scholarly journals De novo design of transmembrane β barrels

Science ◽  
2021 ◽  
Vol 371 (6531) ◽  
pp. eabc8182
Author(s):  
Anastassia A. Vorobieva ◽  
Paul White ◽  
Binyong Liang ◽  
Jim E. Horne ◽  
Asim K. Bera ◽  
...  
Keyword(s):  
Single Molecule ◽  
Lipid Membranes ◽  
Computational Models ◽  
De Novo ◽  
Computational Design ◽  
X Ray ◽  
Custom Design ◽  
Wide Range ◽  
Pore Properties ◽  
Β Sheet

Transmembrane β-barrel proteins (TMBs) are of great interest for single-molecule analytical technologies because they can spontaneously fold and insert into membranes and form stable pores, but the range of pore properties that can be achieved by repurposing natural TMBs is limited. We leverage the power of de novo computational design coupled with a “hypothesis, design, and test” approach to determine TMB design principles, notably, the importance of negative design to slow β-sheet assembly. We design new eight-stranded TMBs, with no homology to known TMBs, that insert and fold reversibly into synthetic lipid membranes and have nuclear magnetic resonance and x-ray crystal structures very similar to the computational models. These advances should enable the custom design of pores for a wide range of applications.

scholarly journals De novo design of transmembrane β-barrels

2020 ◽  
Author(s):  
Anastassia A. Vorobieva ◽  
Paul White ◽  
Binyong Liang ◽  
Jim E Horne ◽  
Asim K. Bera ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Single Molecule ◽  
First Principles ◽  
Lipid Membranes ◽  
De Novo ◽  
Computational Design ◽  
Protein Sequencing ◽  
Naturally Occurring ◽  
Transmembrane Pores ◽  
Almost All

AbstractThe ability of naturally occurring transmembrane β-barrel proteins (TMBs) to spontaneously insert into lipid bilayers and form stable transmembrane pores is a remarkable feat of protein evolution and has been exploited in biotechnology for applications ranging from single molecule DNA and protein sequencing to biomimetic filtration membranes. Because it has not been possible to design TMBs from first principles, these efforts have relied on re-engineering of naturally occurring TMBs that generally have a biological function very different from that desired. Here we leverage the power of de novo computational design coupled with a “hypothesis, design and test” approach to determine principles underlying TMB structure and folding, and find that, unlike almost all other classes of protein, locally destabilizing sequences in both the β-turns and β-strands facilitate TMB expression and global folding by modulating the kinetics of folding and the competition between soluble misfolding and proper folding into the lipid bilayer. We use these principles to design new eight stranded TMBs with sequences unrelated to any known TMB and show that they insert and fold into detergent micelles and synthetic lipid membranes. The designed proteins fold more rapidly and reversibly in lipid membranes than the TMB domain of the model native protein OmpA, and high resolution NMR and X-ray crystal structures of one of the designs are very close to the computational model. The ability to design TMBs from first principles opens the door to custom design of TMBs for biotechnology and demonstrates the value of de novo design to investigate basic protein folding problems that are otherwise hidden by evolutionary history.One sentence summarySuccess in de novo design of transmembrane β-barrels reveals geometric and sequence constraints on the fold and paves the way to design of custom pores for sequencing and other single-molecule analytical applications.

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 Surface Engineering of Top7 to Facilitate Structure Determination

2022 ◽  
Vol 23 (2) ◽  
pp. 701
Author(s):  
Yuki Ito ◽  
Takuya Araki ◽  
Shota Shiga ◽  
Hiroyuki Konno ◽  
Koki Makabe
Keyword(s):  
Structure Determination ◽  
Surface Engineering ◽  
De Novo ◽  
Crystal Packing ◽  
Structure Model ◽  
X Ray ◽  
Hexahistidine Tag ◽  
Model Protein ◽  
Β Sheet ◽  
Packing Interactions

Top7 is a de novo designed protein whose amino acid sequence has no evolutional trace. Such a property makes Top7 a suitable scaffold for studying the pure nature of protein and protein engineering applications. To use Top7 as an engineering scaffold, we initially attempted structure determination and found that crystals of our construct, which lacked the terminal hexahistidine tag, showed weak diffraction in X-ray structure determination. Thus, we decided to introduce surface residue mutations to facilitate crystal structure determination. The resulting surface mutants, Top7sm1 and Top7sm2, crystallized easily and diffracted to the resolution around 1.7 Å. Despite the improved data, we could not finalize the structures due to high R values. Although we could not identify the origin of the high R values of the surface mutants, we found that all the structures shared common packing architecture with consecutive intermolecular β-sheet formation aligned in one direction. Thus, we mutated the intermolecular interface to disrupt the intermolecular β-sheet formation, expecting to form a new crystal packing. The resulting mutant, Top7sm2-I68R, formed new crystal packing interactions as intended and diffracted to the resolution of 1.4 Å. The surface mutations contributed to crystal packing and high resolution. We finalized the structure model with the R/Rfree values of 0.20/0.24. Top7sm2-I68R can be a useful model protein due to its convenient structure determination.

scholarly journals A defined structural unit enables de novo design of small-molecule–binding proteins

Science ◽  
2020 ◽  
Vol 369 (6508) ◽  
pp. 1227-1233 ◽  
Author(s):  
Nicholas F. Polizzi ◽  
William F. DeGrado
Keyword(s):  
Protein Structure ◽  
Amino Acid ◽  
Small Molecules ◽  
Structural Unit ◽  
De Novo ◽  
Computational Design ◽  
De Novo Design ◽  
X Ray ◽  
X Ray Crystallography ◽  
Chemical Groups

The de novo design of proteins that bind highly functionalized small molecules represents a great challenge. To enable computational design of binders, we developed a unit of protein structure—a van der Mer (vdM)—that maps the backbone of each amino acid to statistically preferred positions of interacting chemical groups. Using vdMs, we designed six de novo proteins to bind the drug apixaban; two bound with low and submicromolar affinity. X-ray crystallography and mutagenesis confirmed a structure with a precisely designed cavity that forms favorable interactions in the drug–protein complex. vdMs may enable design of functional proteins for applications in sensing, medicine, and catalysis.

scholarly journals The structural ensemble of a Holliday junction determined by X-ray scattering interference

2020 ◽  
Vol 48 (14) ◽  
pp. 8090-8098
Author(s):  
Thomas Zettl ◽  
Xuesong Shi ◽  
Steve Bonilla ◽  
Steffen M Sedlak ◽  
Jan Lipfert ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genetic Recombination ◽  
Resonance Energy ◽  
Holliday Junction ◽  
Full Description ◽  
Conformational Ensemble ◽  
X Ray ◽  
X Ray Scattering ◽  
Wide Range ◽  
Ray Scattering

Abstract The DNA four-way (Holliday) junction is the central intermediate of genetic recombination, yet key aspects of its conformational and thermodynamic properties remain unclear. While multiple experimental approaches have been used to characterize the canonical X-shape conformers under specific ionic conditions, the complete conformational ensemble of this motif, especially at low ionic conditions, remains largely undetermined. In line with previous studies, our single-molecule Förster resonance energy transfer (smFRET) measurements of junction dynamics revealed transitions between two states under high salt conditions, but smFRET could not determine whether there are fast and unresolvable transitions between distinct conformations or a broad ensemble of related states under low and intermediate salt conditions. We therefore used an emerging technique, X-ray scattering interferometry (XSI), to directly probe the conformational ensemble of the Holliday junction across a wide range of ionic conditions. Our results demonstrated that the four-way junction adopts an out-of-plane geometry under low ionic conditions and revealed a conformational state at intermediate ionic conditions previously undetected by other methods. Our results provide critical information to build toward a full description of the conformational landscape of the Holliday junction and underscore the utility of XSI for probing conformational ensembles under a wide range of solution conditions.

scholarly journals Purification, crystallization and preliminary X-ray diffraction analysis of theN-acetyltransferase SAV0826 fromStaphylococcus aureus

2014 ◽  
Vol 70 (2) ◽  
pp. 211-214
Author(s):  
Parul Srivastava ◽  
Yogesh B. Khandokar ◽  
Jade K. Forwood
Keyword(s):  
Diffraction Analysis ◽  
Single Molecule ◽  
Protein Function ◽  
Bacterial Species ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Wide Range ◽  
Antibiotic Drug

Staphylococcus aureusis a prevalent microorganism that is capable of causing a wide range of infections and diseases. Several strains of this bacterial species have developed antibiotic resistance to methicillin and vancomycin, and higher death rates are still being reported each year owing to multidrug-resistant strains. Certain GCN5-relatedN-acetyltransferases (GNATs) exhibit a broad substrate range, including aminoglycosides, histones, other proteins and serotonin, and have been implicated in antibiotic drug resistance. Here, the expression, purification, crystallization and preliminary X-ray diffraction analysis of a GNAT fromS. aureus(SaNAT) are reported. SaNAT was recombinantly expressed and crystallized by the hanging-drop vapour-diffusion method at 296 K, and the crystals diffracted to 1.7 Å resolution on the MX2 beamline at the Australian Synchrotron. The crystals belonged to space groupP43212, with unit-cell parametersa=b= 84.86,c= 49.06 Å, α = β = γ = 90°. A single molecule is likely to be present in the asymmetric unit. A full structural and functional analysis is currently being undertaken to provide novel insights into the protein function, which in turn may provide a basis for drug design.

scholarly journals Comparative genome analyses of four rice-infecting Rhizoctonia solani isolates reveal extensive enrichment of homogalacturonan modification genes

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Da-Young Lee ◽  
Jongbum Jeon ◽  
Ki-Tae Kim ◽  
Kyeongchae Cheong ◽  
Hyeunjeong Song ◽  
...  
Keyword(s):  
Rhizoctonia Solani ◽  
Single Molecule ◽  
De Novo ◽  
Average Length ◽  
Gene Clusters ◽  
Anastomosis Group ◽  
Secreted Proteins ◽  
Geographical Regions ◽  
Wide Range ◽  
Fungal Genomes

Abstract Background Plant pathogenic isolates of Rhizoctonia solani anastomosis group 1-intraspecific group IA (AG1-IA) infect a wide range of crops causing diseases such as rice sheath blight (ShB). ShB has become a serious disease in rice production worldwide. Additional genome sequences of the rice-infecting R. solani isolates from different geographical regions will facilitate the identification of important pathogenicity-related genes in the fungus. Results Rice-infecting R. solani isolates B2 (USA), ADB (India), WGL (India), and YN-7 (China) were selected for whole-genome sequencing. Single-Molecule Real-Time (SMRT) and Illumina sequencing were used for de novo sequencing of the B2 genome. The genomes of the other three isolates were then sequenced with Illumina technology and assembled using the B2 genome as a reference. The four genomes ranged from 38.9 to 45.0 Mbp in size, contained 9715 to 11,505 protein-coding genes, and shared 5812 conserved orthogroups. The proportion of transposable elements (TEs) and average length of TE sequences in the B2 genome was nearly 3 times and 2 times greater, respectively, than those of ADB, WGL and YN-7. Although 818 to 888 putative secreted proteins were identified in the four isolates, only 30% of them were predicted to be small secreted proteins, which is a smaller proportion than what is usually found in the genomes of cereal necrotrophic fungi. Despite a lack of putative secondary metabolite biosynthesis gene clusters, the rice-infecting R. solani genomes were predicted to contain the most carbohydrate-active enzyme (CAZyme) genes among all 27 fungal genomes used in the comparative analysis. Specifically, extensive enrichment of pectin/homogalacturonan modification genes were found in all four rice-infecting R. solani genomes. Conclusion Four R. solani genomes were sequenced, annotated, and compared to other fungal genomes to identify distinctive genomic features that may contribute to the pathogenicity of rice-infecting R. solani. Our analyses provided evidence that genomic conservation of R. solani genomes among neighboring AGs was more diversified than among AG1-IA isolates and the presence of numerous predicted pectin modification genes in the rice-infecting R. solani genomes that may contribute to the wide host range and virulence of this necrotrophic fungal pathogen.

scholarly journals Caulobacter crescentus Hfq structure reveals a conserved mechanism of RNA annealing regulation

2019 ◽  
Vol 116 (22) ◽  
pp. 10978-10987 ◽  
Author(s):  
Andrew Santiago-Frangos ◽  
Kathrin S. Fröhlich ◽  
Jeliazko R. Jeliazkov ◽  
Ewelina M. Małecka ◽  
Giada Marino ◽  
...  
Keyword(s):  
Computational Models ◽  
Rna Binding ◽  
De Novo ◽  
Caulobacter Crescentus ◽  
X Ray ◽  
The Core ◽  
Chaperone Protein ◽  
Crystal Contacts ◽  
Conserved Core

We have solved the X-ray crystal structure of the RNA chaperone protein Hfq from the alpha-proteobacterium Caulobacter crescentus to 2.15-Å resolution, resolving the conserved core of the protein and the entire C-terminal domain (CTD). The structure reveals that the CTD of neighboring hexamers pack in crystal contacts, and that the acidic residues at the C-terminal tip of the protein interact with positive residues on the rim of Hfq, as has been recently proposed for a mechanism of modulating RNA binding. De novo computational models predict a similar docking of the acidic tip residues against the core of Hfq. We also show that C. crescentus Hfq has sRNA binding and RNA annealing activities and is capable of facilitating the annealing of certain Escherichia coli sRNA:mRNA pairs in vivo. Finally, we describe how the Hfq CTD and its acidic tip residues provide a mechanism to modulate annealing activity and substrate specificity in various bacteria.

scholarly journals ACID: a free tool for drug repurposing using consensus inverse docking strategy

2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Fan Wang ◽  
Feng-Xu Wu ◽  
Cheng-Zhang Li ◽  
Chen-Yang Jia ◽  
Sun-Wen Su ◽  
...  
Keyword(s):  
Success Rate ◽  
Single Molecule ◽  
In Silico ◽  
Drug Target ◽  
Computational Models ◽  
De Novo ◽  
Web Server ◽  
Drug Repurposing ◽  
Promising Alternative ◽  
Computational Approaches

AbstractDrug repurposing offers a promising alternative to dramatically shorten the process of traditional de novo development of a drug. These efforts leverage the fact that a single molecule can act on multiple targets and could be beneficial to indications where the additional targets are relevant. Hence, extensive research efforts have been directed toward developing drug based computational approaches. However, many drug based approaches are known to incur low successful rates, due to incomplete modeling of drug-target interactions. There are also many technical limitations to transform theoretical computational models into practical use. Drug based approaches may, thus, still face challenges for drug repurposing task. Upon this challenge, we developed a consensus inverse docking (CID) workflow, which has a ~ 10% enhancement in success rate compared with current best method. Besides, an easily accessible web server named auto in silico consensus inverse docking (ACID) was designed based on this workflow (http://chemyang.ccnu.edu.cn/ccb/server/ACID).

Single-molecule studies of amyloid proteins: from biophysical properties to diagnostic perspectives

2020 ◽  
Vol 53 ◽  
Author(s):  
Jinming Wu ◽  
Chan Cao ◽  
Rolf Antonie Loch ◽  
Ann Tiiman ◽  
Jinghui Luo
Keyword(s):  
Neurodegenerative Diseases ◽  
Single Molecule ◽  
Lipid Membranes ◽  
Feasibility Studies ◽  
Diagnostic Tools ◽  
Amyloid Proteins ◽  
Self Assembling ◽  
Ensemble Techniques ◽  
Wide Range ◽  
Single Molecule Studies

Abstract In neurodegenerative diseases, a wide range of amyloid proteins or peptides such as amyloid-beta and α-synuclein fail to keep native functional conformations, followed by misfolding and self-assembling into a diverse array of aggregates. The aggregates further exert toxicity leading to the dysfunction, degeneration and loss of cells in the affected organs. Due to the disordered structure of the amyloid proteins, endogenous molecules, such as lipids, are prone to interact with amyloid proteins at a low concentration and influence amyloid cytotoxicity. The heterogeneity of amyloid proteinscomplicates the understanding of the amyloid cytotoxicity when relying only on conventional bulk and ensemble techniques. As complementary tools, single-molecule techniques (SMTs) provide novel insights into the different subpopulations of a heterogeneous amyloid mixture as well as the cytotoxicity, in particular as involved in lipid membranes. This review focuses on the recent advances of a series of SMTs, including single-molecule fluorescence imaging, single-molecule force spectroscopy and single-nanopore electrical recording, for the understanding of the amyloid molecular mechanism. The working principles, benefits and limitations of each technique are discussed and compared in amyloid protein related studies.. We also discuss why SMTs show great potential and are worthy of further investigation with feasibility studies as diagnostic tools of neurodegenerative diseases and which limitations are to be addressed.

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