Determination of the molecular mass and dimensions of membrane proteins by size exclusion chromatography

The Medical Research Council, Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 0XY, UK.
Methods (Impact Factor: 3.65). 11/2008; 46(2):62-72. DOI: 10.1016/j.ymeth.2008.10.020
Source: PubMed


Size exclusion chromatography is an established technique for the determination of hydrodynamic volumes of proteins or protein complexes. When applied to membrane proteins, the contribution of the detergent micelle, which is required to keep the protein soluble in the aqueous phase, needs to be determined to obtain accurate measurements for the protein. In a detergent series, in which the detergents differ only by the length of the alkyl chain, the contribution of the detergent micelle to the hydrodynamic volume is variable, whereas the contribution of the protein is constant. By using this approach, several parameters of membrane proteins can be estimated by extrapolation, such as the radius at the midpoint of the membrane, the average radius, the Stokes radius, and the excluded volume. The molecular mass of the protein can be determined by two independent measurements that arise from the behaviour of the free detergent micelle and protein-detergent micelle during size exclusion chromatography and the determination of the detergent-protein ratio. Determining the dimensions of protein-detergent micelles may facilitate membrane protein purification and crystallization by defining the accessibility of the protein surface.

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    • "A more plausible explanation would involve a Fos14-dependent dissociation of important lipids that may play a crucial role in the receptor ligand binding activity. Alternatively, considering the length of the alkyl chain of Fos14, another possibility detailed in a recent report [45] would be related to the size and shape of the Fos14 micelle surrounding MT1 that would hinder the melatonin-binding site and thus interfere with the binding assay. Further investigations are therefore needed to understand the actual effect of Fos14 on MT1 activity and would be very helpful to evaluate the potential interest of this detergent for the study of other GPCRs. "
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    ABSTRACT: The human melatonin MT1 receptor-belonging to the large family of G protein-coupled receptors (GPCRs)-plays a key role in circadian rhythm regulation and is notably involved in sleep disorders and depression. Structural and functional information at the molecular level are highly desired for fine characterization of this receptor; however, adequate techniques for isolating soluble MT1 material suitable for biochemical and biophysical studies remain lacking. Here we describe the evaluation of a panel of constructs and host systems for the production of recombinant human MT1 receptors, and the screening of different conditions for their solubilization and purification. Our findings resulted in the establishment of an original strategy using a mixture of Fos14 and CHAPS detergents to extract and purify a recombinant human MT1 from Pichia pastoris membranes. This procedure enabled the recovery of relatively pure, monomeric and ligand-binding active MT1 receptor in the near-milligram range. A comparative study based on extensive ligand-binding characterization highlighted a very close correlation between the pharmacological profiles of MT1 purified from yeast and the same receptor present in mammalian cell membranes. The high quality of the purified MT1 was further confirmed by its ability to activate its cognate Gαi protein partner when reconstituted in lipid discs, thus opening novel paths to investigate this receptor by biochemical and biophysical approaches.
    PLoS ONE 06/2014; 9(6):e100616. DOI:10.1371/journal.pone.0100616 · 3.23 Impact Factor
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    • "This unit describes how to combine HPLC-SEC and SLS-UV-RI to evaluate molecular mass and the physicochemical heterogeneity of purified membrane protein-detergent complexes. The general theory and application of SEC-SLS-UV-RI for biopolymers has been reviewed previously (Hayashi et al., 1989; Wyatt, 1991, 1993; Wen et al., 1996; Folta-Stogniew and Williams, 1999; Folta-Stogniew, 2006; Kunji et al., 2008; Slotboom et al., 2008; also see UNIT 20.6) and will receive only minimal discussion here. The Basic Protocol describes (1) instrumentation requirements and set up of the system; (2) column and buffer selection; (3) sample preparation, data collection and data processing; and (4) determination of the molar mass of the PDC and its protein and detergent components. "
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    ABSTRACT: Determination of the oligomeric state of integral membrane proteins in detergent solutions is a challenging task because the amount of detergent associated with the protein is typically unknown and unpredictable. Methods that estimate the molecular weight of proteins from their hydrodynamic properties in solution are not suitable for detergent-solubilized membrane proteins. However, size-exclusion chromatography (SEC) performed in combination with analyses of static light scattering (SLS), ultraviolet absorbance (UV), and refractive index (RI) provides a universal method for determination of the molar masses of biopolymers and protein-detergent complexes. The light scattered by a protein is directly proportional to its molecular mass, irrespective of shape, and any additional contributions due to bound detergent molecules can be quantitatively accounted for by the additional combined analysis of ultraviolet absorbance and refractive index information. The primary intention of this unit is to describe how to apply the combination of high-performance liquid chromatography SEC and SLS-UV-RI to evaluate molecular mass and the physicochemical heterogeneity of purified membrane protein-detergent complexes.
    Current protocols in protein science / editorial board, John E. Coligan ... [et al.] 04/2012; Chapter 29:Unit 29.5.1-12. DOI:10.1002/0471140864.ps2905s68
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    • "For this reason, the precise detection of proteins in the presence of other formulations with high sensitivity and specifity is essential for disease diagnostics, drug screening, and other applications [4,5]. The most widely used method for the detection and analysis of proteins and their aggregates in formulations is size exclusion chromatography (SEC) [5]. Although useful for the determination of molecular-weights of proteins, its application to the quantitative determination of protein concentrations is more difficult. "
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    ABSTRACT: A highly sensitive and molecular size-selective method for the detection of proteins using heteroliganded gold nanoislands and localized surface plasmon resonance (LSPR) is described. Two different heteroligands with different chain lengths (3-mercaptopionicacid and decanethiol) were used in fabricating nanoholes for the size-dependent separation of a protein in comparison with its aggregate. Their ratios on gold nanoisland were optimized for the sensitive detection of superoxide dismutase (SOD1). This protein has been implicated in the pathology of amyotrophic lateral sclerosis (ALS). Upon exposure of the optimized gold nanoisland to a solution of SOD1 and aggregates thereof, changes in the LSPR spectra were observed which are attributed to the size-selective and covalent chemical binding of SOD1 to the nanoholes. With a lower detection limit of 1.0 ng/ml, the method can be used to selectively detect SOD1 in the presence of aggregates at the molecular level.
    Nanoscale Research Letters 04/2011; 6(1):336. DOI:10.1186/1556-276X-6-336 · 2.78 Impact Factor
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