Improving extraction and post-purification concentration of membrane proteins

The Analyst ◽  
2018 ◽  
Vol 143 (6) ◽  
pp. 1378-1386 ◽  
Author(s):  
Hasin Feroz ◽  
HyeYoung Kwon ◽  
Jing Peng ◽  
Hyeonji Oh ◽  
Bryan Ferlez ◽  
...  
Keyword(s):  
Pharmaceutical Industry ◽  
Membrane Proteins ◽  
Drug Targets ◽  
Functional Protein ◽  
High Yields ◽  
Important Drug

Membrane proteins (MPs), despite being critically important drug targets for the pharmaceutical industry, are difficult to study due to challenges in obtaining high yields of functional protein.

Structural biology and structure–function relationships of membrane proteins

2018 ◽  
Vol 47 (1) ◽  
pp. 47-61 ◽  
Author(s):  
Rosana Reis ◽  
Isabel Moraes
Keyword(s):  
Membrane Proteins ◽  
Structure Function ◽  
Structural Biology ◽  
Drug Targets ◽  
Three Dimensional ◽  
Data Bank ◽  
Technical Advances ◽  
Medical Importance ◽  
G Protein Coupled ◽  
Important Drug

Abstract The study of structure–function relationships of membrane proteins (MPs) has been one of the major goals in the field of structural biology. Many Noble Prizes regarding remarkable accomplishments in MP structure determination and biochemistry have been awarded over the last few decades. Mutations or improper folding of these proteins are associated with numerous serious illnesses. Therefore, as important drug targets, the study of their primary sequence and three-dimensional fold, combined with cell-based assays, provides vital information about their structure–function relationships. Today, this information is vital to drug discovery and medicine. In the last two decades, many have been the technical advances and breakthroughs in the field of MP structural biology that have contributed to an exponential growth in the number of unique MP structures in the Protein Data Bank. Nevertheless, given the medical importance and many unanswered questions, it will never be an excess of MP structures, regardless of the method used. Owing to the extension of the field, in this brief review, we will only focus on structure–function relationships of the three most significant pharmaceutical classes: G protein-coupled receptors, ion channels and transporters.

Direct ligand screening against membrane proteins on live cells enabled by DNA-Programmed Affinity Labelling

2021 ◽  
Author(s):  
Yiran Huang ◽  
Yuqing Deng ◽  
Jianfu Zhang ◽  
Meng Ling ◽  
Xiaoyu Li
Keyword(s):  
Membrane Proteins ◽  
Drug Targets ◽  
Live Cells ◽  
Ligand Screening ◽  
Ligand Discovery ◽  
Affinity Labelling ◽  
Hydrophobic Nature ◽  
Important Drug

Membrane proteins are are important drug targets; however, ligand discovery for membrane proteins is highly challenging due to their hydrophobic nature. We show that membrane proteins may be specifically labelled...

Discovery of an Unexpected Similarity in Ligand Binding Between BRD4 and PPARγ

2019 ◽  
Author(s):  
Lina Humbeck ◽  
Jette Pretzel ◽  
Saskia Spitzer ◽  
Oliver Koch
Keyword(s):  
Cancer Therapy ◽  
Drug Targets ◽  
Synergistic Effects ◽  
Complex Structure ◽  
Peroxisome Proliferator ◽  
Resistance Development ◽  
Peroxisome Proliferator Activated Receptor ◽  
Starting Point ◽  
Fundamental Research ◽  
Important Drug

Knowledge about interrelationships between different proteins is crucial in fundamental research for the elucidation of protein networks and pathways. Furthermore, it is especially critical in chemical biology to identify further key regulators of a disease and to take advantage of polypharmacology effects. A comprehensive scaffold-based analysis uncovered an unexpected relationship between bromodomain-containing protein 4 (BRD4) and peroxisome-proliferator activated receptor gamma (PPARγ). They are both important drug targets for cancer therapy and many more important diseases. Both proteins share binding site similarities near a common hydrophobic subpocket which should allow the design of a polypharmacology-based ligand targeting both proteins. Such a dual-BRD4-PPARγ-modulator could show synergistic effects with a higher efficacy or delayed resistance development in, for example, cancer therapy. Thereon, a complex structure of sulfasalazine was obtained that involves two bromodomains and could be a potential starting point for the design of a bivalent BRD4 inhibitor.

scholarly journals Crystal structure of SARS-CoV-2 main protease in complex with a Chinese herb inhibitor shikonin

2020 ◽  
Author(s):  
Jian Li ◽  
Xuelan Zhou ◽  
Yan Zhang ◽  
Fanglin Zhong ◽  
Cheng Lin ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Drug Targets ◽  
Chinese Herb ◽  
Binding Modes ◽  
High Potency ◽  
Main Protease ◽  
Covalent Inhibitors ◽  
Catalytic Dyad ◽  
Important Drug

AbstractMain protease (Mpro, also known as 3CLpro) has a major role in the replication of coronavirus life cycle and is one of the most important drug targets for anticoronavirus agents. Here we report the crystal structure of main protease of SARS-CoV-2 bound to a previously identified Chinese herb inhibitor shikonin at 2.45 angstrom resolution. Although the structure revealed here shares similar overall structure with other published structures, there are several key differences which highlight potential features that could be exploited. The catalytic dyad His41-Cys145 undergoes dramatic conformational changes, and the structure reveals an unusual arrangement of oxyanion loop stabilized by the substrate. Binding to shikonin and binding of covalent inhibitors show different binding modes, suggesting a diversity in inhibitor binding. As we learn more about different binding modes and their structure-function relationships, it is probable that we can design more effective and specific drugs with high potency that can serve as effect SARS-CoV-2 anti-viral agents.

scholarly journals Membrane proteins: always an insoluble problem?

2016 ◽  
Vol 44 (3) ◽  
pp. 790-795 ◽  
Author(s):  
Andrea E. Rawlings
Keyword(s):  
Membrane Proteins ◽  
Lipid Membranes ◽  
Drug Targets ◽  
Functional Characterization ◽  
Water Soluble ◽  
High Yield ◽  
Cellular Processes ◽  
Native Sequence ◽  
Protein Research ◽  
New Methodologies

Membrane proteins play crucial roles in cellular processes and are often important pharmacological drug targets. The hydrophobic properties of these proteins make full structural and functional characterization challenging because of the need to use detergents or other solubilizing agents when extracting them from their native lipid membranes. To aid membrane protein research, new methodologies are required to allow these proteins to be expressed and purified cheaply, easily, in high yield and to provide water soluble proteins for subsequent study. This mini review focuses on the relatively new area of water soluble membrane proteins and in particular two innovative approaches: the redesign of membrane proteins to yield water soluble variants and how adding solubilizing fusion proteins can help to overcome these challenges. This review also looks at naturally occurring membrane proteins, which are able to exist as stable, functional, water soluble assemblies with no alteration to their native sequence.

Database Study on the Expression and Purification of Membrane Proteins

2021 ◽  
Vol 28 ◽  
Author(s):  
Chen-Yan china Zhang ◽  
Shi-Qi Zhao ◽  
Shi-Long Zhang ◽  
Li-Heng Luo ◽  
Ding-Chang Liu ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Expression ◽  
Drug Targets ◽  
Expression System ◽  
Data Bank ◽  
Hek293 Cells ◽  
Expression And Purification ◽  
Beta Barrel ◽  
Membrane Protein Expression

: Membrane proteins are crucial for biological processes, and many of them are important to drug targets. Understanding the three-dimensional structures of membrane proteins are essential to evaluate their bio function and drug design. High-purity membrane proteins are important for structural determination. Membrane proteins have low yields and are difficult to purify because they tend to aggregate. We summarized membrane protein expression systems, vectors, tags, and detergents, which have deposited in the Protein Data Bank (PDB) in recent four-and-a-half years. Escherichia coli is the most expression system for membrane proteins, and HEK293 cells are the most commonly cell lines for human membrane protein expression. The most frequently vectors are pFastBac1 for alpha-helical membrane proteins, pET28a for beta-barrel membrane proteins, and pTRC99a for monotopic membrane proteins. The most used tag for membrane proteins is the 6×His-tag. FLAG commonly used for alpha-helical membrane proteins, Strep and GST for beta-barrel and monotopic membrane proteins, respectively. The detergents and their concentrations used for alpha-helical, beta-barrel, and monotopic membrane proteins are different, and DDM is commonly used for membrane protein purification. It can guide the expression and purification of membrane proteins, thus contributing to their structure and bio function studying.

scholarly journals Thiol-to-amine cyclization reaction enables screening of large libraries of macrocyclic compounds and the generation of sub-kilodalton ligands

2019 ◽  
Vol 5 (8) ◽  
pp. eaaw2851 ◽  
Author(s):  
S. S. Kale ◽  
M. Bergeron-Brlek ◽  
Y. Wu ◽  
M. G. Kumar ◽  
M. V. Pham ◽  
...  
Keyword(s):  
Drug Development ◽  
Structure Analysis ◽  
Drug Targets ◽  
Macrocyclic Ligands ◽  
Cyclization Reaction ◽  
Thrombin Inhibitor ◽  
Macrocyclic Compounds ◽  
Cyclization Reactions ◽  
X Ray ◽  
High Yields

Macrocyclic compounds are an attractive modality for drug development, but the limited availability of large, structurally diverse macrocyclic libraries hampers the discovery of leads. Here, we describe the discovery of efficient macrocyclization reactions based on thiol-to-amine ligations using bis-electrophiles, their application to synthesize and screen large libraries of macrocyclic compounds, and the identification of potent small macrocyclic ligands. The thiol-to-amine cyclization reactions showed unexpectedly high yields for a wide substrate range, which obviated product purification and enabled the generation and screening of an 8988 macrocycle library with a comparatively small effort. X-ray structure analysis of an identified thrombin inhibitor (Ki = 42 ± 5 nM) revealed a snug fit with the target, validating the strategy of screening large libraries with a high skeletal diversity. The approach provides a route for screening large sub-kilodalton macrocyclic libraries and may be applied to many challenging drug targets.

scholarly journals Crystal Structures of Four Novel Thiophene/Phenyl-piperidine Hybrid Chalcones

2014 ◽  
Vol 70 (a1) ◽  
pp. C1020-C1020
Author(s):  
Masood Parvez ◽  
Muhammad Bakhtiar ◽  
Muhammad Baqir ◽  
Muhammad Zia-ur-Rehman
Keyword(s):  
Hydrogen Bonding ◽  
Pharmaceutical Industry ◽  
Hydrogen Bonds ◽  
Crystal Structures ◽  
Benzene Ring ◽  
Drug Targets ◽  
Important Class ◽  
Hydrogen Bonding Interactions ◽  
Benzene Rings ◽  
Type C

Chalcones constitute an important class of bioactive drug targets in the pharmaceutical industry that includes anti-ulcerative drug sofalcone. In continuation of our work, the crystal structures of four closely related 1-phenyl-piperidine based chalcones will be presented. I: C19 H21NOS, MW = 311.43, T = 173(2) K, λ = 0.71073 Å, Orthorhombic, P b c a, a = 10.1045(4), b = 10.5358(4), c = 30.6337(12) Å, V = 3261.2(2) Å3, Z = 8, Dc = 1.269 Mg/m3, F (000) = 1328, R [I>2σ(I)] = 0.059. II: C18H19NOS, MW = 297.40, T = 173(2) K, λ = 1.54178 Å, Orthorhombic, P b c a, a = 8.9236(2), b = 11.0227(2), c = 30.8168(6) Å, V = 3031.21(11) Å3 Z = 8, Dc = 1.303 Mg/m3, F (000) = 1264, R [I>2σ(I)] = 0.035. III: C18H19NOS, MW = 297.40, T = 173(2) K, λ = 1.54178 Å, Orthorhombic, P b c a, a = 8.82990(10), b = 11.0061(2), c = 31.2106(5) Å, V = 3033.13(8) Å3, Z = 8, Dc = 1.303 Mg/m3, F (000) = 1264, R [I>2σ(I)] = 0.048. IV: C18H18ClNOS, MW = 331.84, T = 173(2) K, λ = 0.71073 Å, Monoclinic, P 21/c, a = 14.1037(4), b = 11.3153(3), c = 10.1290(2) Å, β = 101.1367(14)0, V = 1586.02(7) Å3, Z = 4, Dc = 1.390 Mg/m3, F (000) = 696, R [I>2σ(I)] = 0.038. The crystals of I, II and III are isomorphous. In all structures, the piperidine rings are in chair conformations, thiophene rings are essentially planar and the C=C bonds in the prop-2-en-1-one fragment adopt E-conformation. All crystal structures are devoid of any classical hydrogen bonds. However, non-classical hydrogen bonding interactions of the type C---H...O in compounds II, III and IV link the molecules into chains extended along the b-axis. Moreover, C---H...Cg interactions involving thiophene rings in I and III and benzene ring in IV and π...π interactions between benzene rings lying about inversion centers are present in II and III.

scholarly journals Understanding the yeast host cell response to recombinant membrane protein production

2011 ◽  
Vol 39 (3) ◽  
pp. 719-723 ◽  
Author(s):  
Zharain Bawa ◽  
Charlotte E. Bland ◽  
Nicklas Bonander ◽  
Nagamani Bora ◽  
Stephanie P. Cartwright ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Drug Targets ◽  
Large Scale ◽  
Protein Production ◽  
Molecular Mechanisms ◽  
New Drugs ◽  
Host Cells ◽  
Host Cell Response ◽  
Wide Range

Membrane proteins are drug targets for a wide range of diseases. Having access to appropriate samples for further research underpins the pharmaceutical industry's strategy for developing new drugs. This is typically achieved by synthesizing a protein of interest in host cells that can be cultured on a large scale, allowing the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process. Even in these cells, the production of human membrane proteins can be plagued by low functional yields; we wish to understand why. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast host strains. By relieving the bottlenecks to recombinant membrane protein production in yeast, we aim to contribute to the drug discovery pipeline, while providing insight into translational processes.

Meeting report: membrane proteins in health and disease

2002 ◽  
Vol 80 (5) ◽  
pp. v-xi ◽  
Author(s):  
James D Young ◽  
Joseph R Casey ◽  
Reinhart A.F Reithmeier
Keyword(s):  
Membrane Proteins ◽  
Drug Targets ◽  
Ph Regulation ◽  
Meeting Report ◽  
Inherited Disease ◽  
Waste Products ◽  
Wide Range ◽  
Bicarbonate Transporters ◽  
Health And Disease ◽  
Biology Symposium

This article summarizes the scientific presentations made at a Canadian Society of Biochemistry and Molecular & Cellular Biology Symposium on "Membrane Proteins in Health and Diseases" and two satellite meetings on "Bicarbonate Transporters" and "Nucleoside Transporters" held in Banff, Alberta, 20–24 March 2002. Membrane proteins are encoded by about 1/3 of genes and are involved in a wide range of essential functions, including the transport of nutrients, ions, and waste products across biological membranes. Mutations or changes in the expression of these genes cause an equally wide range of diseases. Membrane proteins are also common drug targets or provide drug entry mechanisms. The importance of membrane proteins in biology and medicine was highlighted by the presentations made at this exciting meeting by an international group of experts.Key words: bicarbonate, genomics, inherited disease, nucleosides, organelles, pH regulation, structural biology, trafficking, transporters.

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