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A ratiometric NMR pH sensing strategy based on a slow-proton-exchange (SPE) mechanism.

ABSTRACT: Real time and non-invasive detection of pH in live biological systems is crucial for understanding the physiological role of acid-base homeostasis and for detecting pathological conditions associated with pH imbalance. One method to achieve in vivo pH monitoring is NMR. Conventional NMR methods, however, mainly utilize molecular sensors displaying pH-dependent chemical shift changes, which are vulnerable to multiple pH-independent factors. Here, we present a novel ratiometric strategy for sensitive and accurate pH sensing based on a small synthetic molecule, SPE1, which exhibits exceptionally slow proton exchange on the NMR time scale. Each protonation state of the sensor displays distinct NMR signals and the ratio of these signals affords precise pH values. In contrast to standard NMR methods, this ratiometric mechanism is not based on a chemical shift change, and SPE1 binds protons with high selectivity, resulting in accurate measurements. SPE1 was used to measure the pH in a single oocyte as well as in bacterial cultures, demonstrating the versatility of this method and establishing the foundation for broad biological applications.

SUBMITTER: Perruchoud LH

PROVIDER: S-EPMC6054103 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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A ratiometric NMR pH sensing strategy based on a slow-proton-exchange (SPE) mechanism.

Perruchoud L H LH Jones M D MD Sutrisno A A Zamble D B DB Simpson A J AJ Zhang X-A XA

Chemical science 20150720 11

Real time and non-invasive detection of pH in live biological systems is crucial for understanding the physiological role of acid-base homeostasis and for detecting pathological conditions associated with pH imbalance. One method to achieve <i>in vivo</i> pH monitoring is NMR. Conventional NMR methods, however, mainly utilize molecular sensors displaying pH-dependent chemical shift changes, which are vulnerable to multiple pH-independent factors. Here, we present a novel ratiometric strategy for ...[more]

PMID: 30090248

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Project description:The acid-activated proton channel formed by the influenza M2 protein is important for the life cycle of the virus. A single histidine, His37, in the M2 transmembrane domain (M2TM) is responsible for pH activation and proton selectivity of the channel. Recent studies suggested three models for how His37 mediates proton transport: a shuttle mechanism involving His37 protonation and deprotonation, a H-bonded imidazole-imidazolium dimer model, and a transporter model involving large protein conformational changes in synchrony with proton conduction. Using magic-angle-spinning (MAS) solid-state NMR spectroscopy, we examined the proton exchange and backbone conformational dynamics of M2TM in a virus-envelope-mimetic membrane. At physiological temperature and pH, (15)N NMR spectra show fast exchange of the imidazole (15)N between protonated and unprotonated states. To quantify the proton exchange rates, we measured the (15)N T(2) relaxation times and simulated them for chemical-shift exchange and fluctuating N-H dipolar fields under (1)H decoupling and MAS. The exchange rate is 4.5 × 10(5) s(-1) for N?1 and 1.0 × 10(5) s(-1) for N?2, which are approximately synchronized with the recently reported imidazole reorientation. Binding of the antiviral drug amantadine suppressed both proton exchange and ring motion, thus interfering with the proton transfer mechanism. By measuring the relative concentrations of neutral and cationic His as a function of pH, we determined the four pK(a) values of the His37 tetrad in the viral membrane. Fitting the proton current curve using the charge-state populations from these pK(a)'s, we obtained the relative conductance of the five charge states, which showed that the +3 channel has the highest time-averaged unitary conductance. At physiologically relevant pH, 2D correlation spectra indicated that the neutral and cationic histidines do not have close contacts, ruling out the H-bonded dimer model. Moreover, a narrowly distributed nonideal helical structure coexists with a broadly distributed ideal helical conformation without interchange on the sub-10 ms time scale, thus excluding the transporter model in the viral membrane. These data support the shuttle mechanism of proton conduction, whose essential steps involve His-water proton exchange facilitated by imidazole ring reorientations.

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A ratiometric NMR pH sensing strategy based on a slow-proton-exchange (SPE) mechanism.

Publications

A ratiometric NMR pH sensing strategy based on a slow-proton-exchange (SPE) mechanism.

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