4 FUNCTIONAL STUDIES
4.8 Catalysis and Sensing
The main applications of synthetic transport systems that open and close in response to chemical stimulation are catalysis and sensing. Whereas sensing applications made much progress over the last few years, the more challenging catalytic systems remained nearly unexplored. The only reported assay to study catalysis that takes place on the way across vesicle membranes uses entrapped HPTS acetate (Ac-HPTS) as fluorogenic substrate (Fig. 17a).32,33 Esterolysis during substrate efflux is detectable following HPTS fluorescence. This assay is compatible with Michaelis-Menten kinetics to extract KM and kcat. From there, catalytic efficiency and proficiency can be determined as in solution to report catalysis with the most relevant ground-state and transition-state stabilization. The assay holds for the determination of salt-rate profiles which are needed to extract n’, a value similar to the Hill coefficient reporting on the number of operational ion pairs, the maximal efficiencies (kcat/KM)MAX and from there the maximal transition-state stabilization.59 Combination with safranin O as external potential sensitive probe (Section 4.4) in valinomycin-polarized vesicles is possible as well (Fig. 17a).
The voltage dependence of Michaelis-Menten kinetics can be used to determine long-range steering effects of membrane potentials to drive the substrate into the catalytic pore and to accelerate product release on the other side of the membrane.32
Catalytic pores have so far been beyond the reach of single-cannel conductance measurements. However, covalent modifications within engineered biological ion channels and pores have been observed routinely on the single-molecule level. Initially, these techniques were developed for structural studies of biological systems. Best known is SCAM, the substituted-cysteine-accessibility method, which measures the “jump” in single-channel conductance when small, charged methanethiosulfonates react with cysteines in the ion-conducting pathway.23 Other realized examples include thermal cis-trans isomerization, photodeprotection, disulfide exchange and polymerization, and nucleophilic substitutions.23,61,64 Detection of reactions on the single-molecule level is particularly attractive to study single reactive intermediates (Fig. 17b).61 For this purpose, the conductance-levels for the pore-substrate and pore-product conjugate (or, in future applications towards catalysis, pore-substrate and -product complexes) are identified first.
Any new conductance level appearing during a reaction between the substrate and
pore-product levels originates from a reactive intermediate and can be used to explore their stability with regard to pH and so on.
a)
substrate PI
PE
outside inside
catalytic pore V
K+ V
PE
-O3S
-O3S SO3 -O
products
-O3S
-O3S SO3 -O OH
O -+ O
current (pA)
time (ms)
closed pore-substrate pore-product
t1 t2 stimulation
pore-intermediate 1 b)
pore-intermediate 2
Figure 17. Techniques to study (a) catalytic pores in unpolarized or polarized vesicles with entrapped fluorogenic substrates and (b) covalent modifications of ion channels and pores in
single-channel conductance experiments.
Reactions can be very generally detected with synthetic transport systems (indicated by reaction arrows in Fig. 16, a-d). The only condition is that the substrate and/or product activate or inactivate the transport systems, and that the IC50 or EC50 of substrate and product are sufficiently different. Then changing ability of the reaction mixture to inactivate or activate the transport system can be easily followed as described in Section 4.7. Baseline discrimination is preferable to detect reactions as “on-off” or “off-on” events. This technique has been used to produce fluorometric or colorimetric assays for enzymes as different as acetate kinase, aldolase, apyrase, cholesterol oxidase, citrate lyase, DNA exonuclease, DNA polymerase, elastase, esterase, ficin, galactosyltransferase, galactosidase, heparinase, hexokinase, hyaluronidase, invertase, lactate oxidase, papain, phosphatase, phosphofructokinase, pronase, RNase, phytase, subtilisin, transaminase, triose phosphate isomerase (TIM) or tyrosinase.23,60
This general detectability of the activity of enzymes demonstrated that synthetic transport systems can be used as multianalyte biosensors. Realized examples include cholesterol, citrate, glucose, lactate, lactose, phytate, polyphenol and sucrose sensors. In these sensing systems, the
c)
exonuclease nucleotides
current (pA)
time (ms)
closed open
gene
analyte
signal generators
enzymes
substrate product
signal transducers synthetic pores
CPPs DNA signal
amplifiers product-amplifier conjugate
signal detectors fluorescence
color, CD current
antibody
antigen
blocker
G, C, A, T
pore-C pore-T pore-A pore-G b)
a)
Figure 18. Biosensing (a), immunosensing (b) and stochastic sensing (c) as leading techniques for sensing applications of synthetic (and bioengineered) transport systems. (a) In
biosensing, enzymes are used to generate an analyte specific signal that can be further amplified by bifunctional in-/activators for transduction by the transporter and detection with
the techniques described in Section 3. (b) For immunosensing, antibodies (or aptamers) are coupled with the transporters, whereas (c) stochastic sensing aims to use the characteristic
fingerprint from lifetime and conductances of analyte-pore complexes for, e.g., gene sequencing.
synthetic transporters act as signal transducers, whereas the enzymes generate the signal by selectively recognizing and converting the analyte of interest in a complex matrix (Fig. 18a). To
generalize the use of synthetic transport systems as biosensors, signal amplifiers have been introduced. Signal amplifiers are bifunctional molecules that, on the one hand, covalently capture the product of the enzymatic signal generation and, on the other hand, activate or inactivate the signal transducer.23,44,60
Besides biosensing, the second technique developed to sense with transport systems is immunosensing.23 Several possibilities to attach either antigen or antibody to pore transducers have been reported, one of the most inspired technique is to attach the antigen of choice to a common blocker. Binding of the bulky and hydrophilic antibody then produces a soluble complex that unplugs the pore (Fig. 18b). As general as antibodies but much easier to prepare, DNA aptamers have been widely considered as advantageous alternative to immunosensors.
Recently, a technique that uses DNA aptamers as counterion activated ion carriers in LUVs to both generate and transduce the analyte-specific signal has been introduced.
The third technique to sense with transport systems is stochastic sensing.23,61,62 In this approach, the unique lifetime and conductance of single pore-analyte complexes is thought to produce a unique fingerprint that will reveal its presence in complex matrices. Today, it is quite clear that stochastic sensing is not applicable to complex matrices because any specific signatures are hidden behind very big noise. However, stochastic “sensing” remains a very attractive technique to address specific problems in relatively pure mixtures. The proposal to sequence single genes with pores has attracted much attention.62 Whereas it remains to be shown that the velocity of a single ss-DNA rushing through a pore can be slowed down to reach single-base resolution, the use of exonucleases is promising to bypass this intrinsic challenge (Fig. 18c). The technique envisions to attach exonuclease to the pore and record the produced nucleotide monophosphates according to the characteristic lifetime and conductance of their complex formed transiently in the pore. In this case, single nucleotide discrimination and detectability of exonuclease activity are confirmed.
4.9 Photosynthesis
The central transport in artificial photosynthesis is transmembrane electron transfer in response to irradiation with light. This transmembrane charge separation either polarizes the membrane (electrogenic photosynthesis) or is compensated by passive anion antiport or cation symport (electroneutral photosynthesis).
O3S
EDTA+. irradiation time (min) light
Figure 19. The HPTS assay and the Hurst assay to detect artificial photosynthesis. (a) In the HPTS assay, internal quinone reduction with light produces a proton gradient, which is detected by the pH-sensitive HPTS. (b) In the Hurst assay, internal cobalt reduction with light is detected by a change in color. (c) Electrogenic and electroneutral photosynthesis can be discriminated in
the Hurst assay by the sensitivity toward the proton carrier FCCP. (d) Hill analysis of dose response curves delivers the basic information on the photosystem.
The leading techniques to monitor artificial photosynthesis in lipid bilayer membranes are the HPTS assay and the Hurst assay (Fig. 19).9,65-67 In both assays, an external electron donor
with appropriate redox potential, usually a tertiary amine such as ETDA, is combined with an internal electron acceptor with appropriate redox potential. To convert photonic into chemical energy, reduction of the internal acceptor by the external donor must be “uphill”, i.e., thermodynamically unfavorable. In the HPTS assay, the internal acceptor is a quinone, which consumes two protons during its reduction with two electrons to a hydroquinone (Fig. 19a).65,66 The resulting increase in internal pH in response to irradiation with light is detected by the pH-sensitive HPTS (see Section 3).
The HPTS assay is elegant because artificial photosynthesis produces a pH gradient as in biology. However, the assay requires high selectivity of the photosystems because already minor leaks will cause the pH gradient to collapse. This also means that the HPTS works only with electrogenic photosystems. However, the pH gradient produced with quinone reduction has been coupled with ATP synthase to convert light into ATP by artificial photosystems in vesicles.
In the Hurst assay, internal Co3+(bipy)3 is used as electron acceptor, and the change in color during cobalt reduction with light is used to monitor photosynthesis (Fig. 19b).9,67 Operating exclusively with relatively large molecules, the Hurst assay is more robust than the HPTS assay. With the Hurst assay, electrogenic and electroneutral photosynthesis can be discriminated with the addition of external FCCP to enable proton-coupled electron pumping.
Increasing activity in the presence of FCCP thus identifies electrogenic photosynthesis, insensitivity to FCCP is found for electroneutral photosynthesis (Fig. 19c). As for other synthetic transport systems, artificial photosystems are best characterized in dose response curves, with Hill analysis informing on activity (EC50), thermodynamic stability and supramolecular nature of the photosystem (n, Fig. 19d, see Section 4.2).
5 EPILOGUE
This contribution aims to give an introductory summary to standard techniques used by and recommended for supramolecular chemists for the study of transport across bilayer membranes. Emphasis is on fluorescence techniques because they are most easily accessible for the non-expert and can be used to address most pertinent questions. The complementary information accessible with the more specialized planar bilayer conductance experiments is described as well. Emphasis is on the creation and identification of functional relevance, including topics such as ion selectivity, voltage gating, ligand gating, blockages, catalysis,
sensing and photosynthesis.
The text is highly simplified. Meaningful design, execution and interpretation of transport experiments is often an enriching and stimulating but also a very challenging adventure that requires highest attention. Transport across bilayer membranes occurs in heterogeneous multiphasic environment and depends on numerous parameters. Multiple regions such as the aqueous phase, the highly charged head group region and the nonpolar region of lipid tails exist and physicochemical properties change at each interfacial region. Particular attention is required to determine the importance of the not directly related delivery and partitioning to the final activity, with dichotomic approaches being arguably the most relevant techniques to tackle the problem (Section 4.1). In general, failure to detect activity doesn’t necessarily mean that the synthetic transport system doesn’t exist, interesting activities with high selectivities can be the most difficult ones to detect.
Many more functional characteristics beyond the discussed selection exist. For instance, the influence of the composition of the membrane on activity has been almost completely ignored. This includes important topics such as membrane fluidity, phase transition, thickness (and hydrophobic matching), surface potentials 0 (and Gouy-Chapman theory),20,22 partitioning, heterogeneity (“rafts”) or swelling and shrinking in response to stress.
Neglected in this contribution are mechanistic and structural studies. This was done because the primary goal in research focusing on the creation of significant function is obviously to obtain experimental evidence for the desired significant function. Compared to any unique responsiveness of any synthetic transport system to physical or chemical stimulation, mechanisms of transport are simply less important, particularly considering that the different mechanisms (carrier, channel, pore, detergent) are often interchangeable depending on experimental conditions.16 To reiterate the fundamentals, ion channel activity is demonstrated with single-channel conductances, ion carriers work in the U-tube (decreasing activity with decreasing membrane fluidity does not prove carriers, this can also be a simple partitioning effect).20
Structural studies are not included because they are usually obsolete and often misleading.
Most significant synthetic transport systems are minority populations. Beware of overimplications from low-sensitivity, low-selectivity and static techniques such as NMR, IR, X-ray or TEM data, they have caused much damage to the field! In this context, the most relevant structural data come from functional studies (Hill analysis, etc). Relevant structural studies
operate under conditions relevant for function (M to nM concentrations, in membranes) and can specifically address a specific question in a selective manner. For example, FRET from a labeled transport system into an acceptor in the membrane can correctly inform on the role of partitioning and positioning in key processes such as activity, voltage gating, ligand gating35 or blockage.35 Fluorescence depth quenching with DOXYL labeled membranes is the most powerful tool to determine specific location, orientation and repositioning in the membrane during these processes.20,34,36 Other selective and sensitive method such as CD spectroscopy or isothermal titration calorimetry (ITC) are routinely used in meaningful structural studies, for example to complement insights on interactions with the membranes, self-assembly of active structures and the formation of host-guest complexes during ligand gating and blockage.
The heart of the matter with synthetic transporters is to design and identify “smart systems” that respond to chemical or physical stimulation in a unique and significant manner.
The techniques available to identify such significant functions can never be used without creative thinking and applying grand principles of chemistry in a unique context. As a result, the techniques used are under constant development, never routine, calling for constant improvisation and inspired innovation, depending on the system studied and the questions asked.
This makes the use of transmembrane transport detection techniques very demanding and at the same time very entertaining and satisfactory.
Acknowledgment. We thank the University of Geneva and the Swiss NSF for financial support.
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Glossary
Activator Compound that activates a transport system, without mechanistic implications (compare ligand gating)
AMFE Anomalous mole fraction effect; deviation from linear dependence of activity on the mole fraction of ions
Antiport Transport of ions of identical charge in opposite directions
Black lipid membrane Planar unilamellar bilayer of micrometer diameter (appears black), used in single-channel conductance (or BLM) experiments
Blockage Inactivation of a transport system by a blocker (inactivator), usually implying non-destructive binding to active structures Bulk liquid membrane Organic phase in U-tube experiments, usually chloroform
CF assay Reports the release of self-quenched 5(6)-carboxyfluorescein from LUVs as fluorescence recovery
Detergents In micellar form, detergents lyse lipid bilayers
Dichotomic behavior Complementary responsiveness of comparable systems, important to exclude artefacts
Dose response curve Dependence of activity on the concentration of individual components of the system (transporter, activators, inactivators, vesicles)
EC50 Effective concentration needed to reach 50% of the maximal activity, related to dissociation constant (KD)
Eisenman topology Used for the eleven selectivity topologies of group one cations Electroneutral Transport process that does not polarize the membrane
Electrogenic Transport process that separates charges and produces a membrane potential
Flip-flop Reversible transversal diffusion of lipids, mediated by flippases (can imply micellar defects in lipid bilayers)
Gating charge Defines voltage gating from exponential dependence of activity on membrane potentials
Hill coefficient Informs on cooperativity, stoichiometry and thermodynamic stability of the transport system
Hille diameter Inner diameter of transporters, from single-channel conductance HPTS assay Reports with 8-hydroxy-1,3,6-pyrenetrisulfonate (pyranine) on pH
changes within vesicles
Hofmeister series Anion selectivity sequence dominated by dehydration only
IC50 Inhibitory concentration needed to reduce maximal activity to 50%, related to dissociation constant (KD)
Inactivator Compound that inactivates transport systems, without mechanistic implications (compare blockage)
Ion carrier Moves together with the ion across the membrane; active in the U-tube
Ion channel Transports ions without moving much, has small single-channel conductance
Ligand gating Activation of a transport system by a ligand (activator), usually implying non-destructive binding to the active structure
Lucigenin assay Reports on the chloride concentration within vesicles
LUVs Large unilamellar vesicles, spherical lipid bilayers with a diameter of 100-200 nm
Lysis Destruction of lipid bilayers by detergent micelles into mixed disc
Lysis Destruction of lipid bilayers by detergent micelles into mixed disc