Static light scattering to characterize membrane proteins in detergent solution

Slotboom, Dirk Jan; Duurkens, Ria H.; Olieman, Kees; Erkens, Guus B.

doi:10.1016/j.ymeth.2008.06.012

Cited by 166 publications

(185 citation statements)

References 22 publications

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“…We expected that this property should be maintained in Na V Sp1p and used two independent methods to probe Na V Sp1p oligomerization (Table 1): glutaraldehyde (GTA) chemical cross-linking (37,38) and size-exclusion chromatography coupled with triple detector analysis (SEC-TDA) (39). SDS-PAGE analysis of GTA cross-linked Na V Sp1p solubilized in DDM revealed the presence of three cross-linked products that corresponded to dimers, trimers, and tetramers, but did not indicate higher-order species ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…This methodology, in which changes in refractive index, ultraviolet absorption, and static angle light scattering are measured, allows determination of the absolute molecular weight of the protein component of the protein-detergent micelle (39,40) and has been recently applied to determine the association states of a variety of membrane proteins in different detergents (41)(42)(43). SEC-TDA of Na V Sp1p (Fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Voltage-gated sodium channel (Na_V) protein dissection creates a set of functional pore-only proteins

Shaya

Kreir

Robbins³

et al. 2011

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

133

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Many voltage-gated ion channel (VGIC) superfamily members contain six-transmembrane segments in which the first four form a voltage-sensing domain (VSD) and the last two form the pore domain (PD). Studies of potassium channels from the VGIC superfamily together with identification of voltage-sensor only proteins have suggested that the VSD and the PD can fold independently. Whether such transmembrane modularity is common to other VGIC superfamily members has remained untested. Here we show, using protein dissection, that the Silicibacter pomeroyi voltagegated sodium channel (Na V Sp1) PD forms a stand-alone, ion selective pore (Na V Sp1p) that is tetrameric, α-helical, and that forms functional, sodium-selective channels when reconstituted into lipid bilayers. Mutation of the Na V Sp1p selectivity filter from LESWSM to LDDWSD, a change similar to that previously shown to alter ion selectivity of the bacterial sodium channel Na V Bh1 (NaChBac), creates a calcium-selective pore-only channel, Ca V Sp1p. We further show that production of PDs can be generalized by making pore-only proteins from two other extremophile Na V s: one from the hydrocarbon degrader Alcanivorax borkumensis (Na V Ab1p), and one from the arsenite oxidizer Alkalilimnicola ehrlichei (Na V Ae1p). Together, our data establish a family of active pore-only ion channels that should be excellent model systems for study of the factors that govern both sodium and calcium selectivity and permeability. Further, our findings suggest that similar dissection approaches may be applicable to a wide range of VGICs and, thus, serve as a means to simplify and accelerate biophysical, structural, and drug development efforts.V oltage-gated sodium channels (Na V s) are large polytopic membrane proteins involved in action potential generation in excitable cells and belong to an ion channel superfamily that includes voltage-gated calcium channels (Ca V s), voltage-gated potassium channels (K V s), and transient receptor potential (TRP) channels (1, 2). Within the voltage-gated ion channel (VGIC) superfamily, Na V s and Ca V s are close relatives (1-3) that share a topology of 24 transmembrane segments organized in four homologous six-transmembrane repeats. These two families are also thought to share some common structure in the ion selectivity filter despite having markedly different ion permeation properties (4). Both are central to human neuromuscular, cardiovascular, and neural physiology. Consequently, they are targets for a host of pharmaceuticals used to treat a diverse set of disorders and remain active targets for drug development (5-7). Recently, single subunit, six-transmembrane segment Na V s have been identified in a large number of bacteria from diverse environments (8, 9). These channels show clear similarities to eukaryotic Na V s and Ca V s (2, 9, 10), suggesting that the prokaryotic channels may have been ancestors of the more complex vertebrate channels.Despite the central importance of Na V s, nothing is known about the high-resolution structure o...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Voltage-gated sodium channel (Na_V) protein dissection creates a set of functional pore-only proteins

Shaya

Kreir

Robbins³

et al. 2011

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

133

View full text Add to dashboard Cite

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“…IMAC-purified protein samples were loaded onto a Superdex 200 column (10/300 GL, GE Healthcare) equilibrated with 50 mM potassium phosphate, pH 7.8, 200 mM NaCl, 0.05% (w/v) DDM, run at 0.5 ml/min. For data analysis, protein was taken to have a refractive index increment (dn/dc) of 0.186 ml/g and DDM of 0.143 ml/g (26). The ratio of DDM to protein was determined by a colorimetric assay of the DDM maltoside (25) as 219 Ϯ 63 DDM per VcSiaQM and 235 Ϯ 93 DDM per HiSiaQM.…”

Section: Methodsmentioning

confidence: 99%

The Membrane Proteins SiaQ and SiaM Form an Essential Stoichiometric Complex in the Sialic Acid Tripartite ATP-independent Periplasmic (TRAP) Transporter SiaPQM (VC1777–1779) from Vibrio cholerae

Mulligan

Leech

Kelly

et al. 2012

Journal of Biological Chemistry

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Background: Vibrio cholerae has a need for sialic acid transport and catabolism for colonization. Results: The VC1777-1779 genes encode a functional tripartite ATP-independent periplasmic (TRAP) transporter for sialic acid in which the membrane subunits form a tight complex. Conclusion: Vibrio cholerae encodes a functional sialic acid TRAP transporter. Significance: This study reveals the route of sialic acid acquisition by an important human pathogen.

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“…2). The weight-averaged molecular mass was calculated based on the measured ⌬LS (light scattering) and C (the concentration of the protein) values (20). The molecular mass of NorM_PS in detergent solution was determined to be 54.7 kDa, which corresponds well to the theoretical monomeric molar mass of 53.0 kDa.…”

Section: Production and Purification Of Norm_ps And Its Variants-mentioning

confidence: 98%

Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri

Nie

Grell

Malviya

et al. 2016

Journal of Biological Chemistry

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Multidrug and toxic compound extrusion (MATE) transportThe ability of all living organisms to protect themselves against toxic substances is essential for their survival. Organisms acquire this ability through evolutionary force (1). Due to the development of scientific knowledge, more and more medical drugs are being developed and applied. As a consequence, general defense mechanisms to resist antibiotics, drugs, and other toxic compounds have been evolved in bacteria (2). Being one of those defense mechanisms, the active extrusion of the toxic compounds from the cells plays an important role (3). In bacteria, the extrusion is often carried out by multidrug transporters, which are integral membrane proteins. They are classified into five distinct families: ATP-binding cassette, multidrug and toxic compound extrusion (MATE), 4 major facilitator superfamily, resistance nodulation division, and small multidrug resistance transporters (4).In 1999, the MATE family proteins were first recognized as a new group of the multidrug transporters, existing in all three domains of life (5). Most members of this family consist of a single chain of 450 -550 amino acid residues and exhibit 12 putative transmembrane helices (TMHs). Up to now, about 900 proteins have been annotated as MATE transporters on the basis of amino acid sequence similarities. Moreover, a subclassification of the MATE family has been suggested, NorM-, DinF-(DNA damage-inducible protein F), and eukaryotic subfamily (4, 6, 7). Various compounds can be recognized by NorM proteins, including dyes, fluoroquinolones, and aminoglycosides (7). As secondary active transporters, MATE proteins utilize transmembrane Na ϩ or H ϩ electrochemical gradients to extrude toxic compounds.So far atomic structures of four MATE transporters have been reported in their putative outward-facing states, in both drug-bound and/or drug-free states. NorM_VC from Vibrio cholerae and NorM_NG from Neisseria gonorrhoeae, are both Na ϩ driven, whereas PfMATE from Pyrococcus furiosus and DinF-BH from Bacillus halodurans are published to be H ϩ -dependent antiporters (8 -11). All these four proteins contain 12 TMHs with intracellular N and C termini, and possess a hydrophobic internal cavity, formed by two symmetric bundles with 6 TMHs each. The two bundles are linked by a cytoplasmic loop between the 6th and 7th TMH. All MATE proteins share about 40% sequence similarity, and they employ the same rockerswitch mechanism for substrate extrusion (12). In addition, the crystal structures have also revealed different locations of cationand substrate-binding sites, suggesting a mechanistic diversity among MATE transporters. In the structure of drug-bound NorM_NG, the binding site is located close to the membraneperiplasm interface, whereas in the structure of PfMATE, the

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Static light scattering to characterize membrane proteins in detergent solution

Cited by 166 publications

References 22 publications

Voltage-gated sodium channel (Na_V) protein dissection creates a set of functional pore-only proteins

Voltage-gated sodium channel (Na_V) protein dissection creates a set of functional pore-only proteins

The Membrane Proteins SiaQ and SiaM Form an Essential Stoichiometric Complex in the Sialic Acid Tripartite ATP-independent Periplasmic (TRAP) Transporter SiaPQM (VC1777–1779) from Vibrio cholerae

Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri

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Static light scattering to characterize membrane proteins in detergent solution

Cited by 166 publications

References 22 publications

Voltage-gated sodium channel (NaV) protein dissection creates a set of functional pore-only proteins

Voltage-gated sodium channel (NaV) protein dissection creates a set of functional pore-only proteins

The Membrane Proteins SiaQ and SiaM Form an Essential Stoichiometric Complex in the Sialic Acid Tripartite ATP-independent Periplasmic (TRAP) Transporter SiaPQM (VC1777–1779) from Vibrio cholerae

Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri

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Voltage-gated sodium channel (Na_V) protein dissection creates a set of functional pore-only proteins

Voltage-gated sodium channel (Na_V) protein dissection creates a set of functional pore-only proteins