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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"Characterizing AMB-PEG Loaded with Free AMB separated by SEC using a Superdex 75 column, both AMB-PEG formulations had a retention time of 20 minutes (Fig 4B), suggesting that they were significantly smaller than the smallest globular protein standard possessing a mass of 6.5 kDa. This method of size determination via the utilization of these molecular weight standards is thus possibly inaccurate as it assumes that the standard and our sample possess highly similar structures [35,36]. Since AMB-PEG and globular proteins have largely different chemical compositions and polarity, this is a possible explanation for the difference in estimated size observed during SEC. "
[Show abstract][Hide abstract]ABSTRACT: Amphotericin B (AMB) is a highly hydrophobic antifungal, whose use is limited by its toxicity and poor solubility. To improve its solubility, AMB was reacted with a functionalized polyethylene glycol (PEG), yielding soluble complex AmB-PEG formulations that theoretically comprise of chemically conjugated AMB-PEG and free AMB that is physically associated with the conjugate. Reverse-phase chromatography and size exclusion chromatography methods using HPLC were developed to separate conjugated AMB-PEG and free AmB, enabling the further characterization of these formulations. Using HPLC and dynamic light scattering analyses, it was observed that the AMB-PEG 2 formulation, having a higher molar ratio of 2 AMB: 1 PEG, possesses more free AMB and has relatively larger particle diameters compared to the AMB-PEG 1 formulation, that consists of 1 AMB: 1 PEG. The identity of the conjugate was also verified using mass spectrometry. AMB-PEG 2 demonstrates improved antifungal efficacy relative to AMB-PEG 1, without a concurrent increase in in vitro toxicity to mammalian cells, implying that the additional loading of free AMB in the AMB-PEG formulation can potentially increase its therapeutic index. Compared to unconjugated AMB, AMB-PEG formulations are less toxic to mammalian cells in vitro, even though their MIC50 values are comparatively higher in a variety of fungal strains tested. Our in vitro results suggest that AMB-PEG 2 formulations are two times less toxic than unconjugated AMB with antifungal efficacy on Candida albicans and Cryptococcus neoformans.
"Mass estimation by SEC is based on a comparison of the elution behaviour of the protein of interest with that of standard proteins, which are globular and water-soluble. Since detergent binding substantially alters a protein's elution behaviour  and, thus, its apparent size, analysing membrane proteins with SEC will provide, at best, a very crude size estimate. Nevertheless, with careful sample preparation, information on the coexistence of different oligomeric species can be deduced from SEC elution profiles even without knowledge of exact masses  . "
[Show abstract][Hide abstract]ABSTRACT: Background: In vitro investigations of membrane proteins usually depend on detergents for protein solubilisation and stabilisation. The amount of detergent bound to a membrane protein is relevant to successful experiment design and data analysis but is often unknown. Triple-detection size-exclusion chromatography enables simultaneous separation of protein/detergent complexes and protein-free detergent micelles and determination of their molar masses in a straightforward and absolute manner. Size-exclusion chromatography is used to separate different species, while ultraviolet absorbance, static light scattering, and refractive index measurements allow molar mass determination of protein and detergent components.
Results: We refined standard experimental and data-analysis procedures for challenging membrane-protein samples that elude routine approaches. The general procedures including preparatory steps, measurements, and data analysis for the characterisation of both routine and complex samples in difficult solvents such as concentrated denaturant solutions are demonstrated. The applicability of the protocol but also its limitations and possible solutions are discussed, and an extensive troubleshooting section is provided.
Conclusions: We established and validated a protocol for triple-detection size-exclusion chromatography that enables the inexperienced user to perform and analyse measurements of well-behaved protein/detergent complexes. More experienced users are provided with an example of a more sophisticated analysis procedure allowing mass determination under challenging separation conditions.
Keywords: Membrane proteins, Detergent micelles, Absolute mass determination, Multiple detection, Static light scattering
Full-text · Article · Feb 2016 · Biological Procedures Online
"For XRD, maintaining soluble monodispersed protein is crucial for successful crystallization, whereas for solution-state NMR a polydisperse sample will unduly confound what is likely an already challenging resonance assignment process. SEC may be used to confirm homogeneity of a purified/solubilized POI, monodispersity and oligomeric state of the POI, and as such is frequently used as an analytical technique to validate sample integrity (Kunji et al. 2008), rather than " just " for purification as discussed above. Hydrodynamics techniques, such as dynamic light scattering (Neale et al. 2013 ), sedimentation velocity analytical ultracentrifugation (Ebel 2011 ), or pulsed-field gradient diffusion NMR (Liebau et al. 2015), are highly valuable alternatives or corroborative techniques for SEC-based confirmation of sample mondispersity. "
[Show abstract][Hide abstract]ABSTRACT: Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles, owing to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and (or) amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through the introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10-15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM).
No preview · Article · Jan 2016 · Biochemistry and Cell Biology