De Novo Design of a Highly Stable Ovoid TIM Barrel:Unlocking Pocket Shape towards Functional Design

The ability to finely control the structure of protein folds is an important prerequisite to functional protein design. The TIM barrelfold is an important target for these efforts as it is highly enriched for diverse functions in nature. Although a TIM barrel proteinhas been designed de novo, the ability to finely alter the curvature of the central beta barrel and the overall architecture of the foldremains elusive, limiting its utility for functional design. Here, we report the de novo design of a TIM barrel with ovoid (twofold)symmetry, drawing inspiration from natural beta and TIM barrels with ovoid curvature. We use an autoregressive backbonesampling strategy to implement our hypothesis for elongated barrel curvature, followed by an iterative enrichment sequencedesign protocol to obtain sequences which yield a high proportion of successfully folding designs. Designed sequences arehighly stable and fold to the designed barrel curvature as determined by a 2.1Å resolution crystal structure. The designs showrobustness to drastic mutations, retaining high melting temperatures even when multiple charged residues are buried in thehydrophobic core or when the hydrophobic core is ablated to alanine. As a scaffold with a greater capacity for hosting diversehydrogen bonding networks and installation of binding pockets or active sites, the ovoid TIM barrel represents a major steptowards the de novo design of functional TIM barrels.

Biodesign Research to Advance the Principles and Applications of Biosystems Design

Over the course of civilization,humans have increasingly expanded their freedom to live a better life.In comparison with the primitive society,our modern society has many more choices of life-supporting resources,such as yearround food supply,permanent shelters,diverse energy sources,and effective preventive and curing medicine.However,our society is currently still heavily relying on the resources provided by Mother Nature,which cannot meet the future global needs in terms of both quantity and quality under the pressure of population growth,natural resource reduction,and environmental deterioration.For example,the food sources originating from plants,animals,or microbes do not have the nutrition balance for optimal human health[1–3].Climate change and environmental deterioration threaten the food security[4–6].Increasingly,infectious diseases(e.g.,HIV/AIDS),genetic diseases(e.g.,cancer),and improper lifestyle-related disorders(e.g.,obesity)become more prevalent and remain challenging to be prevented,controlled,and cured.Conventional medical technologies and modern medicine development are also meeting the ceiling.

Fragment antigen binding domains (F_(ab)s) as tools to study assembly-line polyketide synthases

The crystallization of proteins remains a bottleneck in our fundamental understanding of their functions.Therefore,discovering tools that aid crystallization is crucial.In this review,the versatility of fragment-antigen binding domains(F_(ab)s)as protein crystallization chaperones is discussed.F_(ab)s have aided the crystallization of membrane-bound and soluble proteins as well as RNA.The ability to bind three F_(ab)s onto a single protein target has demonstrated their potential for crystallization of challenging proteins.We describe a high-throughput workflow for identifying F_(ab)s to aid the crystallization of a protein of interest(POI)by leveraging phage display technologies and differential scanning fluorimetry(DSF).This workflow has proven to be especially effective in our structural studies of assembly-line polyketide synthases(PKSs),which harbor flexible domains and assume transient conformations.PKSs are of interest to us due to their ability to synthesize an unusually broad range of medicinally relevant compounds.Despite years of research studying these megasynthases,their overall topology has remained elusive.One F ab in particular,1B2,has successfully enabled X-ray crystallographic and single particle cryo-electron microscopic(cryoEM)analyses of multiple modules from distinct assembly-line PKSs.Its use has not only facilitated multidomain protein crystallization but has also enhanced particle quality via cryoEM,thereby enabling the visualization of intact PKS modules at near-atomic(3–5Å)resolution.The identification of PKS-binding F_(ab)s can be expected to continue playing a key role in furthering our knowledge of polyketide biosynthesis on assembly-line PKSs.

MRBLES 2.0: High-throughput generation of chemically functionalized spectrally and magnetically encoded hydrogel beads using a simple single-layer microfluidic device

The widespread adoption of bead-based multiplexed bioassays requires the ability to easily synthesize encoded microspheres and conjugate analytes of interest to their surface.Here,we present a simple method(MRBLEs 2.0)for the efficient high-throughput generation of microspheres with ratiometric barcode lanthanide encoding(MRBLEs)that bear functional groups for downstream surface bioconjugation.Bead production in MRBLEs 2.0 relies on the manual mixing of lanthanide/polymer mixtures(each of which comprises a unique spectral code)followed by droplet generation using single-layer,parallel flow-focusing devices and the off-chip batch polymerization of droplets into beads.To streamline downstream analyte coupling,MRBLEs 2.0 crosslinks copolymers bearing functional groups on the bead surface during bead generation.Using the MRBLEs 2.0 pipeline,we generate monodisperse MRBLEs containing 48 distinct well-resolved spectral codes with high throughput(>150,000/min and can be boosted to 450,000/min).We further demonstrate the efficient conjugation of oligonucleotides and entire proteins to carboxyl MRBLEs and of biotin to amino MRBLEs.Finally,we show that MRBLEs can also be magnetized via the simultaneous incorporation of magnetic nanoparticles with only a minor decrease in the potential code space.With the advantages of dramatically simplified device fabrication,elimination of the need for custom-made equipment,and the ability to produce spectrally and magnetically encoded beads with direct surface functionalization with high throughput,MRBLEs 2.0 can be directly applied by many labs towards a wide variety of downstream assays,from basic biology to diagnostics and other translational research.