Polymeric Nonelectrolytes to Probe Pore Geometry: Application to the α-Toxin Transmembrane Channel

Merzlyak, Petr G.; Yuldasheva, Liliya N.; Rodrigues, Cláudio Gabriel; Carneiro, C. M. M.; Krasilnikov, Oleg V.; Bezrukov, Sergey M.

doi:10.1016/s0006-3495(99)77133-x

Cited by 116 publications

(123 citation statements)

References 46 publications

Supporting

Mentioning

112

Contrasting

Order By: Relevance

“…PEGs behave approximately like spherical molecules in aqueous solutions (34,35), and PEG molecules of different sizes have been widely used to estimate the diameter of artificial and natural ion channels (36 -38). Estimates of the hydrated diameter of PEG-5 and PEG-20 are 41-60 Å (39) and ϳ130 Å (40), respectively.…”

Section: Discussionmentioning

confidence: 99%

Surface Accessibility and Conformational Changes in the N-terminal Domain of Type I Inositol Trisphosphate Receptors

Anyatonwu

Joseph

2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

To identify surface-accessible residues and monitor conformational changes of the type I inositol 1,4,5-trisphosphate receptor protein in membranes, we have introduced 10 cysteine substitutions into the N-terminal ligand-binding domain. The reactivity of these mutants with progressively larger maleimidepolyethylene glycol derivatives (MPEG) was measured using a gel shift assay of tryptic fragments. The results indicate that the mutations fall into four categories as follows: sites that are highly accessible based on reactivity with the largest 20-kDa MPEG (S2C); sites that are moderately accessible based on reactivity only with 5-kDa MPEG (S6C, S7C, A189C, and S277C); sites whose accessibility is markedly enhanced by Ca 2؉ (S171C, S277C, and A575C); and sites that are inaccessible irrespective of incubation conditions (S217C, A245C, and S436C). The stimulation of accessibility induced by Ca 2؉ at the S277C site occurred with an EC 50 of 0.8 M and was mimicked by Sr 2؉ but not Ba 2؉ . Inositol 1,4,5-trisphosphate alone did not affect reactivity of any of the mutants in the presence or absence of Ca 2؉ . The data are interpreted using crystal structures and EM reconstructions of the receptor. Our data identify N-terminal regions of the protein that become exposed upon Ca 2؉ binding and suggest possible orientations of the suppressor and ligand-binding domains that have implications for the mechanism of gating of the channel.Inositol 1,4,5-trisphosphate receptors (IP 3 R) 2 are ligandgated channels important in Ca 2ϩ signaling triggered by diverse cellular stimuli (1). Three different isoforms are present that share 60 -70% sequence homology (2-4). The type I isoform is also subject to alternative splicing at three sites (5). IP 3 R channels are tetrameric with each monomer organized into four distinct domains as follows: an N-terminal suppressor domain, a core ligand-binding domain (LBD), a regulatory domain, and a C-terminal channel domain (2-4). Deletion mutagenesis mapped the suppressor domain to amino acids 1-223, and IP 3 binding studies showed that this domain reduces the IP 3 binding affinity for the receptor (6). The crystal structure of the suppressor domain shows that it folds into a variant of a ␤-trefoil structure (7). The core LBD (amino acids 224 -604) has also been crystallized and shown to fold into two distinct domains: a ␤-trefoil and a ␣-helical domain, with residues from both domains contributing to the IP 3 -binding site. The regulatory domain spans amino acids 600 -2220 and contains sites for numerous channel modulators (2). Ca 2ϩ release from the receptor occurs in the channel domain (amino acids 2250 -2700) consisting of the six transmembrane (TM) segments, of which the TM5 and TM6 form the channel pore (8, 9).The two principal modulators of the channel are IP 3 and Ca 2ϩ . Conformational changes induced by these ligands are likely to be critical to the mechanism of channel function. IP 3 binding in the N-terminal domain results in channel gating in the C-terminal domain. Conformational cha...

show abstract

Section: Discussionmentioning

confidence: 99%

Surface Accessibility and Conformational Changes in the N-terminal Domain of Type I Inositol Trisphosphate Receptors

Anyatonwu

Joseph

2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…This linearity cannot be expected, however, to extend to arbitrarily high stresses. At sufficiently high concentrations, excluded solute can be expected to exchange with preferentially included water on exposed macromolecular surfaces (18,19). Additionally, osmotic stress will eventually deform the macromolecule and distort states a and b; their individual N s and N w would change with ⌸ to give a distorted estimate of ⌬N ew ab .…”

Section: Thermodynamic Constraints Link the Different Perspectivesmentioning

confidence: 99%

Osmotic stress, crowding, preferential hydration, and binding: A comparison of perspectives

Parsegian

Rand

Rau

2000

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

472

606

View full text Add to dashboard Cite

There has been much confusion recently about the relative merits of different approaches, osmotic stress, preferential interaction, and crowding, to describe the indirect effect of solutes on macromolecular conformations and reactions. To strengthen all interpretations of measurements and to forestall further unnecessary conceptual or linguistic confusion, we show here how the different perspectives all can be reconciled. Our approach is through the Gibbs-Duhem relation, the universal constraint on the number of ways it is possible to change the temperature, pressure, and chemical potentials of the several components in any thermodynamically defined system. From this general Gibbs-Duhem equation, it is possible to see the equivalence of the different perspectives and even to show the precise identity of the more specialized equations that the different approaches use.

show abstract

“…The diameter of an ion channel can be estimated from the dependence of the single-channel conductance on the size of nonelectrolytes present in the membrane bathing solutions (71)(72)(73)(74). The effect of glycerol, PEG 300, and PEG 3350 on the conductance of small and large channels formed by P178 is shown (Figs.…”

Section: Estimation Of the Size Of 60-and 600-ps Channelsmentioning

confidence: 99%

Lipid Dependence of the Channel Properties of a Colicin E1-Lipid Toroidal Pore

Sobko

Kotova

Antonenko

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

Colicin E1 belongs to a group of bacteriocins whose cytotoxicity toward Escherichia coli is exerted through formation of ion channels that depolarize the cytoplasmic membrane. The lipid dependence of colicin single-channel conductance demonstrated intimate involvement of lipid in the structure of this channel. The colicin formed "small" conductance 60-picosiemens (pS) channels, with properties similar to those previously characterized, in 1,2-dieicosenoyl-snglycero-3-phosphocholine (C20) or thinner membranes, whereas it formed a novel "large" conductance 600-pS state in thicker 1,2-dierucoyl-sn-glycero-3-phosphocholine (C22) bilayers. Both channel states were anion-selective and voltage-gated and displayed a requirement for acidic pH. Lipids having negative spontaneous curvature inhibited the formation of both channels but increased the ratio of open 600 pS to 60 pS conductance states. Different diameters of small and large channels, 12 and 16 Å , were determined from the dependence of single-channel conductance on the size of nonelectrolyte solute probes. Colicin-induced lipid "flip-flop" and the decrease in anion selectivity of the channel in the presence of negatively charged lipids implied a significant contribution of lipid to the structure of the channel, most readily described as toroidal organization of lipid and protein to form the channel pore.The lipid environment of membrane proteins that defines the local distribution of polarity, dielectric constant, and steric geometry has a central role in the determination of protein structure and function. For ion channels, lipids impose the following constraints: (i) structure-function is sensitive to the matching of hydrophobic thickness of the protein and lipid bilayer (1-10); (ii) opening of channels (e.g. mechano-sensitive, can be dependent on bilayer tension) (11), which manifests itself in a dependence on acyl chain length (12); (iii) spontaneous curvature (SC) 2 of lipids linked to the lateral pressure of the bilayer can modulate ion channel activity (13), as demonstrated for the KcsA channel (14) and mechano-sensitive MscL channels (12, 15); and (iv) many channel proteins require anionic lipids for function (16 -19).Channel-forming proteins can require specific lipids, presumably because of a combination of charge and curvature parameters, to form active channels (e.g. anionic lipid bound between the transmembrane ␣-helices and necessary for gating of the KcsA channel (20, 21)). The lantibiotic nisin provides a well defined example of lipid participation in peptide pore formation, in which the peptidoglycan precursor lipid II is an intrinsic component of the pore formed by this antimicrobial peptide (22). The proposed structure of this pore contains 5-8 nisin molecules and an identical number of lipid II molecules (23). Participation of lipid in the formation of a channel wall was hypothesized for cholesteroldependent cytolysins (24,25).Colicins are plasmid-encoded bacteriocins that are cytotoxic to Escherichia coli and related strains. Their modes of ...

show abstract

Polymeric Nonelectrolytes to Probe Pore Geometry: Application to the α-Toxin Transmembrane Channel

Cited by 116 publications

References 46 publications

Surface Accessibility and Conformational Changes in the N-terminal Domain of Type I Inositol Trisphosphate Receptors

Surface Accessibility and Conformational Changes in the N-terminal Domain of Type I Inositol Trisphosphate Receptors

Osmotic stress, crowding, preferential hydration, and binding: A comparison of perspectives

Lipid Dependence of the Channel Properties of a Colicin E1-Lipid Toroidal Pore

Contact Info

Product

Resources

About