HPLC‐SEC Characterization of Membrane Protein‐Detergent Complexes

Korepanova, Alla; Matayoshi, Edmund D.

doi:10.1002/0471140864.ps2905s68

Cited by 15 publications

(14 citation statements)

References 15 publications

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“…1 D ) indicated that the protein was of high monodispersity, with an apparent M r of ∼500 kDa. Although the subunit structure of membrane proteins cannot be determined by gel filtration ( 24 ), the similar size obtained by BN/PAGE supported that the TMEM16F protein existed as a homodimer.…”

Section: Resultsmentioning

confidence: 94%

Single-molecule analysis of phospholipid scrambling by TMEM16F

Watanabe

Sakuragi

Noji

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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SignificanceMembrane proteins that translocate various compounds across biological membranes play important roles in maintaining intracellular homeostasis in cells. Scramblases are membrane proteins that translocate phospholipids between the inner and outer leaflets of the plasma membrane. However, the mechanisms through which these scramblases translocate phospholipids carrying a hydrophilic residue across the hydrophobic membranes are still unclear. Here, we developed a microsystem containing lipid-bilayer membrane arrays with asymmetrically distributed fluorescent phospholipids. Using this system, we measured phospholipid transport mediated by the TMEM16F scramblase at the single-molecule level, thereby enabling characterization of the biophysical features of phospholipid transport.

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Section: Resultsmentioning

confidence: 94%

Single-molecule analysis of phospholipid scrambling by TMEM16F

Watanabe

Sakuragi

Noji

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…The presence of all expected proteins in the elution peaks was checked by immuno-blotting. Interpreting these large apparent molecular masses is however challenging, as the extracellular domain of DivIB, DivIC, FtsL, PBP2x are elongated [23,47,52,53] and the estimation of the molecular mass of proteins from their hydrodynamic properties in solution is not suitable for detergent-solubilized membrane proteins [54]. However, DivIB and the H-DivIB/DivIC/FtsL complex eluted in two peaks, indicating that these entities could form dimers.…”

Section: Resultsmentioning

confidence: 99%

Reconstitution of Membrane Protein Complexes Involved in Pneumococcal Septal Cell Wall Assembly

et al. 2013

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The synthesis of peptidoglycan, the major component of the bacterial cell wall, is essential to cell survival, yet its mechanism remains poorly understood. In the present work, we have isolated several membrane protein complexes consisting of the late division proteins of Streptococcus pneumoniae: DivIB, DivIC, FtsL, PBP2x and FtsW, or subsets thereof. We have co-expressed membrane proteins from S. pneumoniae in Escherichia coli. By combining two successive affinity chromatography steps, we obtained membrane protein complexes with a very good purity. These complexes are functional, as indicated by the retained activity of PBP2x to bind a fluorescent derivative of penicillin and to hydrolyze the substrate analogue S2d. Moreover, we have evidenced the stabilizing role of protein-protein interactions within each complex. This work paves the way for a complete reconstitution of peptidoglycan synthesis in vitro, which will be critical to the elucidation of its intricate regulation mechanisms.

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“…By far the most common applications are establishing whether a purified protein is monomeric or oligomeric and the degree of oligomerization, and assessing aggregates 3,10,11,17,31,36,37,38 . The ability to do so for detergentsolubilized membrane proteins that cannot be characterized by traditional means is especially prized, and detailed protocols for this have been published 31,39,40,41,42,43 . Other common applications include establishing the degree of post-translational modification and polydispersity of glycoprotein, lipoproteins and similar conjugates 4,31,44,45,46,47 ; the formation (or lack thereof) and absolute stoichiometry (as opposed to stoichiometric ratio) of heterocomplexes including protein-protein, protein-nucleic acid and protein-polysaccharide complexes 24,46,48,49,50,51,52 ; determining the monomer-dimer equilibrium dissociation constant 49,53,54 ; and evaluating protein conformation 55,56 .…”

Section: Introductionmentioning

confidence: 99%

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Some

Amartely

Tsadok³

et al. 2019

JoVE

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Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in solution, is a relative technique that relies on the elution volume of the analyte to estimate molecular weight. When the protein is not globular or undergoes non-ideal column interactions, the calibration curve based on protein standards is invalid, and the molecular weight determined from elution volume is incorrect. Multi-angle light scattering (MALS) is an absolute technique that determines the molecular weight of an analyte in solution from basic physical equations. The combination of SEC for separation with MALS for analysis constitutes a versatile, reliable means for characterizing solutions of one or more protein species including monomers, native oligomers or aggregates, and heterocomplexes. Since the measurement is performed at each elution volume, SEC-MALS can determine if an eluting peak is homogeneous or heterogeneous and distinguish between a fixed molecular weight distribution versus dynamic equilibrium. Analysis of modified proteins such as glycoproteins or lipoproteins, or conjugates such as detergent-solubilized membrane proteins, is also possible. Hence, SEC-MALS is a critical tool for the protein chemist who must confirm the biophysical properties and solution behavior of molecules produced for biological or biotechnological research. This protocol for SEC-MALS analyzes the molecular weight and size of pure protein monomers and aggregates. The data acquired serve as a foundation for further SEC-MALS analyses including those of complexes, glycoproteins and surfactant-bound membrane proteins.

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HPLC‐SEC Characterization of Membrane Protein‐Detergent Complexes

Cited by 15 publications

References 15 publications

Single-molecule analysis of phospholipid scrambling by TMEM16F

Single-molecule analysis of phospholipid scrambling by TMEM16F

Reconstitution of Membrane Protein Complexes Involved in Pneumococcal Septal Cell Wall Assembly

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

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