Free-boundary Electrophoresis of Sodium Dodecyl Sulfate-Protein Polypeptide Complexes with Special Reference to SDS-Polyacrylamide Gel Electrophoresis

Shirahama, Keishiro; Tsujii, Kaoru; Takagi, Toshio

doi:10.1093/oxfordjournals.jbchem.a130398

Cited by 282 publications

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“…The "necklace and bead structure" of protein-surfactant complexes has two possibilities for a dominant mode of interaction ( Fig. 1): (1) The protein wraps around the micelle and (2) the micelles nucleate on the protein hydrophobic sites (Shirahama et al 1974;Chen and Teixeira 1986;Guo et al 1990;Turro et al 1995). Although we can not prove or disprove this model, we can evaluate the consistency of either model with our observed data.…”

Section: ␣-Helices (Closed) ␣-Helices (Open) → ␤-Strandmentioning

confidence: 81%

“…Several models have been proposed for the structure of protein-surfactant complexes in SDS over its critical micelle concentration (CMC): (1) a correlated "necklace and bead" model in which clusters with a micelle-like structure are stabilized by protein ( Fig. 1; Shirahama et al 1974;Guo et al 1990;Turro et al 1995); (2) "rod-like" prolate elliposidal surfactant aggregate with a semi-minor axis of ∼18 Å, corresponding to the surfactant chain length (Reynolds and Tanford 1970b;Tanford 1980); and (3) a flexible capped helical cylinder micelle with the protein wrapping around the micelle (Lundahl et al 1986). A small-angle neutron scattering study of BSA and ovalbumin complexes with SDS led to the conclusion that a necklace and bead structure, composed of protein-surfactant aggregates, accounted for the scattering behavior of these systems (Chen and Teixeira 1986;Guo et al 1990), which was confirmed by NMR experiments by Turro et al (1995).…”

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confidence: 99%

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Effect of sodium dodecyl sulfate on folding and thermal stability of acid‐denatured cytochrome c: A spectroscopic approach

Keiderling

2004

Protein Science

102

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The molten globule (MG) state can be an intermediate in the protein folding pathway; thus, its detailed description can help understanding protein folding. Sodium dodecyl sulfate (SDS), an anionic surfactant that is commonly used to mimic hydrophobic binding environments such as cell membranes, is known to denature some native state proteins, including horse cytochrome c (cyt c). In this article, refolding of acid denatured cyt c is studied under the influence of SDS to form MG-like states at both low concentration and above the critical micelle concentration using Fourier transform Infrared (FTIR) and ultraviolet and visible absorption as well as fluorescence and circular dichroism (CD). Thermal denaturation monitored with FTIR and CD shows distinct final high temperature states starting from MG-like states formed with different SDS/protein ratios. The results suggest that the SDS/protein ratio as well as the actual SDS (or protein) concentration affects structure and its thermal stability. Thermal denaturation monitored with CD and FTIR for cyt c at neutral pH but denatured with SDS showed that at a high SDS/protein ratio, the thermal behavior of MG-like states formed at low and neutral pH are quite similar. Based on the results obtained, the merits of two models of the protein-surfactant structure are discussed for different SDS concentrations.

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Section: ␣-Helices (Closed) ␣-Helices (Open) → ␤-Strandmentioning

confidence: 81%

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confidence: 99%

Effect of sodium dodecyl sulfate on folding and thermal stability of acid‐denatured cytochrome c: A spectroscopic approach

Keiderling

2004

Protein Science

102

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“…The latter finding presumably arises from a change in the balance of the charge and frictional forces known to underlie Y 0 as M r is increased (22). In certain systems, the relationship of Y 0 to M r among typical reference proteins may be approximated by monotonic or polynomial functions (23); in others, Y 0 values among globular, water-soluble polypeptides are nearly independent of M r (22,(25)(26)(27).…”

Section: Resultsmentioning

confidence: 99%

Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts

Rath

Cunningham

Deber

2013

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

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SDS/PAGE is universally used in biochemistry, cell biology, and immunology to resolve minute protein amounts readily from tissue and cell extracts. Although molecular weights of watersoluble proteins are reliably determined from their SDS/PAGE mobility, most helical membrane proteins, which comprise 20-30% of the human genome and the majority of drug targets, migrate to positions that have for decades been unpredictably slower or faster than their actual formula weight, often confounding their identification. Using de novo designed transmembrane-mimetic polypeptides that match the composition of helical membranespanning sequences, we quantitate anomalous SDS/PAGE fractionation of helical membrane proteins by comparing the relative mobilities of these polypeptides with typical water-soluble reference proteins on Laemmli gels. We find that both the net charge and effective molecular size of the migrating particles of transmembrane-mimetic species exceed those of the corresponding reference proteins and that gel acrylamide concentration dictates the impact of these two factors on the direction and magnitude of anomalous migration. Algorithms we derived from these data compensate for this differential effect of acrylamide concentration on the SDS/PAGE mobility of a variety of natural membrane proteins. Our results provide a unique means to predict anomalous migration of membrane proteins, thereby facilitating straightforward determination of their molecular weights via SDS/PAGE. gel mobility | protein migration | protein identification | apparent size | immunoblotting L aemmli's system for polyacrylamide gel protein electrophoresis in the presence of the detergent SDS (SDS/PAGE) is one of the most cited methodological papers in life sciences (1). The facility with which SDS/PAGE resolves minute amounts of proteins revolutionized the analysis of tissue and cell extracts, resulting in "overnight" adoption of the technique in biochemistry, cell biology, immunology, and virology (2). Considered "the single most useful analytical tool to study protein molecules" (3), SDS/ PAGE is routinely used for simultaneous determination of protein heterogeneity and molecular weight in applications ranging from diagnosis of hereditary red cell membrane disorders to evaluation of recombinant protein expression and purification procedures. Protein analysis by SDS/PAGE is relatively simple, affordable, and rapid (4): A buffer containing a tracking dye and SDS is added to the sample of interest, the mixture is applied to a polyacrylamide gel, and a potential difference is used to drive the dye and the resulting anionic particle composed of protein and dodecyl sulfate (DS) through the gel. The distance traveled by the protein/DS particle from the top of the gel is then divided by that of the dye to obtain relative migration (R f ), and molecular weight [as relative molecular mass (M r )] determined by comparison of this value with a logarithmic plot derived from the R f s and M r s of reference proteins.Fractionation on SDS/PAGE is contro...

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“…evidence for SDS/protein complexes taking the form of spherical SDS micelle-like structures distributed along an extended polypeptide backbone, the so-called necklace model (Shirahama et al, 1974;Chen and Teixeira, 1986;Ibel et al, 1990;. None of this work, however, makes any attempt to approach the conditions of SDSPAGE and as it is known from cryo-transmission electron microscopy (CTEM) that detergent assemblies display different morphologies in different conditions (Magid et al, 1990;Vinson et al, 1991 ;Zana and Talmon, 1993), several candidate models have been proposed for SDS/protein complexes (e.g.…”

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confidence: 99%

Evidence for Sodium Dodecyl Sulfate/Protein Complexes Adopting a Necklace Structure

Samsó

Daban

Hansen³

et al. 1995

European Journal of Biochemistry

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Structural analysis by cryo-electron microscopy and small-angle X-ray scattering of ten sodium dodecyl sulfate/protein complexes in 25 mM Tris/HCI, 0.192 M glycine, pH 8.3, showed necklace-like structures of spherical micelles dispersed along the unfolded peptide chain. The micelles of most SDS/protein complexes had a constant diameter (= 6.2 nm), slightly larger than pure SDS micelles (-5.7 nm), all micelles possessing a degree of surface roughness. The micelle-associated polypeptide is mostly situated at the interface of the sulfate head groups and hydrocarbon core, intruding into the core rather than outward from the surface. Proteins with a molecular mass less than about 20 kDa formed complexes with a single SDS micelle. Multi-micellar SDSIprotein complexes had centre-to-centre intermicellar distances in the range 7.0-12.0 nm. Our findings on the constancy of micellar size, number of micelles/complex, and the relationship between the degree of occupancy of micelles and a polypeptide's molecular mass, have enabled us to speculate on the correlation between the electrophoretic mobility of a polypeptide in SDS/PAGE and its molecular mass. The anomalous electrophoretic behaviour observed for the sodium dodecyl sulfate/histone H5 complex is accounted for by the large micelle of its complex.

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Free-boundary Electrophoresis of Sodium Dodecyl Sulfate-Protein Polypeptide Complexes with Special Reference to SDS-Polyacrylamide Gel Electrophoresis

Cited by 282 publications

References 0 publications

Effect of sodium dodecyl sulfate on folding and thermal stability of acid‐denatured cytochrome c: A spectroscopic approach

Effect of sodium dodecyl sulfate on folding and thermal stability of acid‐denatured cytochrome c: A spectroscopic approach

Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts

Evidence for Sodium Dodecyl Sulfate/Protein Complexes Adopting a Necklace Structure

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