Dicarboxylic acid analogs of Gramicidin A: Dimerization kinetics and single channel properties

Apell, Hans-Jürgen; Bamberg, Ernst; Alpes, H.

doi:10.1007/bf01868893

Cited by 15 publications

(17 citation statements)

References 24 publications

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“…Benzenesulfonamides are well-known ligands for carbonic anhydrase II (CA, EC 4.2.1.1), a Zn 2+ -containing protein (71)(72)(73)(74)(75)(76)(77)(78). 2 This assumption is very rough since we would expect the negatively charged product 10 to partition significantly less in the membrane due its increased solubility in water compared to the neutrally "charged" starting material 3. The yields shown in Figure 3B are, therefore, likely to be an underestimate due to the likely difference in partitioning between 3 and 10 in the membrane.…”

Section: Resultsmentioning

confidence: 99%

“…At least eight reasons make this peptide attractive as a practical molecular platform for the development of ion channelbased nanoprobes: (1) its ion conducting properties can measur- (2,4,26,43,100,101).…”

Section: Introductionmentioning

confidence: 99%

“…ably amplify a signal by monitoring a flux of ions through a single pore; (2) its current levels are distinct and quantized, which facilitate the measurement of changes in conductance on a single pore level; (3) its C-terminus is conducive to derivatization without compromising function (2,3,(9)(10)(11)(12)(44)(45)(46)(47)(48); (4) its availability in gram quantities from commercial sources makes it possible to generate tailored derivatives on practical scales; (5) its self-incorporation into lipid bilayers makes its use straightforward as opposed to most ion channel proteins that require incorporation into bilayers through proteoliposome fusion (49)(50)(51)(52); (6) its electrophysiological characteristics are well explored, which makes it an excellent model for fundamental biophysical studies of pores in membranes; (7) its size is nanoscale, which is conducive to miniaturized device designs and may enable parallel assays developed in a high density format; and (8) its activity is localized in a membrane, a property useful for applications where control of location is important. Figure 1A shows the sequence and helical structure of gA. Gramicidin A reversibly dimerizes in a bilayer ( Figure 1B), forming an N-terminus to N-terminus dimeric structure held together by a network of hydrogen bonds (53)(54)(55).…”

Section: Introductionmentioning

confidence: 99%

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Chemically Reactive Derivatives of Gramicidin A for Developing Ion Channel-Based Nanoprobes

et al. 2008

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Ion channel-forming peptides and proteins offer tremendous opportunities for fundamental and applied studies of function on individual molecules. An ongoing challenge in ion channel research is the lack of simple and accessible synthetic methods to engineer pores with tailored chemical and physical properties. This paper describes a practical synthetic route to rapidly generate C-terminally modified derivatives of gramicidin A (gA), an ion channel-forming peptide, through the use of two chemically reactive gA-based building blocks. These amine- and azide-containing building blocks can react readily with typical substrates for amidation and 1,3-dipolar cycloaddition ("click") reactions to present molecules with desired structure and functionality near the opening of a gA pore. These derivatives of gA are stable under typical aqueous conditions for ion channel recordings and retain characteristic single ion channel conductance properties in planar lipid bilayers. Additionally, the synthetic methods described here afford useful quantities of these gA derivatives in good purity and yield with minimal purification. We demonstrate that derivatives of gA can be used for studying, in situ, a change in conductance through a channel upon performing a "click" reaction on an azide moiety attached to the gA pore. We also demonstrate that these gA-based building blocks can be used to construct sensors for the recognition of specific protein-ligand binding interactions in solution. This widely accessible, enabling synthetic methodology represents a powerful new tool to study relationships between chemical structure and function on the single molecule level.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Chemically Reactive Derivatives of Gramicidin A for Developing Ion Channel-Based Nanoprobes

et al. 2008

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“…Other backbone alterations include the appendage of a titratable acidic group of variable length at the N-terminus (3), which produces pH-sensitive channels, a lipid group at the C-terminus (89), which stabilizes the channel, and various changes in the length of the peptide (95,103, 124,153,163). The structurally conservative deletion mutant , des-L-VaJ 1 -D-VaI 8 gramicidin A, had a decreased channel lifetime as expected for a mismatch between channel length, and bilayer thickness , but unexpectedly a 35% lower conductance in 1 M KCI, which was ascribed to a decrease in the rate of second ion entry (measured by NMR) and thus of the quasi-knockoff kinetics (163).…”

Section: Initial Modelsmentioning

confidence: 99%

The Use of Physical Methods in Determining Gramicidin Channel Structure and Function

Busath

1993

Annu. Rev. Physiol.

124

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The various details provided by physical methods have been integrated into a coherent picture of the cation transport process. The free energy profile describes simultaneously the thermodynamic and kinetic factors that govern conductance. Localization of ion binding sites near each end of the channel by NMR and X-ray crystallographic techniques demonstrates that there is an energy barrier separating the two ends. There is no a priori reason to suspect any barrier to ion entry into the channel, and recent molecular modeling computations confirm this intuition. Therefore, the channel is best described as a two-site, one-barrier channel. However, the movement of ions across the central barrier over a distance of 1.9 nm is really a multistep process best described as Brownian motion (even the short steps from one pair of carbonyls to the next is more diffusive than activated for ions as large as Na+), the entry step is probably best considered as a compound step requiring correction for interfacial polarization and diffusion limitation, the binding sites in the channel ought not be considered to be in equilibrium with the bath, and quasi-knock off behavior (binding of a second ion at the entry facilitates release of a first ion from the exit) is probably the rule at physiological permeant ion concentrations and higher. Progress will probably focus on channel kinetics. The channel backbone probably undergoes conformational changes as the ions pass which, according to solid state NMR results, may last long enough to provide the channel with a sort of memory and which may give rise to excess single channel noise. These conformational changes are further reflected in shifts in side chain positions which, in turn, may be partly responsible for changes in the single-channel lifetime, which appears to be exquisitely sensitive to side chain-lipid interactions. Reasonable explanations for the impermeance of divalent cations, anions, and medium-sized organic cations such as guanidinium have proven elusive at first, but it now appears that divalent cations bind water too tightly, anions bind water in an orientation that produces unfavorable contacts between water oxygens and peptide carbonyl oxygens at the channel entry, and iminium ions bind strongly to the flexible peptide carbonyls at the channel entry.

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“…7 (Heck-9 There exist gramicidin A analogues, for example N-succinylgramicidin A, which form channels with a lower conductance than gramicidin A channels and w~th superlinear characteristics at all permeant ion activities (Apell, Bamberg & Alpes, 1979). These channels thus behave as if the major barrier for ion movement is indeed the translocation step through the channel interior.…”

Section: Single-channel Conductance As a Function Of Permeant Ion Actmentioning

confidence: 99%

The gramicidin a channel: A review of its permeability characteristics with special reference to the single-file aspect of transport

1981

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Gramicidin A forms univalent cation-selective channels of approximately 4 A diameter in phospholipid bilayer membranes. The transport of ions and water throughout most of the channel length is by a single-file process; that is, cations and water molecules cannot pass each other within the channel. The implications of this single-file mode of transport for ion movement are considered. In particular, we show that there is no significant electrostatic barrier to ion movement between the energy wells at the two ends of the channel. The rate of ion translocation (e.g., Na+ or Cs+) through the channel between these wells is limited by the necessity for an ion to move six water molecules in single file along with it; this also limits the maximum possible value for channel conductance. At all attainable concentrations of NaCl, the gramicidin A channel never contains more than one sodium ion, whereas even at 0.1 M CsCl, some channels contain two cesium ions. There is no necessity to postulate more than two ion-binding sites in the channel or occupancy of the channel by more than two ions at any time.

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Dicarboxylic acid analogs of Gramicidin A: Dimerization kinetics and single channel properties

Cited by 15 publications

References 24 publications

Chemically Reactive Derivatives of Gramicidin A for Developing Ion Channel-Based Nanoprobes

Chemically Reactive Derivatives of Gramicidin A for Developing Ion Channel-Based Nanoprobes

The Use of Physical Methods in Determining Gramicidin Channel Structure and Function

The gramicidin a channel: A review of its permeability characteristics with special reference to the single-file aspect of transport

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