Tetra Detector Analysis of Membrane Proteins

Miercke, Larry J. W.; Robbins, Rebecca A.; Stroud, Robert M.

doi:10.1002/0471140864.ps2910s77

Cited by 7 publications

(8 citation statements)

References 32 publications

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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)

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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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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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“…This includes protein and detergent as well as any post-translational modifications such as glycosylation. By using a co-polymer analysis, the amount of protein, glycosylation, protein-associated detergent as well as the free detergent micelles can be calculated [171,178,179].…”

Section: Assessing the Quality Of Purified Mpsmentioning

confidence: 99%

Changes in Membrane Protein Structural Biology

Birch

Cheruvara

Gamage

et al. 2020

Biology

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Membrane proteins are essential components of many biochemical processes and are important pharmaceutical targets. Membrane protein structural biology provides the molecular rationale for these biochemical process as well as being a highly useful tool for drug discovery. Unfortunately, membrane protein structural biology is a difficult area of study due to low protein yields and high levels of instability especially when membrane proteins are removed from their native environments. Despite this instability, membrane protein structural biology has made great leaps over the last fifteen years. Today, the landscape is almost unrecognisable. The numbers of available atomic resolution structures have increased 10-fold though advances in crystallography and more recently by cryo-electron microscopy. These advances in structural biology were achieved through the efforts of many researchers around the world as well as initiatives such as the Membrane Protein Laboratory (MPL) at Diamond Light Source. The MPL has helped, provided access to and contributed to advances in protein production, sample preparation and data collection. Together, these advances have enabled higher resolution structures, from less material, at a greater rate, from a more diverse range of membrane protein targets. Despite this success, significant challenges remain. Here, we review the progress made and highlight current and future challenges that will be overcome.

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“…However, when solubilised in lauryldimethylamine-N-oxide (LDAO), SaMscL shows a tetrameric arrangement that is corroborated with data from analytical ultracentrifugation, SEC-MALS, and X-ray crystallography. In addition, the method can also measure the amount of “free” detergent micelles in the sample that are crucial to consider prior to crystallisation trials [62,63,64,65,66,67,68] and other assays.…”

Section: Characterisation Of Membrane Proteins By Size-exclusion Cmentioning

confidence: 99%

Selection of Biophysical Methods for Characterisation of Membrane Proteins

Kwan

Reis

Siligardi

et al. 2019

IJMS

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Over the years, there have been many developments and advances in the field of integral membrane protein research. As important pharmaceutical targets, it is paramount to understand the mechanisms of action that govern their structure–function relationships. However, the study of integral membrane proteins is still incredibly challenging, mostly due to their low expression and instability once extracted from the native biological membrane. Nevertheless, milligrams of pure, stable, and functional protein are always required for biochemical and structural studies. Many modern biophysical tools are available today that provide critical information regarding to the characterisation and behaviour of integral membrane proteins in solution. These biophysical approaches play an important role in both basic research and in early-stage drug discovery processes. In this review, it is not our objective to present a comprehensive list of all existing biophysical methods, but a selection of the most useful and easily applied to basic integral membrane protein research.

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Tetra Detector Analysis of Membrane Proteins

Cited by 7 publications

References 32 publications

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

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

Changes in Membrane Protein Structural Biology

Selection of Biophysical Methods for Characterisation of Membrane Proteins

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