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Article ; Online: Fluorescence-Detection Size-Exclusion Chromatography-Based Thermostability Assay for Membrane Proteins.

Yao, Hebang / Cai, Hongmin / Li, Dianfan

Methods in molecular biology (Clifton, N.J.)

2022 Volume 2564, Page(s) 299–315

Abstract: Green fluorescent proteins (GFPs) have lightened up almost every aspect of biological research including protein sciences. In the field of membrane protein structural biology, GFPs have been used widely to monitor membrane protein localization, ... ...

Abstract	Green fluorescent proteins (GFPs) have lightened up almost every aspect of biological research including protein sciences. In the field of membrane protein structural biology, GFPs have been used widely to monitor membrane protein localization, expression level, the purification process and yield, and the stability inside the cells and in the test tube. Of particular interest is the fluorescence-detector size-exclusion chromatography-based thermostability assay (FSEC-TS). By simple heating and FSEC, the generally applicable method allows rapid assessment of the thermostability of GFP-fused membrane proteins without purification. Here we describe the experimental details and some typical results for the FSEC-TS method.
MeSH term(s)	Chromatography, Gel ; Green Fluorescent Proteins/metabolism ; Membrane Proteins/metabolism
Chemical Substances	Membrane Proteins ; Green Fluorescent Proteins (147336-22-9)
Language	English
Publishing date	2022-09-15
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-2667-2_16
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Article ; Online: The phosphatidylglycerol phosphate synthase PgsA utilizes a trifurcated amphipathic cavity for catalysis at the membrane-cytosol interface.

Yang, Bowei / Yao, Hebang / Li, Dianfan / Liu, Zhenfeng

Current research in structural biology

2021 Volume 3, Page(s) 312–323

Abstract: Phosphatidylglycerol is a crucial phospholipid found ubiquitously in biological membranes of prokaryotic and eukaryotic cells. The phosphatidylglycerol phosphate (PGP) synthase (PgsA), a membrane-embedded enzyme, catalyzes the primary reaction of ... ...

Abstract	Phosphatidylglycerol is a crucial phospholipid found ubiquitously in biological membranes of prokaryotic and eukaryotic cells. The phosphatidylglycerol phosphate (PGP) synthase (PgsA), a membrane-embedded enzyme, catalyzes the primary reaction of phosphatidylglycerol biosynthesis. Mutations in
Language	English
Publishing date	2021-11-23
Publishing country	Netherlands
Document type	Journal Article
ISSN	2665-928X
ISSN (online)	2665-928X
DOI	10.1016/j.crstbi.2021.11.005
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Article ; Online: The phosphatidylglycerol phosphate synthase PgsA utilizes a trifurcated amphipathic cavity for catalysis at the membrane-cytosol interface

Bowei Yang / Hebang Yao / Dianfan Li / Zhenfeng Liu

Current Research in Structural Biology, Vol 3, Iss , Pp 312-

2021 Volume 323

Abstract	Phosphatidylglycerol is a crucial phospholipid found ubiquitously in biological membranes of prokaryotic and eukaryotic cells. The phosphatidylglycerol phosphate (PGP) synthase (PgsA), a membrane-embedded enzyme, catalyzes the primary reaction of phosphatidylglycerol biosynthesis. Mutations in pgsA frequently correlate with daptomycin resistance in Staphylococcus aureus and other prevalent infectious pathogens. Here we report the crystal structures of S. aureus PgsA (SaPgsA) captured at two distinct states of the catalytic process, with lipid substrate (cytidine diphosphate-diacylglycerol, CDP-DAG) or product (PGP) bound to the active site within a trifurcated amphipathic cavity. The hydrophilic head groups of CDP-DAG and PGP occupy two different pockets in the cavity, inducing local conformational changes. An elongated membrane-exposed surface groove accommodates the fatty acyl chains of CDP-DAG/PGP and opens a lateral portal for lipid entry/release. Remarkably, the daptomycin resistance-related mutations mostly cluster around the active site, causing reduction of enzymatic activity. Our results provide detailed mechanistic insights into the dynamic catalytic process of PgsA and structural frameworks beneficial for development of antimicrobial agents targeting PgsA from pathogenic bacteria.
Keywords	Phosphatidylglycerol ; Synthase ; Staphylococcus aureus ; Daptomycin resistance ; Membrane protein ; Crystal structure ; Biology (General) ; QH301-705.5
Language	English
Publishing date	2021-01-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
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Article ; Online: Thermostabilization of Membrane Proteins by Consensus Mutation: A Case Study for a Fungal Δ8-7 Sterol Isomerase.

Yao, Hebang / Cai, Hongmin / Li, Dianfan

Journal of molecular biology

2020 Volume 432, Issue 18, Page(s) 5162–5183

Abstract: Membrane proteins are generally challenging to work with because of their notorious instability. Protein engineering has been used increasingly to thermostabilize labile membrane proteins such as G-protein-coupled receptors for structural and functional ... ...

Abstract	Membrane proteins are generally challenging to work with because of their notorious instability. Protein engineering has been used increasingly to thermostabilize labile membrane proteins such as G-protein-coupled receptors for structural and functional studies in recent years. Two major strategies exist. Scanning mutagenesis systematically eliminates destabilizing residues, whereas the consensus approach assembles mutants with the most frequent residues among selected homologs, bridging sequence conservation with stability. Here, we applied the consensus concept to stabilize a fungal homolog of the human sterol Δ8-7 isomerase, a 26.4 kDa protein with five transmembrane helices. The isomerase is also called emopamil-binding protein (EBP), as it binds this anti-ischemic drug with high affinity. The wild-type had an apparent melting temperature (T
MeSH term(s)	Chromatography, Gel ; Enzyme Stability ; Humans ; Models, Molecular ; Mutagenesis, Site-Directed ; Mutation ; Protein Conformation ; Protein Engineering ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/growth & development ; Saccharomyces cerevisiae Proteins/chemistry ; Saccharomyces cerevisiae Proteins/genetics ; Saccharomyces cerevisiae Proteins/metabolism ; Steroid Isomerases/chemistry ; Steroid Isomerases/genetics ; Steroid Isomerases/metabolism ; Thermodynamics
Chemical Substances	Saccharomyces cerevisiae Proteins ; Steroid Isomerases (EC 5.3.3.-) ; delta(8)-delta(7)-sterol isomerase (EC 5.3.3.-) ; EBP protein, human (EC 5.3.3.5)
Language	English
Publishing date	2020-02-24
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	80229-3
ISSN	1089-8638 ; 0022-2836
ISSN (online)	1089-8638
ISSN	0022-2836
DOI	10.1016/j.jmb.2020.02.015
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Article ; Online: A high-affinity RBD-targeting nanobody improves fusion partner's potency against SARS-CoV-2.

Hebang Yao / Hongmin Cai / Tingting Li / Bingjie Zhou / Wenming Qin / Dimitri Lavillette / Dianfan Li

PLoS Pathogens, Vol 17, Iss 3, p e

2021 Volume 1009328

Abstract: A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research has been devoted to the development of neutralizing antibodies, including llama-derived single-chain ... ...

Abstract	A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research has been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and 'greasy' site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.
Keywords	Immunologic diseases. Allergy ; RC581-607 ; Biology (General) ; QH301-705.5
Subject code	572
Language	English
Publishing date	2021-03-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: A high-affinity RBD-targeting nanobody improves fusion partner's potency against SARS-CoV-2.

Yao, Hebang / Cai, Hongmin / Li, Tingting / Zhou, Bingjie / Qin, Wenming / Lavillette, Dimitri / Li, Dianfan

PLoS pathogens

2021 Volume 17, Issue 3, Page(s) e1009328

Abstract	A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research has been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and 'greasy' site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.
MeSH term(s)	Angiotensin-Converting Enzyme 2/immunology ; Antibodies, Neutralizing/chemistry ; Antibodies, Neutralizing/genetics ; Antibodies, Neutralizing/immunology ; Antibodies, Viral/chemistry ; Antibodies, Viral/genetics ; Antibodies, Viral/immunology ; Antibody Affinity ; Binding Sites ; Crystallography, X-Ray ; HEK293 Cells ; Humans ; Models, Molecular ; Recombinant Fusion Proteins/chemistry ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/immunology ; SARS-CoV-2/immunology ; Single-Domain Antibodies/chemistry ; Single-Domain Antibodies/genetics ; Single-Domain Antibodies/immunology
Chemical Substances	Antibodies, Neutralizing ; Antibodies, Viral ; Recombinant Fusion Proteins ; Single-Domain Antibodies ; ACE2 protein, human (EC 3.4.17.23) ; Angiotensin-Converting Enzyme 2 (EC 3.4.17.23)
Language	English
Publishing date	2021-03-03
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2205412-1
ISSN	1553-7374 ; 1553-7374
ISSN (online)	1553-7374
ISSN	1553-7374
DOI	10.1371/journal.ppat.1009328
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Article ; Online: A potent and broad-spectrum neutralizing nanobody for SARS-CoV-2 viruses, including all major Omicron strains.

Yao, Hebang / Wang, Hongyang / Zhang, Zhaoyong / Lu, Yuchi / Zhang, Zhiying / Zhang, Yu / Xiong, Xinyi / Wang, Yanqun / Wang, Zhizhi / Yang, Haitao / Zhao, Jincun / Xu, Wenqing

MedComm

2023 Volume 4, Issue 6, Page(s) e397

Abstract: SARS-CoV-2 viruses are highly transmissible and immune evasive. It is critical to develop broad-spectrum prophylactic and therapeutic antibodies for potential future pandemics. Here, we used the phage display method to discover nanobodies (Nbs) for ... ...

Abstract	SARS-CoV-2 viruses are highly transmissible and immune evasive. It is critical to develop broad-spectrum prophylactic and therapeutic antibodies for potential future pandemics. Here, we used the phage display method to discover nanobodies (Nbs) for neutralizing SARS-CoV-2 viruses especially Omicron strains. The leading nanobody (Nb), namely, Nb4, with excellent physicochemical properties, can neutralize Delta and Omicron subtypes, including BA.1, BA.1.1 (BA.1 + R346K), BA.2, BA.5, BQ.1, and XBB.1. The crystal structure of Nb4 in complex with the receptor-binding domain (RBD) of BA.1 Spike protein reveals that Nb4 interacts with an epitope on the RBD overlapping with the receptor-binding motif, and thus competes with angiotensin-converting enzyme 2 (ACE2) binding. Nb4 is expected to be effective for neutralizing most recent Omicron variants, since the epitopes are evolutionarily conserved among them. Indeed, trivalent Nb4 interacts with the XBB1.5 Spike protein with low nM affinity and competes for ACE2 binding. Prophylactic and therapeutic experiments in mice indicated that Nb4 could reduce the Omicron virus loads in the lung. In particular, in prophylactic experiments, intranasal administration of multivalent Nb4 completely protected mice from Omicron infection. Taken together, these results demonstrated that Nb4 could serve as a potent and broad-spectrum prophylactic and therapeutic Nb for COVID-19.
Language	English
Publishing date	2023-10-26
Publishing country	China
Document type	Journal Article
ISSN	2688-2663
ISSN (online)	2688-2663
DOI	10.1002/mco2.397
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Article ; Online: High-level heterologous expression of the human transmembrane sterol Δ8,Δ7-isomerase in Pichia pastoris.

Cai, Hongmin / Yao, Hebang / Li, Tingting / Tang, Yannan / Li, Dianfan

Protein expression and purification

2019 Volume 164, Page(s) 105463

Abstract: Recombinant expression of human membrane proteins in large quantities remains a major challenge. Expression host is an important variable to screen for high-level production of membrane proteins. Using the green fluorescent protein (GFP) as a reporter, ... ...

Abstract	Recombinant expression of human membrane proteins in large quantities remains a major challenge. Expression host is an important variable to screen for high-level production of membrane proteins. Using the green fluorescent protein (GFP) as a reporter, we screened the expression of a human multi-pass membrane protein called sterol Δ8-Δ7 isomerase in three different hosts: Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. The expression of the His-tagged isomerase was exceptionally high in P. pastoris, reaching ~200 mg L
MeSH term(s)	Chromatography, Gel ; Gene Expression ; Humans ; Pichia/genetics ; Recombinant Proteins/chemistry ; Recombinant Proteins/genetics ; Solubility ; Steroid Isomerases/chemistry ; Steroid Isomerases/genetics
Chemical Substances	Recombinant Proteins ; Steroid Isomerases (EC 5.3.3.-) ; EBP protein, human (EC 5.3.3.5)
Language	English
Publishing date	2019-08-02
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1055455-5
ISSN	1096-0279 ; 1046-5928
ISSN (online)	1096-0279
ISSN	1046-5928
DOI	10.1016/j.pep.2019.105463
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Article ; Online: An improved fluorescent tag and its nanobodies for membrane protein expression, stability assay, and purification

Hongmin Cai / Hebang Yao / Tingting Li / Cedric A. J. Hutter / Yanfang Li / Yannan Tang / Markus A. Seeger / Dianfan Li

Communications Biology, Vol 3, Iss 1, Pp 1-

2020 Volume 16

Abstract: In this work, the authors demonstrate that a coral thermostable GFP (TGP) is an improved tag compared to conventional GFPs. In addition to reporting melting point stability at temperatures near 90 °C, its fusion also helps increase expression levels of ... ...

Abstract	In this work, the authors demonstrate that a coral thermostable GFP (TGP) is an improved tag compared to conventional GFPs. In addition to reporting melting point stability at temperatures near 90 °C, its fusion also helps increase expression levels of test membrane proteins. They further generated synthetic nanobodies against TGP to facilitate purification.
Keywords	Biology (General) ; QH301-705.5
Language	English
Publishing date	2020-12-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: An improved fluorescent tag and its nanobodies for membrane protein expression, stability assay, and purification.

Cai, Hongmin / Yao, Hebang / Li, Tingting / Hutter, Cedric A J / Li, Yanfang / Tang, Yannan / Seeger, Markus A / Li, Dianfan

Communications biology

2020 Volume 3, Issue 1, Page(s) 753

Abstract: Green fluorescent proteins (GFPs) are widely used to monitor membrane protein expression, purification, and stability. An ideal reporter should be stable itself and provide high sensitivity and yield. Here, we demonstrate that a coral (Galaxea ... ...

Abstract	Green fluorescent proteins (GFPs) are widely used to monitor membrane protein expression, purification, and stability. An ideal reporter should be stable itself and provide high sensitivity and yield. Here, we demonstrate that a coral (Galaxea fascicularis) thermostable GFP (TGP) is by such reasons an improved tag compared to the conventional jellyfish GFPs. TGP faithfully reports membrane protein stability at temperatures near 90 °C (20-min heating). By contrast, the limit for the two popular GFPs is 64 °C and 74 °C. Replacing GFPs with TGP increases yield for all four test membrane proteins in four expression systems. To establish TGP as an affinity tag for membrane protein purification, several high-affinity synthetic nanobodies (sybodies), including a non-competing pair, are generated, and the crystal structure of one complex is solved. Given these advantages, we anticipate that TGP becomes a widely used tool for membrane protein structural studies.
MeSH term(s)	Chromatography, Affinity ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Gene Expression ; Genes, Reporter ; Green Fluorescent Proteins/chemistry ; Luminescent Proteins ; Membrane Proteins/chemistry ; Membrane Proteins/genetics ; Membrane Proteins/isolation & purification ; Models, Molecular ; Protein Conformation ; Protein Stability ; Recombinant Proteins/chemistry ; Recombinant Proteins/genetics ; Recombinant Proteins/isolation & purification ; Recombinant Proteins/metabolism ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism ; Single-Domain Antibodies/chemistry
Chemical Substances	Luminescent Proteins ; Membrane Proteins ; Recombinant Proteins ; Single-Domain Antibodies ; Green Fluorescent Proteins (147336-22-9)
Language	English
Publishing date	2020-12-10
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2399-3642
ISSN (online)	2399-3642
DOI	10.1038/s42003-020-01478-z
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