Simultaneous Optical and Electrical Recording of Single Gramicidin Channels

Borisenko, Vitali; Lougheed, Tyler; Hesse, Jan; Füreder-Kitzmüller, E.; Fertig, Niels; Behrends, Jan C.; Woolley, G. Andrew; Schütz, Gerhard J.

doi:10.1016/s0006-3495(03)74881-4

Cited by 125 publications

(107 citation statements)

References 51 publications

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“…Combined optical and electrical measurements are consistent with all gA dimers in a bilayer being conducting (24) [a subset may be nonconducting (31)]. The effects of manipulations that alter the activation energy for channel formation (ΔG ‡ 1 ) and dissociation (ΔG ‡ −1 ), and the equilibrium free energy (ΔG Fig.…”

supporting

confidence: 52%

Linear rate-equilibrium relations arising from ion channel-bilayer energetic coupling

Greisen

Lum

Ashrafuzzaman

et al. 2011

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

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Linear rate-equilibrium (RE) relations, also known as linear free energy relations, are widely observed in chemical reactions, including protein folding, enzymatic catalysis, and channel gating. Despite the widespread occurrence of linear RE relations, the principles underlying the linear relation between changes in activation and equilibrium energy in macromolecular reactions remain enigmatic. When examining amphiphile regulation of gramicidin channel gating in lipid bilayers, we noted that the gating process could be described by a linear RE relation with a simple geometric interpretation. This description is possible because the gating process provides a well-understood reaction, in which structural changes in a bilayer-embedded model protein can be studied at the singlemolecule level. It is thus possible to obtain quantitative information about the energetics of the reaction transition state and its position on a spatial coordinate. It turns out that the linear RE relation for the gramicidin monomer-dimer reaction can be understood, and the quantitative relation between changes in activation energy and equilibrium energy can be interpreted, by considering the effects of amphiphiles on the changes in bilayer elastic energy associated with channel gating. We are not aware that a similar simple mechanistic explanation of a linear RE relation has been provided for a chemical reaction in a macromolecule. RE relations generally should be useful for examining how amphiphile-induced changes in bilayer properties modulate membrane protein folding and function, and for distinguishing between direct (e.g., due to binding) and indirect (bilayer-mediated) effects.Brønsted | linear free energy relationships | phi value | bilayer elasticity I t is a common observation that manipulations that alter the energetics of a reaction:alter the rate constants (k 1 and k −1 ) and equilibrium constant (K eq ¼ k 1 ∕k −1 ), such that lnfk 1 g is a linear function of lnfK eq g. This linearity is interpreted to mean that, for a series of manipulations of the reaction, the activation free energy (ΔG ‡ ) varies as a linear function of the equilibrium free energy (ΔG 0 ):where a is a slope factor and b a constant (1-3). Such linear relations, known as linear rate-equilibrium (RE) relations (4), linear free energy relations (5), or linear rate-equilibrium free energy relations (6), have been observed and studied since the early 1900s. First observed in acid-based reactions (7-9), they have been identified in a wide range of reactions (1-3). Leffler proposed, based on studies of covalent reactions, that the slope factor a (Eq. 2) identifies the position of the transition state on a reaction coordinate, where S and P are positioned at 0 and 1, respectively (5). Marcus and coworkers deduced the principles underlying linear RE relations in electron transfer processes using a model in which the transfer is described as a one-step transition between two harmonic energy wells (10, 11). Fersht and coworkers used linear RE relations associated with p...

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supporting

confidence: 52%

Linear rate-equilibrium relations arising from ion channel-bilayer energetic coupling

Greisen

Lum

Ashrafuzzaman

et al. 2011

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

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“…More recently, the low-force response of single Holliday junctions was tracked using a hybrid instrument combining FRET and optical tweezers to map out the 2D folding landscape 90 . smFRET was also combined with the single channel recording of a simple dimeric gramicidin channel 91,92 .…”

Section: Advanced Smfret Techniquesmentioning

confidence: 99%

A practical guide to single-molecule FRET

2008

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Despite the explosive growth in the biological applications of single molecule methods over the last decade, these techniques have thus far been practiced mostly by researchers who are biophysically oriented. This is partly because of the lack of commercial instruments in many cases and also because of the perceived steep learning curve and need for expensive equipments. We wish to provide a practical guide to using Förster (or Fluorescence) Resonance Energy Transfer (FRET) at the single molecule level, focusing on the study of immobilized molecules that allow measurements of single molecule reaction trajectories from about 1 millisecond to many minutes. An instrument can be built at a reasonable cost using various off-the-shelf components and operated reliably using current well-established protocols and freely available software.The future holds the promise of personalized DNA sequencing and high throughput screening for pathogens at affordable cost and viable time. These promises are riding high on a surge of single-molecule based technologies that enable us to manipulate and probe individual molecules. Using this approach, several important biological riddles that have intrigued scientists for a long time are coming under the microscope. As Feynman famously said "It is very easy to answer many of these fundamental biological questions; you just look at the thing!" 1 . Single-molecule methods are allowing us to do just that 2,3 . They may one day become an elementary tool for characterizing proteins, signaling pathways or any biological phenomenon. In the hopes of facilitating this objective, we provide a brief, but practical guide for single-molecule FRET 4 measurements (smFRET) [5][6][7] , one of the most general and adaptable single-molecule techniques. Since its humble beginning under nonaqueous conditions in 1996 8 , smFRET has rapidly developed to answer fundamental questions about replication, recombination, transcription, translation, RNA folding and catalysis, non-canonical DNA dynamics, protein folding and conformational changes, various motor proteins, membrane fusion proteins, ion channels, signal transduction, to name just a few, and the list keeps growing at a fast pace. Since it is not the purpose of this review to survey the vast literature on such studies, we refer the reader to reviews in the field and the references therein 6, 9-13 .Correspondence to: Taekjip Ha. Competing interests statement:The authors declare no competing financial interests. In FRET measurements, the extent of non-radiative energy transfer between two fluorescent dye molecules, termed donor and acceptor, reports the intervening distance which can be estimated from the ratio of acceptor to total emission intensity ( Fig. 1) 4,14,15 . This efficiency of energy transfer, E is given as E = [1 + (R/R 0 ) 6 ] −1 , where R is the inter-dye distance and R 0 is the Förster radius at which E = 0.5 (Fig. 1a). Conformational dynamics of single molecules can be observed in real-time by tracking FRET changes (Fig. 1b). The ad...

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“…These dyes have been used in single molecule spectroscopy (2,3), in protein profiling (4), and for quantifying protein conformational changes in living cells (5,6). Site-specific protein labeling in these applications was accomplished through reaction with cysteine, introduced into proteins at the desired protein sites using molecular biology approaches.…”

Section: Introductionmentioning

confidence: 99%

Simple One-Pot Preparation of Water-Soluble, Cysteine-Reactive Cyanine and Merocyanine Dyes for Biological Imaging

2007

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A simple one-pot-procedure for preparation of protein-reactive, water soluble merocyanine and cyanine dyes has been developed. The 1-(3-ammoniopropyl)-2,3,3-trimethyl-3H-indolium-5-sulfonate bromide (1) was used as a common starting intermediate. The method allows easy preparation of dyes with chloro and iodoacetamide side chains for covalent attachment to cysteine. By placing a sulfonato group directly on the dye fluorophore system, dyes with high fluorescence quantum yields in water were generated. Both iodo-and chloroacetamido-derivatives were shown to be useful in protein labeling. Less reactive chloroacetamides will be preferential for selective labeling of the most reactive cysteines.

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Simultaneous Optical and Electrical Recording of Single Gramicidin Channels

Cited by 125 publications

References 51 publications

Linear rate-equilibrium relations arising from ion channel-bilayer energetic coupling

Linear rate-equilibrium relations arising from ion channel-bilayer energetic coupling

A practical guide to single-molecule FRET

Simple One-Pot Preparation of Water-Soluble, Cysteine-Reactive Cyanine and Merocyanine Dyes for Biological Imaging

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