface N doping and 3D support. The resulting Co-N-CAs exhibit 3D hierarchical, interconnected macro- and bimodal meso-porosity (6.3 nm and <4 nm), high surface area (1383 m(2) g(-1)), and highly dispersed, semi-exposured CoOx nanoparticles (diameter of 12.5 nm). The surface doping of N, semi-exposured configuration of CoOx nanoparticles and the penetrated complementary pores (<4 nm) in the carbon walls provide highly accessibility between electroactive components and electrolytes to improve reactivity. With their tailored architecture, the Co-N-CAs show superior electrocatalytic oxygen reduction (ORR) activities comparable to the commercially Pt/C catalysts, high specific capacitance (433 F g(-1)), excellent lithium storage (938 mAh g(-1)), and outstanding durability, making them very promising for advanced energy conversion and storage. In addition, the presented strategy can be extended to fabricate other metal oxide- and N-co-doped carbon aerogels for diverse energy-related applications.

Oligomeric nanoparticles functionalized with NIR-emitting CdTe/CdS QDs and folate for tumor-targeted imaging.

We report herein the facile surface-functionalization of one type of biocompatible, oligomeric nanoparticles 1-NPs with NIR-emitting CdTe/CdS QDs and folate for tumor-targeted imaging in vivo. The -NH2 and -SH groups of cysteine residues on the 1-NPs were utilized to covalently conjugate CdTe/CdS QDs and Mal-FA to prepare the hybrid nanoparticles 1-NPs-QDs-FA. As-prepared 1-NPs-QDs-FA showed NIR-fluorescence emission at 734 nm, selective uptake by FR-overexpressing tumor cells in vitro, and selective FR-overexpressing tumor-targeted imaging in vivo. This first example of oligomeric/inorganic hybrid nanoparticles provides people with new type of biomaterials for tumor-targeted imaging with high selectivity.

Using magnetic resonance imaging to study enzymatic hydrogelation.

Herein, we report, for the first time, the use of MRI methods to study enzymatic hydrogelation. Supramolecular hydrogels have been exploited as biomaterials for many applications. However, behaviors of the water molecules encapsulated in hydrogels have not been fully understood. In this work, we designed a precursor 1 which could self-assemble into nanofibers and form hydrogel I (gel I) upon the catalysis of phosphatase. The differences of mechanic property, pore size, water diffusion rate, and magnetic resonance relaxation times T1 and T2 of gel I containing different concentrations of 1 were systematically studied and analyzed. T1, T2, and diffusion-weighted (1)H MR images from gel I phantoms were obtained at 9.4 T. Analyses of the MRI data uncovered how the density of the nanofiber networks affects the relaxation behaviors of the water protons encapsulated in such hydrogels. Rheological analyses and cryo-TEM observations showed increased gel elasticities with increased concentrations of 1 while the pore sizes of gel I decreased. This also resulted in an increase in the proton relaxation rate (i.e., shortened T1, T2, and apparent diffusion coefficient (ADC)) for the water encapsulated in the hydrogel. With MRI, our study provides a new in vitro method to potentially mimic and study in vivo diseases that involve fibrous aggregates.

Fluorescent switch for fast and selective detection of mercury (II) ions in vitro and in living cells and a simple device for its removal.

A water-soluble, biocompatible, and fluorescent chemosensor (1) for label-free, simple, and fast detection of mercury ions (Hg(2+)) in aqueous solutions and in HepG2 cells with high selectivity is reported herein. Chelation of 1 with Hg(2+) results in the disappearance of its fluorescence emission at 350 nm and the appearance of a new emission at 405 nm. Selectivity and interference studies indicated that 1 could be selectively chelated by Hg(2+) without interference from other metal ions. Insight into the mechanisms responsible for its fluorescence effect was gained from ultrafast transient absorption spectroscopy. With these properties, 1 was successfully applied for imaging Hg(2+) in living cells and for removing Hg(2+) from river water. Moreover, we also constructed a simple device for fast and effective removal of Hg(2+) from contaminated liquid samples.

Peptide-based nanostructures for cancer diagnosis and therapy.

Peptide-based nanoparticles (pep-NPs) are emerging as promising imaging and therapeutic agents against cancer due to their biocompatibility and tunability. Optimized design of the peptide sequence and moderate conjugation of the sequence with extraneous molecules are crucial to the performance of the inherent properties of pep-NPs such as nanostructure formation and ability of drug delivery. Meanwhile, the peptide sequences based on natural/unnatural amino acids could be utilized for designing nanostructures susceptive/resistant to specific enzymes. Herein, we firstly summarize the basic peptide structures to provide a whole image of pep-NPs. Subsequently, the diagnostic strategies based on different imaging modalities and recent therapeutic applications of pep-NPs for cancer are reviewed.

Labeling thiols on proteins, living cells, and tissues with enhanced emission induced by FRET.

Using N-(2-Aminoethyl)maleimide-cysteine(StBu) (Mal-Cys) as a medium, protein thiols were converted into N-terminal cysteines. After a biocompatible condensation reaction between the N-terminal cysteine and fluorescent probe 2-cyanobenzothiazole-Gly-Gly-Gly-fluorescein isothiocyanate (CBT-GGG-FITC), a new fluorogenic structure Luciferin-GGG-FITC was obtained. The latter exhibits near one order of magnitude (7 folds) enhanced fluorescence emission compared to the precursor moiety due to fluorescence resonance energy transfer (FRET) effect between the newly formed luciferin structure and the FITC motif. Theoretical investigations revealed the underlying mechanism that satisfactorily explained the experimental results. With this method, enhanced fluorescence imaging of thiols on proteins, outer membranes of living cells, translocation of membrane proteins, and endothelial cell layers of small arteries was successfully achieved.