The role of local and remote amino acid substitutions for optimizing fluorescence in bacteriophytochromes: A case study on iRFP

Bacteriophytochromes are promising tools for tissue microscopy and imaging due to their fluorescence in the near-infrared region. These applications require optimization of the originally low fluorescence quantum yields via genetic engineering. Factors that favour fluorescence over other non-radiative excited state decay channels are yet poorly understood. In this work we employed resonance Raman and fluorescence spectroscopy to analyse the consequences of multiple amino acid substitutions on fluorescence of the iRFP713 benchmark protein. Two groups of mutations distinguishing iRFP from its precursor, the PAS-GAF domain of the bacteriophytochrome P2 from Rhodopseudomonas palustris, have qualitatively different effects on the biliverdin cofactor, which exists in a fluorescent (state II) and a non-fluorescent conformer (state I). Substitution of three critical amino acids in the chromophore binding pocket increases the intrinsic fluorescence quantum yield of state II from 1.7 to 5.0% due to slight structural changes of the tetrapyrrole chromophore. Whereas these changes are accompanied by an enrichment of state II from ~40 to ~50%, a major shift to ~88% is achieved by remote amino acid substitutions. Additionally, an increase of the intrinsic fluorescence quantum yield of this conformer by ~34% is achieved. The present results have important implications for future design strategies of biofluorophores.DFG, 221545957, SFB 1078: Proteinfunktion durch ProtonierungsdynamikDFG, 53182490, EXC 314: Unifying Concepts in Catalysi

Control of Gastric H,K-ATPase Activity by Cations, Voltage and Intracellular pH Analyzed by Voltage Clamp Fluorometry in Xenopus Oocytes

Whereas electrogenic partial reactions of the Na,K-ATPase have been studied in depth, much less is known about the influence of the membrane potential on the electroneutrally operating gastric H,K-ATPase. In this work, we investigated site-specifically fluorescence-labeled H,K-ATPase expressed in Xenopus oocytes by voltage clamp fluorometry to monitor the voltage-dependent distribution between E1P and E2P states and measured Rb+ uptake under various ionic and pH conditions. The steady-state E1P/E2P distribution, as indicated by the voltage-dependent fluorescence amplitudes and the Rb+ uptake activity were highly sensitive to small changes in intracellular pH, whereas even large extracellular pH changes affected neither the E1P/E2P distribution nor transport activity. Notably, intracellular acidification by approximately 0.5 pH units shifted V0.5, the voltage, at which the E1P/E2P ratio is 50∶50, by −100 mV. This was paralleled by an approximately two-fold acceleration of the forward rate constant of the E1P→E2P transition and a similar increase in the rate of steady-state cation transport. The temperature dependence of Rb+ uptake yielded an activation energy of ∼90 kJ/mol, suggesting that ion transport is rate-limited by a major conformational transition. The pronounced sensitivity towards intracellular pH suggests that proton uptake from the cytoplasmic side controls the level of phosphoenzyme entering the E1P→E2P conformational transition, thus limiting ion transport of the gastric H,K-ATPase. These findings highlight the significance of cellular mechanisms contributing to increased proton availability in the cytoplasm of gastric parietal cells. Furthermore, we show that extracellular Na+ profoundly alters the voltage-dependent E1P/E2P distribution indicating that Na+ ions can act as surrogates for protons regarding the E2P→E1P transition. The complexity of the intra- and extracellular cation effects can be rationalized by a kinetic model suggesting that cations reach the binding sites through a rather high-field intra- and a rather low-field extracellular access channel, with fractional electrical distances of ∼0.5 and ∼0.2, respectively

ATP1A2 Mutations in Migraine: Seeing through the Facets of an Ion Pump onto the Neurobiology of Disease

Mutations in four genes have been identified in familial hemiplegic migraine (FHM), from which CACNA1A (FHM type 1) and SCN1A (FHM type 3) code for neuronal voltage-gated calcium or sodium channels, respectively, while ATP1A2 (FHM type 2) encodes the α2 isoform of the Na+,K+-ATPase's catalytic subunit, thus classifying FHM primarily as an ion channel/ion transporter pathology. FHM type 4 is attributed to mutations in the PRRT2 gene, which encodes a proline-rich transmembrane protein of as yet unknown function. The Na+,K+-ATPase maintains the physiological gradients for Na+ and K+ ions and is, therefore, critical for the activity of ion channels and transporters involved neuronal excitability, neurotransmitter uptake or Ca2+ signaling. Strikingly diverse functional abnormalities have been identified for disease-linked ATP1A2 mutations which frequently lead to changes in the enzyme's voltage-dependent properties, kinetics, or apparent cation affinities, but some mutations are truly deleterious for enzyme function and thus cause full haploinsufficiency. Here, we summarize structural and functional data about the Na+,K+-ATPase available to date and an overview is provided about the particular properties of the α2 isoform that explain its physiological relevance in electrically excitable tissues. In addition, current concepts about the neurobiology of migraine, the correlations between primary brain dysfunction and mechanisms of headache pain generation are described, together with insights gained recently from modeling approaches in computational neuroscience. Then, a survey is given about ATP1A2 mutations implicated in migraine cases as documented in the literature with focus on mutations that were described to completely destroy enzyme function, or lead to misfolded or mistargeted protein in particular model cell lines. We also discuss whether or not there are correlations between these most severe mutational effects and clinical phenotypes. Finally, perspectives for future research on the implications of Na+,K+-ATPase mutations in human pathologies are presented.DFG, 53182490, EXC 314: Unifying Concepts in Catalysi

Parameters characterizing the voltage dependence of the E1P↔E2P conformational transition.

<p>Parameters from fits of a Boltzmann-type function to the data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033645#pone-0033645-g002" target="_blank">Fig. 2C</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033645#pone-0033645-g005" target="_blank">5A,B</a> (<i>V_0.5</i> and <i>z_q</i>) and parameters characterizing the voltage dependence of forward and backward rate constants <i>k_f</i> and <i>k_b</i> from data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033645#pone-0033645-g002" target="_blank">Figure 2E,F</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033645#pone-0033645-g005" target="_blank">5D,E,G,H</a> (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033645#pone.0033645.s003" target="_blank">Appendix S1</a></b>).</p

Effects of intracellular acidification on the E₁P/E₂P distribution and Rb⁺ transport.

<p>(A–E) Fluorescence responses of TMRM-labeled HKαS806C/βWT under extracellular K⁺-free conditions (90 mM extracellular Na⁺) upon voltage jumps from −40 mV to potentials between −180 mV and +60 mV in −20 mV steps. Recordings in (A–D) originated from a single oocyte (A) at pH_ex 7.4, (B) after 1 min in presence of 40 mM Na-butyrate (pH_ex 7.4), and after 1 min (C) and 2 min (D) washout of butyrate (pH_ex 7.4 buffer). (E) Fluorescence responses from a different cell in pH_ex 5.5 buffer. (F) Voltage dependence of stationary fluorescence amplitudes <i>1-ΔF/F</i> from the recordings in (A–E) at pH_ex 7.4 (▪), pH_ex 7.4+40 mM butyrate (□), and after 1 min (⋄) or 2 min (▵) washout of butyrate. Data at pH_ex 5.5 (•) are also shown. Fits of a Boltzmann-type function are superimposed to each data set, and the fluorescence amplitudes were normalized to saturation values from the fits. (G) Reciprocal time constants (<i>τ⁻¹</i>) from fits of a single exponential function to fluorescence changes under K⁺-free conditions. Data obtained at pH_ex 7.4 in the presence of 40 mM butyrate (♦) are compared to those in butyrate-free solutions at pH_ex 5.5 (□) and at pH_ex 7.4 (▪). Data are means±S.E. from 12–17 oocytes. (H) H,K-ATPase-mediated Rb⁺ uptake (at 5 mM Rb⁺) measured on individual cells by atomic absorption spectroscopy in the absence (gray) or presence (black) of 10 µM SCH28080 at different pH_ex and ionic conditions. Results from non-injected and HKαS806C/βWT-expressing oocytes at pH_ex 5.5, pH_ex 7.4, and pH_ex 7.4+40 mM butyrate are shown. Data are means±S.E. from three experiments on different cell batches with 15–20 oocytes per condition, and normalized to the Rb⁺ uptake of HKαS806C/βWT at pH_ex 5.5 (mean specific activities of 15.4, 23.1 and 39.0 pmol/oocyte/min).</p

Temperature and voltage dependence of Rb⁺ uptake by gastric H,K-ATPase.

<p>(A) H,K-ATPase-mediated Rb⁺ uptake (in pmol/oocyte/min) at 5 mM Rb⁺ and a pH_ex of 7.4 (light gray bars) or 5.5 (gray bars) at temperatures between 18 and 34°C, as indicated. White bars represent Rb⁺ uptake of non-injected control oocytes at each temperature and pH_ex 5.5. The black bar at 34°C shows the residual Rb⁺ uptake at pH_ex 5.5 in the presence of 100 µM SCH28080. Data in each column are means of 20–25 oocytes from oocytes of one cell batch. (B) Arrhenius plot for temperature-dependent Rb⁺ uptakes from data as in (A) at pH_ex 7.4 (▪), and pH_ex 5.5 (○). Data represent means±S.E. of three independent experiments (similar to the one shown in A), after normalization to Rb⁺ uptake at 34°C for each experiment. Activation energies obtained from linear fits to the data (superimposed lines) are given for each pH_ex. (C) Rb⁺ uptake (in pmol/oocyte/min) at 5 mM Rb⁺ and pH_ex 7.4 or 5.5 for oocytes expressing HKαS806C/βWT, which had either been clamped to a membrane potential of −100 mV, or subjected to Rb⁺ uptake without voltage clamping (V_m∼−10 to −20 mV). Black bars represent Rb⁺ uptake of H,K-ATPase-expressing oocytes clamped at −100 mV in the presence of 100 µM SCH28080. Data are means±S.D. from several oocytes of a single batch (numbers stated on each column).</p

Reaction cycle of gastric H,K-ATPase.

<p>(A) Reaction mechanism of gastric H,K-ATPase adapted from the Post-Albers scheme <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033645#pone.0033645-Post1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033645#pone.0033645-Albers1" target="_blank">[2]</a>, which had originally been postulated for the related Na,K-ATPase. Upon intracellular binding of protons to the E₁ conformation (step 1), a phosphointermediate with occluded H⁺ ions (E₁P(H⁺)) is formed (step 2), and after a conformational change to E₂P (step 3), protons dissociate to the extracellular space (step 4). Subsequently, K⁺ ions bind from the extracellular side (step 5) and become occluded, a process which stimulates dephosphorylation (step 6), and after a conformational change from E₂ to E₁ (step 7) the K⁺ ions are intracellularly released (step 8). The gray box indicates the reaction sequence which can be studied by voltage pulses at [K⁺]_ext = 0 in VCF experiments. (B) Pseudo three-state model for the reaction sequence including steps 1 to 4 in (A). A detailed description and analysis of this kinetic scheme is provided in Supporting Information (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033645#pone.0033645.s004" target="_blank">Appendix S2</a></b>).</p