Oligomeric States of Proteins Determined by Size-Exclusion Chromatography Coupled With Light Scattering, Absorbance, and Refractive Index Detectors

Folta‐Stogniew, Ewa

doi:10.1385/1-59745-026-x:97

Cited by 96 publications

(100 citation statements)

References 13 publications

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“…Laser light scattering analyses (34) were performed at the Biophysics Resource of the W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University. Samples were chromatographed on a Superdex 200 column (10/30; GE Healthcare) with detectors for 18-angle static laser light scattering (DAWN-EOS; Wyatt Technology), dynamic light scattering (QELS; Wyatt Technology), refractive index (OPTILAB rEX; Wyatt Technology), and UV absorbance.…”

Section: Laser Light Scattering Determination Of Protein Masses and Hmentioning

confidence: 99%

“…Protein concentration was determined by refractive index and confirmed by UV absorbance (280 nm). Molecular masses were calculated by using ASTRA software (Wyatt Technology) to solve the equation relating scattered light at several angles, concentration, and weight-average molar mass by using the Zimm method (34). The method is accurate to Ϸ3%, based on analysis of standard proteins from 6.5 to 476 kDa.…”

Section: Laser Light Scattering Determination Of Protein Masses and Hmentioning

confidence: 99%

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Assembly of Weibel–Palade body-like tubules from N-terminal domains of von Willebrand factor

Huang

Wang²,

Roth³

et al. 2008

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

144

285

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Lengthwise sections show longitudinal striations, and cross sections reveal closely spaced Ϸ20-nm diameter tubules separated by a less-dense matrix (1). Weibel-Palade bodies are composed almost entirely of von Willebrand factor (VWF) (2, 3), which is a multimeric plasma glycoprotein that can exceed 20 million Da in mass and 4 m in length. Megakaryocytes synthesize large VWF multimers and package them into platelet ␣-granules that are roughly spherical rather than cigar-shaped. Nevertheless, the VWF multimers in ␣-granules are organized into clusters of tubules with dimensions similar to those of VWF tubules in Weibel-Palade bodies (4). VWF is the largest known protein in the blood, and the very largest VWF multimers bind connective tissue and mediate platelet adhesion at sites of vascular injury. Weibel-Palade bodies play a critical role in hemostasis by delivering VWF multimers into the circulation. Defects in VWF multimer structure cause several forms of von Willebrand disease, the most common inherited bleeding disorder worldwide (5).VWF is synthesized as an Ϸ350-kDa precursor with a signal peptide and five kinds of structural domains arranged in the order D1-D2-DЈ-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK ( Fig. 1) (5). In the endoplasmic reticulum (ER), proVWF dimerizes through disulfide bonds between C-terminal CK domains (6-8).The resultant ''tail-to-tail'' proVWF dimers are transported to the Golgi, where the propeptide (domains D1D2) is cleaved by furin and additional ''head-to-head'' disulfide bonds form between D3 domains, yielding multimers that condense into tubules and form Weibel-Palade bodies (6, 9).In fact, the expression of VWF determines the existence and also the cigar-like shape of Weibel-Palade bodies. Without VWF, endothelial cells lack Weibel-Palade bodies (10, 11), and the expression of VWF in other cell types that have a regulated secretory pathway results in the generation of organelles that are indistinguishable from Weibel-Palade bodies (12, 13). This self-organizing behavior depends on a conserved set of Nterminal domains that have the dual function of promoting multimer assembly and directing tubular storage.Multimer assembly (14-16) and tubular packing (12, 17) both depend on the N-terminal D1D2DЈD3 domains and require the acidic pH of the late secretory pathway (13). Furthermore, the tubular morphology of Weibel-Palade bodies is essential for . VWF multimers are held together by intersubunit disulfide bonds between CK domains, which form in the ER, and by intersubunit disulfide bonds between D3 domains, which form in the Golgi. The structures of polypeptides encoded by the plasmids used in these studies are indicated.

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Section: Laser Light Scattering Determination Of Protein Masses and Hmentioning

confidence: 99%

Section: Laser Light Scattering Determination Of Protein Masses and Hmentioning

confidence: 99%

Assembly of Weibel–Palade body-like tubules from N-terminal domains of von Willebrand factor

Huang

Wang²,

Roth³

et al. 2008

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

144

285

View full text Add to dashboard Cite

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“…In doing so we avoid the need to accurately determine the absolute refractive index of the solution (Eq. (4)), and we prevent that small errors in the calibration constants of the instruments are propagated in the molecular mass calculations [6,9,11].…”

Section: Why Use Standards?mentioning

confidence: 99%

“…We routinely only use BSA and aldolase but if very accurate molecular mass determinations are needed then the use of more standards is advisable. It has been noted that albumins may not be suitable standards because they can bind detergent molecules [11]. However, binding of detergent molecules to standard proteins will not affect the determination of the value of K in the three detector method.…”

Section: Why Use Standards?mentioning

confidence: 99%

“…Here we will illustrate the use of the latter technique. Because several groups have reviewed the method of static light scattering for protein analysis in recent years [5][6][7][8][9][10][11] we will focus specifically on issues related to the analysis of membrane proteins. The relevant theory of static light scattering for membrane protein analysis will be presented, followed by an overview of practical aspects illustrated by a number of examples from our lab.…”

Section: Introductionmentioning

confidence: 99%

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Static light scattering to characterize membrane proteins in detergent solution

Slotboom

Duurkens

Olieman

et al. 2008

Methods

166

176

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HPLC‐SEC Characterization of Membrane Protein‐Detergent Complexes

Korepanova

Matayoshi

2012

CP Protein Science

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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.

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Oligomeric States of Proteins Determined by Size-Exclusion Chromatography Coupled With Light Scattering, Absorbance, and Refractive Index Detectors

Cited by 96 publications

References 13 publications

Assembly of Weibel–Palade body-like tubules from N-terminal domains of von Willebrand factor

Assembly of Weibel–Palade body-like tubules from N-terminal domains of von Willebrand factor

Static light scattering to characterize membrane proteins in detergent solution

HPLC‐SEC Characterization of Membrane Protein‐Detergent Complexes

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