ABSTRACT
Polymer-based lipid nanoparticles like styrene-maleic acid lipid particles have revolutionized the study of membrane proteins. More recently, alternative polymers such as poly(diisobutylene-alt-maleic acid) (DIBMA) have been used in this field. DIBMA is commonly synthesized via conventional radical copolymerization. In order to study the influence of its chain length on lipid nanodisc formation and membrane protein extraction, we synthesized DIBMA with molar masses varying from 1.2-12 kDa via RAFT-mediated polymerization. For molar masses in the range of 3-7 kDa, the rate of lipid nanodisc formation was the highest and similar to those of poly(styrene-co-maleic acid) (SMA) and commercially available DIBMA. ZipA solubilization efficiency was significantly higher than for commercially available DIBMA and similar to SMA (circa 75%). Furthermore, RAFT-made DIBMA with a molar mass of 1.2-3.9 kDa showed a much cleaner separation on SDS-PAGE, without the smearing that is typically seen for SMA and commercially available DIBMA.
Subject(s)
Nanoparticles , Polymers , Lipid Bilayers , Lipids , Maleates , Membrane Proteins , Polystyrenes , StyreneABSTRACT
Most membrane proteins function through interactions with other proteins in the phospholipid bilayer, the cytosol or the extracellular milieu. Understanding the molecular basis of these interactions is key to understanding membrane protein function and dysfunction. Here we demonstrate for the first time how a nano-encapsulation method based on styrene maleic acid lipid particles (SMALPs) can be used in combination with native gel electrophoresis to separate membrane protein complexes in their native state. Using four model proteins, we show that this separation method provides an excellent measure of protein quaternary structure, and that the lipid environment surrounding the protein(s) can be probed using mass spectrometry. We also show that the method is complementary to immunoblotting. Finally we show that intact membrane protein-SMALPs extracted from a band on a gel could be visualised using electron microscopy (EM). Taken together these results provide a novel and elegant method for investigating membrane protein complexes in a native state.
Subject(s)
Membrane Proteins/chemistry , Nanotechnology , Native Polyacrylamide Gel Electrophoresis/methods , Blotting, Western , Lipids/chemistry , Mass Spectrometry , Microscopy, Electron , Protein Structure, QuaternaryABSTRACT
Prokaryotic cell division is a highly orchestrated process requiring the formation of a wide range of biomolecular complexes, perhaps the most important of these involving the prokaryotic tubulin homologue FtsZ, a fibre-forming GTPase. FtsZ assembles into a ring (the Z-ring) on the inner surface of the inner membrane at the site of cell division. The Z-ring then acts as a recruitment site for at least ten other proteins which form the division apparatus. One of these proteins, ZapA, acts to enhance lateral associations between FtsZ fibres to form bundles. Previously we have expressed, purified and crystallized ZapA and demonstrated that it exists as a tetramer. We also showed that ZapA binds to FtsZ polymers, strongly promoting their bundling, while inhibiting FtsZ GTPase activity by inducing conformational changes in the bound nucleotide. In the present study we investigate the importance of the tetramerization of ZapA on its function. We generated a number of mutant forms of ZapA with the aim of disrupting the dimer-dimer interface. We show that one of these mutants, I83E, is fully folded and binds to FtsZ, but is a constitutive dimer. Using this mutant we show that tetramerization is a requirement for both FtsZ bundling and GTPase modulation activities.
Subject(s)
Bacterial Proteins/chemistry , Carrier Proteins/chemistry , Cytoskeletal Proteins/chemistry , Escherichia coli Proteins/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cytokinesis/genetics , Cytokinesis/physiology , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Escherichia coli/cytology , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Guanosine Triphosphate/metabolism , Kinetics , Microscopy, Electron, Transmission , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Quaternary , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
It is commonly assumed that a protein must adopt a tertiary structure to achieve its active native state and that regions of a protein that are devoid of alpha-helix or beta-sheet structures are functionally inert. Although extended proline-rich regions are recognized as presenting binding motifs to, for example, Src homology 2 (SH2) and SH3 domains, the idea persists that natively unfolded regions in functional proteins are simply 'spacers' between the folded domains. Such a view has been challenged in recent years and the importance of natively unfolded proteins in biology is now being recognized. In this review, we highlight the role of natively unfolded domains in the field of endocytosis, and show that some important endocytic proteins lack a traditionally folded structure and harbour important binding motifs in their unstructured linker regions.
