scholarly journals Rational construction of compact de novo-designed biliverdin-binding proteins

2018 ◽  
Author(s):  
Molly M. Sheehan ◽  
Michael S. Magaraci ◽  
Ivan A. Kuznetsov ◽  
Joshua A. Mancini ◽  
Goutham Kodali ◽  
...  
Keyword(s):  
Protein Design ◽  
De Novo ◽  
Binding Pocket ◽  
Heptad Repeat ◽  
Hydrophobic Core ◽  
Protein Scaffold ◽  
Self Assembling ◽  
High Resolution Structure ◽  
Rational Construction

Abstract:We report the rational construction of a de novo-designed biliverdin-binding protein by first principles of protein design, informed by energy minimization modeling in Rosetta. The self-assembling tetrahelical bundles bind biliverdin IXa (BV) cofactor auto-catalytically in vitro, similar to photosensory proteins that bind BV (and related bilins, or linear tetrapyrroles) despite lacking sequence and structural homology to the natural counterparts. Upon identifying a suitable site for cofactor ligation to the protein scaffold, stepwise placement of residues stabilized BV within the hydrophobic core. Rosetta modeling was used in the absence of a high-resolution structure to define the structure-function of the binding pocket. Holoprotein formation indeed stabilized BV, resulting in increased far-red BV fluorescence. By removing segments extraneous to cofactor stabilization or bundle stability, the initial 15-kilodalton de novo-designed fluorescence-activating protein (“dFP”) was truncated without altering its optical properties, down to a miniature 10-kilodalton “mini,” in which the protein scaffold extends only a half-heptad repeat beyond the hypothetical position of the bilin D-ring. This work demonstrates how highly compact holoprotein fluorochromes can be rationally constructed using de novo protein design technology and natural cofactors.

scholarly journals Computational redesign of a fluorogen activating protein with Rosetta

2021 ◽  
Vol 17 (11) ◽  
pp. e1009555
Author(s):  
Nina G. Bozhanova ◽  
Joel M. Harp ◽  
Brian J. Bender ◽  
Alexey S. Gavrikov ◽  
Dmitry A. Gorbachev ◽  
...  
Keyword(s):  
Protein Design ◽  
De Novo ◽  
Binding Pocket ◽  
Fluorescent Imaging ◽  
Fine Tuning ◽  
Ligand Docking ◽  
Pose Prediction ◽  
Functional Protein ◽  
Split System

The use of unnatural fluorogenic molecules widely expands the pallet of available genetically encoded fluorescent imaging tools through the design of fluorogen activating proteins (FAPs). While there is already a handful of such probes available, each of them went through laborious cycles of in vitro screening and selection. Computational modeling approaches are evolving incredibly fast right now and are demonstrating great results in many applications, including de novo protein design. It suggests that the easier task of fine-tuning the fluorogen-binding properties of an already functional protein in silico should be readily achievable. To test this hypothesis, we used Rosetta for computational ligand docking followed by protein binding pocket redesign to further improve the previously described FAP DiB1 that is capable of binding to a BODIPY-like dye M739. Despite an inaccurate initial docking of the chromophore, the incorporated mutations nevertheless improved multiple photophysical parameters as well as the overall performance of the tag. The designed protein, DiB-RM, shows higher brightness, localization precision, and apparent photostability in protein-PAINT super-resolution imaging compared to its parental variant DiB1. Moreover, DiB-RM can be cleaved to obtain an efficient split system with enhanced performance compared to a parental DiB-split system. The possible reasons for the inaccurate ligand binding pose prediction and its consequence on the outcome of the design experiment are further discussed.

scholarly journals De novo design of self-assembling helical protein filaments

Science ◽  
2018 ◽  
Vol 362 (6415) ◽  
pp. 705-709 ◽  
Author(s):  
Hao Shen ◽  
Jorge A. Fallas ◽  
Eric Lynch ◽  
William Sheffler ◽  
Bradley Parry ◽  
...  
Keyword(s):  
De Novo ◽  
Computational Design ◽  
Building Blocks ◽  
Repeat Units ◽  
Self Assembling ◽  
Wide Range ◽  
Helical Protein ◽  
Monomeric Proteins

We describe a general computational approach to designing self-assembling helical filaments from monomeric proteins and use this approach to design proteins that assemble into micrometer-scale filaments with a wide range of geometries in vivo and in vitro. Cryo–electron microscopy structures of six designs are close to the computational design models. The filament building blocks are idealized repeat proteins, and thus the diameter of the filaments can be systematically tuned by varying the number of repeat units. The assembly and disassembly of the filaments can be controlled by engineered anchor and capping units built from monomers lacking one of the interaction surfaces. The ability to generate dynamic, highly ordered structures that span micrometers from protein monomers opens up possibilities for the fabrication of new multiscale metamaterials.

scholarly journals Structure of the Lifeact–F-actin complex

PLoS Biology ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. e3000925 ◽  
Author(s):  
Alexander Belyy ◽  
Felipe Merino ◽  
Oleg Sitsel ◽  
Stefan Raunser
Keyword(s):  
Hypervariable Region ◽  
Binding Pocket ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Activity Measurements ◽  
High Resolution Structure ◽  
D Loop ◽  
Hydrophobic Binding

Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact–F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as the hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.

scholarly journals De novo design of potent and resilient hACE2 decoys to neutralize SARS-CoV-2

Science ◽  
2020 ◽  
Vol 370 (6521) ◽  
pp. 1208-1214 ◽  
Author(s):  
Thomas W. Linsky ◽  
Renan Vergara ◽  
Nuria Codina ◽  
Jorgen W. Nelson ◽  
Matthew J. Walker ◽  
...  
Keyword(s):  
Protein Design ◽  
De Novo ◽  
High Specificity ◽  
Spike Protein ◽  
Host Proteins ◽  
Angiotensin Converting Enzyme 2 ◽  
Cryo Electron Microscopy ◽  
De Novo Protein Design ◽  
Single Spike

We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo human angiotensin-converting enzyme 2 (hACE2) decoys to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The best monovalent decoy, CTC-445.2, bound with low nanomolar affinity and high specificity to the receptor-binding domain (RBD) of the spike protein. Cryo–electron microscopy (cryo-EM) showed that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, showed ~10-fold improvement in binding. CTC-445.2d potently neutralized SARS-CoV-2 infection of cells in vitro, and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge.

scholarly journals Design of self-assembling transmembrane helical bundles to elucidate principles required for membrane protein folding and ion transport

2017 ◽  
Vol 372 (1726) ◽  
pp. 20160214 ◽  
Author(s):  
Nathan H. Joh ◽  
Gevorg Grigoryan ◽  
Yibing Wu ◽  
William F. DeGrado
Keyword(s):  
Protein Folding ◽  
Membrane Protein ◽  
Protein Design ◽  
De Novo ◽  
Cell Signalling ◽  
Membrane Protein Folding ◽  
Cellular Processes ◽  
Membrane Pores ◽  
Self Assembling ◽  
And Function

Ion transporters and channels are able to identify and act on specific substrates among myriads of ions and molecules critical to cellular processes, such as homeostasis, cell signalling, nutrient influx and drug efflux. Recently, we designed Rocker, a minimalist model for Zn 2+ /H + co-transport. The success of this effort suggests that de novo membrane protein design has now come of age so as to serve a key approach towards probing the determinants of membrane protein folding, assembly and function. Here, we review general principles that can be used to design membrane proteins, with particular reference to helical assemblies with transport function. We also provide new functional and NMR data that probe the dynamic mechanism of conduction through Rocker. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

scholarly journals Cryo-EM structure and inhibitor design of human IAPP (amylin) fibrils

2020 ◽  
Author(s):  
Qin Cao ◽  
David R. Boyer ◽  
Michael R. Sawaya ◽  
Peng Ge ◽  
David S. Eisenberg
Keyword(s):  
Amyloid Fibrils ◽  
Full Length ◽  
Hydrophobic Core ◽  
Inhibitor Design ◽  
Islet Amyloid ◽  
Fibril Structure ◽  
Starting Point ◽  
High Resolution Structure ◽  
Aggregation Inhibitors

AbstractHuman islet amyloid polypeptide (hIAPP, or amylin) is a 37 amino acid hormone secreted by pancreatic islet β-cells. Aggregation of hIAPP into amyloid fibrils is found in more than 90% of Type-II Diabetes (T2D) patients and is considered to be associated with T2D pathology. Although different models have been proposed, the high resolution structure of hIAPP fibrils is unknown. Here we report the cryo-EM structure of recombinant full-length hIAPP fibrils. The fibril is composed of two symmetrically-related protofilaments with ordered residues 14-37 that meet at a 14-residue central hydrophobic core. Our hIAPP fibril structure (i) supports the previous hypothesis that residues 20-29, especially 23-29 are the primary amyloid core of hIAPP, (ii) suggests a molecular mechanism for the action of the hIAPP hereditary mutation S20G, (iii) explains why the 6 residue substitutions in rodent IAPP prevent aggregation, and (iv) suggests possible regions responsible for the observed hIAPP cross-seeding with β-amyloid. Furthermore, we performed structure-based inhibitor design to generate potential hIAPP aggregation inhibitors via a capping strategy. Four of the designed candidates delay hIAPP aggregation in vitro, providing a starting point for the development of T2D therapeutics and proof-of-concept that the capping strategy can be used on full-length cryo-EM fibril structures.

scholarly journals Structural and functional evaluation of de novo-designed, two-component nanoparticle carriers for HIV Env trimer immunogens

2020 ◽  
Author(s):  
Aleksandar Antanasijevic ◽  
George Ueda ◽  
Philip JM Brouwer ◽  
Jeffrey Copps ◽  
Deli Huang ◽  
...  
Keyword(s):  
De Novo ◽  
Functional Characterization ◽  
Functional Evaluation ◽  
Self Assembling ◽  
Immunization Strategies ◽  
Antigenic Properties ◽  
Two Component ◽  
Env Antigens

AbstractTwo-component, self-assembling nanoparticles represent a versatile platform for multivalent presentation of viral antigens. Nanoparticles of different sizes and geometries can be designed and combined with appropriate antigens to fit the requirements of different immunization strategies. Here, we describe detailed antigenic, structural, and functional characterization of computationally designed tetrahedral, octahedral, and icosahedral nanoparticle immunogens displaying trimeric HIV envelope glycoprotein (Env) ectodomains. Env trimers, based on subtype A (BG505) or consensus group M (ConM) sequences and engineered with SOSIP stabilizing mutations, were fused to the underlying trimeric building block of each nanoparticle. Initial screening yielded one icosahedral and two tetrahedral nanoparticle candidates, capable of presenting twenty or four copies of the Env trimer. A number of analyses, including detailed structural characterization by cryo-EM, demonstrated that the nanoparticle immunogens possessed the intended structural and antigenic properties. Comparing the humoral responses elicited by ConM-SOSIP trimers presented on a two-component tetrahedral nanoparticle to the corresponding soluble protein revealed that multivalent presentation increased the proportion of the overall antibody response directed against autologous neutralizing Ab epitopes present on the ConM-SOSIP trimers.Author SummaryProtein constructs based on soluble ectodomains of HIV glycoprotein (Env) trimers are the basis of many current HIV vaccine platforms. Multivalent antigen display is one strategy applied to improve the immunogenicity of different subunit vaccine candidates. Here, we describe and comprehensively evaluate a library of de novo designed, protein nanoparticles of different geometries for their ability to present trimeric Env antigens. We found three nanoparticle candidates that can stably incorporate model Env trimer on their surface while maintaining its structure and antigenicity. Immunogenicity of the designed nanoparticles is assessed in vitro and in vivo. In addition to introducing a novel set of reagents for multivalent display of Env trimers, this work provides both guiding principles and a detailed experimental roadmap for the generation, characterization, and optimization of Env-presenting, self-assembling nanoparticle immunogens.

scholarly journals Single-component multilayered self-assembling nanoparticles presenting rationally designed glycoprotein trimers as Ebola virus vaccines

2020 ◽  
Author(s):  
Linling He ◽  
Anshul Chaudhary ◽  
Xiaohe Lin ◽  
Cindy Sou ◽  
Sonu Kumar ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Ebola Virus ◽  
Single Component ◽  
Engineered Nanoparticles ◽  
Heptad Repeat ◽  
T Cell Epitopes ◽  
Self Assembling ◽  
Next Generation Sequencing Ngs ◽  
Insight Into

ABSTRACTThe Ebola virus (EBOV) glycoprotein (GP) can be recognized by neutralizing antibodies (NAbs) and is the main target for vaccine design. We first investigated the contribution of the stalk and the heptad repeat 1-C (HR1C) region to GP metastability. Specific stalk and HR1C modifications in a mucin-deleted form (GPΔmuc) increased trimer yield, while alteration of HR1C exerted a more complex effect on thermostability. Crystal structures were determined to validate these rationally designed GPΔmuc trimers in their unliganded state. We then displayed a modified GPΔmuc trimer on engineered nanoparticles with encapsulated locking domains (LD) and a cluster of helper T-cell epitopes. In mice and rabbits, GP trimers and nanoparticles elicited cross-ebolavirus NAbs as well as non-NAbs that enhance in-vitro infection. Next-generation sequencing (NGS) revealed B-cell profiles that are specific to vaccine platforms. This study provides insight into GP metastability and paves the way for development of an effective, pan-ebolavirus nanoparticle vaccine.TEASERWe rationally redesigned the EBOV glycoprotein and engineered single-component multilayered nanoparticles as vaccine candidates

Self-Assembling Protein Scaffold System for Easy in Vitro Coimmobilization of Biocatalytic Cascade Enzymes

ACS Catalysis ◽  
2018 ◽  
Vol 8 (6) ◽  
pp. 5611-5620 ◽  
Author(s):  
Guoqiang Zhang ◽  
Maureen B. Quin ◽  
Claudia Schmidt-Dannert
Keyword(s):  
Protein Scaffold ◽  
Self Assembling

scholarly journals Development and Characterization of Lecithin-based Self-assembling Mixed Polymeric Micellar (saMPMs) Drug Delivery Systems for Curcumin

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Ling-Chun Chen ◽  
Yin-Chen Chen ◽  
Chia-Yu Su ◽  
Wan-Ping Wong ◽  
Ming-Thau Sheu ◽  
...  
Keyword(s):  
Particle Size ◽  
Polymeric Micelles ◽  
Drug Loading ◽  
Physical Stability ◽  
Sodium Deoxycholate ◽  
Hydrophobic Core ◽  
Self Assembling ◽  
Human Disorders

Abstract Self-assembling mixed polymeric micelles (saMPMs) were developed for overcoming major obstacles of poor bioavailability (BA) associated with curcumin delivery. Lecithin added was functioned to enlarge the hydrophobic core of MPMs providing greater solubilization capacity. Amphiphilic polymers (sodium deoxycholate [NaDOC], TPGS, CREMOPHOR, or a PLURONIC series) were examined for potentially self-assembling to form MPMs (saMPMs) with the addition of lecithin. Particle size, size distribution, encapsulation efficacy (E.E.), and drug loading (D.L.) of the mixed micelles were optimally studied for their influences on the physical stability and release of encapsulated drugs. Overall, curcumin:lecithin:NaDOC and curcumin:lecithin:PLURONIC P123 in ratios of 2:1:5 and 5:2:20, respectively, were optimally obtained with a particle size of < 200 nm, an E.E. of >80%, and a D.L. of >10%. The formulated system efficiently stabilized curcumin in phosphate-buffered saline (PBS) at room temperature or 4 °C and in fetal bovine serum or PBS at 37 °C and delayed the in vitro curcumin release. In vivo results further demonstrated that the slow release of curcumin from micelles and prolonged duration increased the curcumin BA followed oral and intravenous administrations in rats. Thus, lecithin-based saMPMs represent an effective curcumin delivery system, and enhancing BA of curcumin can enable its wide applications for treating human disorders.

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