Blue native electrophoresis is used widely for the analysis of non-dissociated protein complexes with respect to composition, oligomeric state and molecular mass. However, the effects of detergent or dye binding on the mass and stability of the integral membrane proteins have not been studied. By comparison with analytical ultracentrifugation, we have evaluated whether the oligomeric state of membrane transport proteins is reflected reliably with blue native electrophoresis. For the analysis we have used two well-characterized transporters, that is, the major facilitator superfamily protein LacS and the phosphotransferase system EII(Mtl). For another member of the major facilitator superfamily, the xyloside transporter XylP from Lactobacillus pentosus, the complete analysis of the quaternary structure determined by analytical ultracentrifugation and freeze-fracture electron microscopy is presented. Our experiments show that during blue native electrophoresis the detergent bound to the proteins is replaced by the amphipathic Coomassie brilliant blue (CBB) dye. The mass of the bound CBB dye was quantified. Provided this additional mass of bound CBB dye is accounted for and care is taken in the choice and concentration of the detergent used, the mass of LacS, XylP and EII(Mtl) and four other membrane (transport) proteins could be deduced within 10 % error. Our data underscore the fact that the oligomeric state of many membrane transport proteins is dimeric.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1006/jmbi.2002.5416 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Key Role of Phosphorylation in Small Heat Shock Protein Regulation via Oligomeric Disaggregation and Functional Activation.
Cells
January 2025
Department of Ophthalmology & Visual Sciences, The University of Michigan, Ann Arbor, MI 48109, USA.
Heat shock proteins (HSPs) are essential molecular chaperones that protect cells by aiding in protein folding and preventing aggregation under stress conditions. Small heat shock proteins (sHSPs), which include members from HSPB1 to HSPB10, are particularly important for cellular stress responses. These proteins share a conserved α-crystallin domain (ACD) critical for their chaperone function, with flexible N- and C-terminal extensions that facilitate oligomer formation.
View Article and Find Full Text PDFPeripheral Evolution of Tanshinone IIA and Cryptotanshinone for Discovery of a Potent and Specific NLRP3 Inflammasome Inhibitor.
J Med Chem
January 2025
College of Pharmaceutical Sciences, State Key Laboratory of Advanced Drug Delivery and Release Systems, Zhejiang University, Hangzhou 310058, China.
Natural products (NPs) continue to serve as an invaluable source in drug discovery, and peripheral evolution of NPs is a highly efficient evolution strategy. Herein, we describe a unified "methyl to amide" peripheral evolution of Tanshinone IIA and Cryptotanshinone for discovery of NLRP3 inflammasome inhibitors. There were 54 compounds designed and prepared, while the chemoinformatic analysis revealed that these evolved NP analogues occupy a unique chemical space.
View Article and Find Full Text PDFFlexible Tail of Antimicrobial Peptide PGLa Facilitates Water Pore Formation in Membranes.
J Phys Chem B
January 2025
Research Center for Analytical Sciences, Tianjin Key Laboratory of Biosensing and Molecular Recognition, State Key Laboratory of Medicinal Chemical, Biology College of Chemistry, Nankai University, Tianjin 300071, China.
PGLa, an antimicrobial peptide (AMP), primarily exerts its antibacterial effects by disrupting bacterial cell membrane integrity. Previous theoretical studies mainly focused on the binding mechanism of PGLa with membranes, while the mechanism of water pore formation induced by PGLa peptides, especially the role of structural flexibility in the process, remains unclear. In this study, using all-atom simulations, we investigated the entire process of membrane deformation caused by the interaction of PGLa with an anionic cell membrane composed of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG).
View Article and Find Full Text PDFDissecting the biophysical mechanisms of oleate hydratase association with membranes.
Front Mol Biosci
January 2025
Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY, United States.
This study investigates the dynamics of oleate hydratase (OhyA), a bacterial flavoenzyme from , and its interactions with lipid membranes, focusing on the factors influencing membrane binding and oligomerization. OhyA catalyzes the hydration of unsaturated fatty acids, playing a key role in bacterial pathogenesis by neutralizing host antimicrobial fatty acids. OhyA binds the membrane bilayer to access membrane-embedded substrates for catalysis, and structural studies have revealed that OhyA forms oligomers on membrane surfaces, stabilized by both protein-protein and protein-lipid interactions.
View Article and Find Full Text PDFGlutamine Synthetase: Diverse Regulation and Functions of an Ancient Enzyme.
Biochemistry
January 2025
Department of Chemistry and Biochemistry, San Francisco State University, 1600 Holloway Avenue, San Francisco, California 94132, United States.
Glutamine synthetase (GS) is a ubiquitous enzyme central to nitrogen metabolism, catalyzing the ATP-dependent formation of glutamine from glutamate and ammonia. Positioned at the intersection of nitrogen metabolism with carbon metabolism, the activity of GS is subject to sophisticated regulation. While the intricate regulatory pathways that govern GS were established long ago, recent work has demonstrated that homologues are controlled by multiple distinct regulatory patterns, such as the metabolite induced oligomeric state formation in archaeal GS by 2-oxoglutarate.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!