Search

Counting viable cells is a universal practice in microbiology. The colony-forming unit (CFU) assay has remained the gold standard to measure viability across disciplines, but it is time-intensive and resource-consuming. Here we describe the geometric viability assay (GVA) that replicates CFU measurements over 6 orders of magnitude while reducing over 10-fold the time and consumables required. GVA computes a sample's viable cell count on the basis of the distribution of embedded colonies growing inside a pipette tip. GVA is compatible with Gram-positive and Gram-negative planktonic bacteria (Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis), biofilms and fungi (Saccharomyces cerevisiae). Laborious CFU experiments such as checkerboard assays, treatment time-courses and drug screens against slow-growing cells are simplified by GVA. The ease and low cost of GVA evinces that it can replace existing viability assays and enable viability measurements at previously impractical scales., (© 2023. The Author(s).)

How dynamic bacterial calcium is regulated, with kinetics faster than typical mechanisms of cellular adaptation, is unknown. We discover bacterial calcium fluctuations are temporal-fractals resulting from a property known as self-organized criticality (SOC). SOC processes are poised at a phase transition separating ordered and chaotic dynamical regimes and are observed in many natural and anthropogenic systems. SOC in bacterial calcium emerges due to calcium channel coupling mediated via membrane voltage. Environmental or genetic perturbations modify calcium dynamics and the critical exponent suggesting a continuum of critical attractors. Moving along this continuum alters the collective information capacity of bacterial populations. We find that the stochastic transition from motile to sessile lifestyle is partially mediated by SOC-governed calcium fluctuations through the regulation of c-di-GMP. In summary, bacteria co-opt the physics of phase transitions to maintain dynamic calcium equilibrium, and this enables cell-autonomous population diversification during surface colonization by leveraging the stochasticity inherent at a boundary between phases.

It began, as with many good things, at a happy hour. Adam Cohen, a young assistant professor asked whether rhodopsins could be used to optically sense voltage. In the heady days of 2009, channel rhodopsin had just been unveiled as a voltage actuator in neurons. Adam had the insight to question whether rhodopsins could be run in reverse; could optical changes in a protein relay the cellular voltage state using light? This was one of the earliest lessons I learned under his mentorship, and the first piece of advice in this retrospective-turning a scientific question or statement on its head can be the basis for many fantastic research projects., Competing Interests: No competing financial interests exist., (Copyright 2021, Mary Ann Liebert, Inc., publishers.)

Changes in bacterial physiology necessarily precede cell death in response to antibiotics. Herein we investigate the early disruption of Ca 2+ homeostasis as a marker for antibiotic response. Using a machine learning framework, we quantify the temporal information encoded in single-cell Ca 2+ dynamics. We find Ca 2+ dynamics distinguish kanamycin sensitive and resistant cells before changes in gross cell phenotypes such as cell growth or protein stability. The onset time (pharmacokinetics) and probability (pharmacodynamics) of these aberrant Ca 2+ dynamics are dose and time-dependent, even at the resolution of single-cells. Of the compounds profiled, we find Ca 2+ dynamics are also an indicator of Polymyxin B activity. In Polymyxin B treated cells, we find aberrant Ca 2+ dynamics precedes the entry of propidium iodide marking membrane permeabilization. Additionally, we find modifying membrane voltage and external Ca 2+ concentration alters the time between these aberrant dynamics and membrane breakdown suggesting a previously unappreciated role of Ca 2+ in the membrane destabilization during Polymyxin B treatment. In conclusion, leveraging live, single-cell, Ca 2+ imaging coupled with machine learning, we have demonstrated the discriminative capacity of Ca 2+ dynamics in identifying antibiotic-resistant bacteria.

Calcium plays numerous critical roles in signaling and homeostasis in eukaryotic cells. Far less is known about calcium signaling in bacteria than in eukaryotic cells, and few genes controlling influx and efflux have been identified. Previous work in Escherichia coli showed that calcium influx was induced by voltage depolarization, which was enhanced by mechanical stimulation, which suggested a role in bacterial mechanosensation. To identify proteins and pathways affecting calcium handling in bacteria, we designed a live-cell screen to monitor calcium dynamics in single cells across a genome-wide knockout panel in E. coli The screen measured cells from the Keio collection of knockouts and quantified calcium transients across the population. Overall, we found 143 gene knockouts that decreased levels of calcium transients and 32 gene knockouts that increased levels of transients. Knockouts of proteins involved in energy production and regulation appeared, as expected, as well as knockouts of proteins of a voltage sink, F 1 F o -ATPase. Knockouts of exopolysaccharide and outer membrane synthesis proteins showed reduced transients which refined our model of electrophysiology-mediated mechanosensation. Additionally, knockouts of proteins associated with DNA repair had reduced calcium transients and voltage. However, acute DNA damage did not affect voltage, and the results suggested that only long-term adaptation to DNA damage decreased membrane potential and calcium transients. Our work showed a distinct separation between the acute and long-term DNA damage responses in bacteria, which also has implications for mitochondrial DNA damage in eukaryotes. IMPORTANCE All eukaryotic cells use calcium as a critical signaling molecule. There is tantalizing evidence that bacteria also use calcium for cellular signaling, but much less is known about the molecular actors and physiological roles. To identify genes regulating cytoplasmic calcium in Escherichia coli , we created a single-cell screen for modulators of calcium dynamics. The genes uncovered in this screen helped refine a model for voltage-mediated bacterial mechanosensation. Additionally, we were able to more carefully dissect the mechanisms of adaptation to long-term DNA damage, which has implications for both bacteria and mitochondria in the face of unrepaired DNA., (Copyright © 2021 American Society for Microbiology.)

Aminoglycosides are broad-spectrum antibiotics whose mechanism of action is under debate. It is widely accepted that membrane voltage potentiates aminoglycoside activity, which is ascribed to voltage-dependent drug uptake. In this paper, we measured the response of Escherichia coli treated with aminoglycosides and discovered that the bactericidal action arises not from the downstream effects of voltage-dependent drug uptake, but rather directly from dysregulated membrane potential. In the absence of voltage, aminoglycosides are taken into cells and exert bacteriostatic effects by inhibiting translation. However, cell killing was immediate upon re-polarization. The hyperpolarization arose from altered ATP flux, which induced a reversal of the F1Fo-ATPase to hydrolyze ATP and generated the deleterious voltage. Heterologous expression of an ATPase inhibitor completely eliminated bactericidal activity, while loss of the F-ATPase reduced the electrophysiological response to aminoglycosides. Our data support a model of voltage-induced death, and separates aminoglycoside bacteriostasis and bactericide in E. coli ., Competing Interests: GB, JK No competing interests declared, (© 2020, Bruni and Kralj.)

Human induced pluripotent stem cell (iPSC)-derived neurons are an attractive substrate for modeling disease, yet the heterogeneity of these cultures presents a challenge for functional characterization by manual patch-clamp electrophysiology. Here, we describe an optimized all-optical electrophysiology, "Optopatch," pipeline for high-throughput functional characterization of human iPSC-derived neuronal cultures. We demonstrate the method in a human iPSC-derived motor neuron (iPSC-MN) model of amyotrophic lateral sclerosis (ALS). In a comparison of iPSC-MNs with an ALS-causing mutation (SOD1 A4V) with their genome-corrected controls, the mutants showed elevated spike rates under weak or no stimulus and greater likelihood of entering depolarization block under strong optogenetic stimulus. We compared these results with numerical simulations of simple conductance-based neuronal models and with literature results in this and other iPSC-based models of ALS. Our data and simulations suggest that deficits in slowly activating potassium channels may underlie the changes in electrophysiology in the SOD1 A4V mutation., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Addition of glucose to starved Saccharomyces cerevisiae initiates collective NADH dynamics termed glycolytic oscillations. Numerous questions remain about the extent to which single cells can oscillate, if oscillations occur in natural conditions, and potential physiological consequences of oscillations. In this paper, we report sustained glycolytic oscillations in single cells without the need for cyanide. Glucose addition to immobilized cells induced pH oscillations that could be imaged with fluorescent sensors. A population of cells had oscillations that were heterogeneous in frequency, start time, stop time, duration and amplitude. These changes in cytoplasmic pH were necessary and sufficient to drive changes in NADH. Oscillators had lower mitochondrial membrane potentials and budded more slowly than non-oscillators. We also uncovered a new type of oscillation during recovery from H 2 O 2 challenge. Our data show that pH in S. cerevisiae changes over several time scales, and that imaging pH offers a new way to measure glycolytic oscillations on individual cells.

Electrically excitable cells harness voltage-coupled calcium influx to transmit intracellular signals, typically studied in neurons and cardiomyocytes. Despite intense study in higher organisms, investigations of voltage and calcium signaling in bacteria have lagged due to their small size and a lack of sensitive tools. Only recently were bacteria shown to modulate their membrane potential on the timescale of seconds, and little is known about the downstream effects from this modulation. In this paper, we report on the effects of electrophysiology in individual bacteria. A genetically encoded calcium sensor expressed in Escherichia coli revealed calcium transients in single cells. A fusion sensor that simultaneously reports voltage and calcium indicated that calcium influx is induced by voltage depolarizations, similar to metazoan action potentials. Cytoplasmic calcium levels and transients increased upon mechanical stimulation with a hydrogel, and single cells altered protein concentrations dependent on the mechanical environment. Blocking voltage and calcium flux altered mechanically induced changes in protein concentration, while inducing calcium flux reproduced these changes. Thus, voltage and calcium relay a bacterial sense of touch and alter cellular lifestyle. Although the calcium effectors remain unknown, these data open a host of new questions about E. coli , including the identity of the underlying molecular players, as well as other signals conveyed by voltage and calcium. These data also provide evidence that dynamic voltage and calcium exists as a signaling modality in the oldest domain of life, and therefore studying electrophysiology beyond canonical electrically excitable cells could yield exciting new findings., Competing Interests: The authors declare no conflict of interest.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising platform for cardiac studies in vitro, and possibly for tissue repair in humans. However, hiPSC-CM cells tend to retain morphology, metabolism, patterns of gene expression, and electrophysiology similar to that of embryonic cardiomyocytes. We grew hiPSC-CM in patterned islands of different sizes and shapes, and measured the effect of island geometry on action potential waveform and calcium dynamics using optical recordings of voltage and calcium from 970 islands of different sizes. hiPSC-CM in larger islands showed electrical and calcium dynamics indicative of greater functional maturity. We then compared transcriptional signatures of the small and large islands against a developmental time course of cardiac differentiation. Although island size had little effect on expression of most genes whose levels differed between hiPSC-CM and adult primary CM, we identified a subset of genes for which island size drove the majority (58%) of the changes associated with functional maturation. Finally, we patterned hiPSC-CM on islands with a variety of shapes to probe the relative contributions of soluble factors, electrical coupling, and direct cell-cell contacts to the functional maturation. Collectively, our data show that optical electrophysiology is a powerful tool for assaying hiPSC-CM maturation, and that island size powerfully drives activation of a subset of genes involved in cardiac maturation.

Introduction: The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative seeks an in vitro test to accurately predict clinical Torsades de Pointes (TdP). We developed a cardiotoxicity assay incorporating simultaneous measurement of the action potential (AP) waveform and Ca(2+) transient (CT) in human iPSC-derived cardiomyocytes (CMs). Concurrent optogenetic pacing provided a well-controlled electrophysiological background., Methods: We used the Optopatch platform for all-optical electrophysiology (Hochbaum et al., 2014). In a monolayer culture, a subset of cells expressed a genetically encoded, calcium and voltage reporter, CaViar (Hou, Kralj, Douglass, Engert, & Cohen, 2014), while others expressed a channelrhodopsin variant, CheRiff. Optical pacing of CheRiff-expressing cells synchronized the syncytium. We screened 12 compounds (11 acute, 1 chronic) to identify electrophysiological (AP rise time, AP50, AP90, beat rate) and CT effects in spontaneously beating and paced cultures (1Hz, 2Hz)., Results: CaViar reported spontaneous and paced APs and CTs with high signal-to-noise ratio and low phototoxicity. Quinidine, flecainide, E-4031, digoxin and cisapride prolonged APs, while verapamil and nifedipine shortened APs. Early after depolarizations (EADs) were elicited by quinidine, flecainide and cisapride. All but four compounds (amiodarone, chromanol, nifedipine, verapamil) prolonged AP rise time. Nifedipine and verapamil decreased CT amplitude, while digoxin increased CT amplitude. Pentamidine prolonged APs after chronic exposure., Discussion: The Optopatch platform provides a robust assay to measure APs and CTs in hiPSC-CMs. This addresses the CiPA mandate and will facilitate comparisons of cell-based assays to human clinical data., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The cardiac action potential (AP) and the consequent cytosolic Ca(2+) transient are key indicators of cardiac function. Natural developmental processes, as well as many drugs and pathologies change the waveform, propagation, or variability (between cells or over time) of these parameters. Here we apply a genetically encoded dual-function calcium and voltage reporter (CaViar) to study the development of the zebrafish heart in vivo between 1.5 and 4 days post fertilization (dpf). We developed a high-sensitivity spinning disk confocal microscope and associated software for simultaneous three-dimensional optical mapping of voltage and calcium. We produced a transgenic zebrafish line expressing CaViar under control of the heart-specific cmlc2 promoter, and applied ion channel blockers at a series of developmental stages to map the maturation of the action potential in vivo. Early in development, the AP initiated via a calcium current through L-type calcium channels. Between 90 and 102 h post fertilization (hpf), the ventricular AP switched to a sodium-driven upswing, while the atrial AP remained calcium driven. In the adult zebrafish heart, a sodium current drives the AP in both the atrium and ventricle. Simultaneous voltage and calcium imaging with genetically encoded reporters provides a new approach for monitoring cardiac development, and the effects of drugs on cardiac function.

All-optical electrophysiology-spatially resolved simultaneous optical perturbation and measurement of membrane voltage-would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk-free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons. In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes.

Development of improved fluorescent voltage indicators is a key challenge in neuroscience, but progress has been hampered by the low throughput of patch-clamp characterization. We introduce a line of non-fluorescent HEK cells that stably express NaV 1.3 and KIR 2.1 and generate spontaneous electrical action potentials. These cells enable rapid, electrode-free screening of speed and sensitivity of voltage sensitive dyes or fluorescent proteins on a standard fluorescence microscope. We screened a small library of mutants of archaerhodopsin 3 (Arch) in spiking HEK cells and identified two mutants with greater voltage-sensitivity than found in previously published Arch voltage indicators.

Archaerhodopsin 3 (AR3) is a light driven proton pump from Halorubrum sodomense that has been used as a genetically targetable neuronal silencer and an effective fluorescent sensor of transmembrane potential. Unlike the more extensively studied bacteriorhodopsin (BR) from Halobacterium salinarum, AR3 readily incorporates into the plasma membrane of both E. coli and mammalian cells. Here, we used near-IR resonance Raman confocal microscopy to study the effects of pH and membrane potential on the AR3 retinal chromophore structure. Measurements were performed both on AR3 reconstituted into E. coli polar lipids and in vivo in E. coli expressing AR3 in the absence and presence of a negative transmembrane potential. The retinal chromophore structure of AR3 is in an all-trans configuration almost identical to BR over the entire pH range from 3 to 11. Small changes are detected in the retinal ethylenic stretching frequency and Schiff Base (SB) hydrogen bonding strength relative to BR which may be related to a different water structure near the SB. In the case of the AR3 mutant D95N, at neutral pH an all-trans retinal O-like species (O(all-trans)) is found. At higher pH a second 13-cis retinal N-like species (N(13-cis)) is detected which is attributed to a slowly decaying intermediate in the red-light photocycle of D95N. However, the amount of N(13-cis) detected is less in E. coli cells but is restored upon addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or sonication, both of which dissipate the normal negative membrane potential. We postulate that these changes are due to the effect of membrane potential on the N(13-cis) to M(13-cis) levels accumulated in the D95N red-light photocycle and on a molecular level by the effects of the electric field on the protonation/deprotonation of the cytoplasmic accessible SB. This mechanism also provides a possible explanation for the observed fluorescence dependence of AR3 and other microbial rhodopsins on transmembrane potential.

Microbial rhodopsins are an important class of light-activated transmembrane proteins whose function is typically studied on bulk samples. Herein, we apply photochromic fluorescence resonance energy transfer to investigate the dynamics of these proteins with sensitivity approaching the single-molecule limit. The brightness of a covalently linked organic fluorophore is modulated by changes in the absorption spectrum of the endogenous retinal chromophore that occur as the molecule undergoes a light-activated photocycle. We studied the photocycles of blue-absorbing proteorhodopsin and sensory rhodopsin II (SRII). Clusters of 2-3 molecules of SRII clearly showed a light-induced photocycle. Single molecules of SRII showed a photocycle upon signal averaging over several illumination cycles., (© 2011 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2011 The American Society of Photobiology.)

Archaerhodopsin-3 (AR3) is a light-driven proton pump from Halorubrum sodomense, but little is known about its photocycle. Recent interest has focused on AR3 because of its ability to serve both as a high-performance, genetically-targetable optical silencer of neuronal activity and as a membrane voltage sensor. We examined light-activated structural changes of the protein, retinal chromophore, and internal water molecules during the photocycle of AR3. Low-temperature and rapid-scan time-resolved FTIR-difference spectroscopy revealed that conformational changes during formation of the K, M, and N photocycle intermediates are similar, although not identical, to bacteriorhodopsin (BR). Positive/negative bands in the region above 3,600 cm( - 1), which have previously been assigned to structural changes of weakly hydrogen bonded internal water molecules, were substantially different between AR3 and BR. This included the absence of positive bands recently associated with a chain of proton transporting water molecules in the cytoplasmic channel and a weakly hydrogen bonded water (W401), which is part of a hydrogen-bonded pentagonal cluster located near the retinal Schiff base. However, many of the broad IR continuum absorption changes below 3,000 cm( - 1) assigned to networks of water molecules involved in proton transport through cytoplasmic and extracellular portions in BR were very similar in AR3. This work and subsequent studies comparing BR and AR3 structural changes will help identify conserved elements in BR-like proton pumps as well as bioengineer AR3 to optimize neural silencing and voltage sensing.

Reliable optical detection of single action potentials in mammalian neurons has been one of the longest-standing challenges in neuroscience. Here we achieved this goal by using the endogenous fluorescence of a microbial rhodopsin protein, Archaerhodopsin 3 (Arch) from Halorubrum sodomense, expressed in cultured rat hippocampal neurons. This genetically encoded voltage indicator exhibited an approximately tenfold improvement in sensitivity and speed over existing protein-based voltage indicators, with a roughly linear twofold increase in brightness between -150 mV and +150 mV and a sub-millisecond response time. Arch detected single electrically triggered action potentials with an optical signal-to-noise ratio >10. Arch(D95N) lacked endogenous proton pumping and had 50% greater sensitivity than wild type but had a slower response (41 ms). Nonetheless, Arch(D95N) also resolved individual action potentials. Microbial rhodopsin-based voltage indicators promise to enable optical interrogation of complex neural circuits and electrophysiology in systems for which electrode-based techniques are challenging.

Bacteria have many voltage- and ligand-gated ion channels, and population-level measurements indicate that membrane potential is important for bacterial survival. However, it has not been possible to probe voltage dynamics in an intact bacterium. Here we developed a method to reveal electrical spiking in Escherichia coli. To probe bacterial membrane potential, we engineered a voltage-sensitive fluorescent protein based on green-absorbing proteorhodopsin. Expression of the proteorhodopsin optical proton sensor (PROPS) in E. coli revealed electrical spiking at up to 1 hertz. Spiking was sensitive to chemical and physical perturbations and coincided with rapid efflux of a small-molecule fluorophore, suggesting that bacterial efflux machinery may be electrically regulated.

Proteorhodopsins (PRs), photoactive retinylidene membrane proteins ubiquitous in marine eubacteria, exhibit light-driven proton transport activity similar to that of the well studied bacteriorhodopsin from halophilic archaea. However, unlike bacteriorhodopsin, PRs have a single highly conserved histidine located near the photoactive site of the protein. Time-resolved Fourier transform IR difference spectroscopy combined with visible absorption spectroscopy, isotope labeling, and electrical measurements of light-induced charge movements reveal participation of His-75 in the proton translocation mechanism of PR. Substitution of His-75 with Ala or Glu perturbed the structure of the photoactive site and resulted in significantly shifted visible absorption spectra. In contrast, His-75 substitution with a positively charged Arg did not shift the visible absorption spectrum of PR. The mutation to Arg also blocks the light-induced proton transfer from the Schiff base to its counterion Asp-97 during the photocycle and the acid-induced protonation of Asp-97 in the dark state of the protein. Isotope labeling of histidine revealed that His-75 undergoes deprotonation during the photocycle in the proton-pumping (high pH) form of PR, a reaction further supported by results from H75E. Finally, all His-75 mutations greatly affect charge movements within the PR and shift its pH dependence to acidic values. A model of the proteorhodopsin proton transport process is proposed as follows: (i) in the dark state His-75 is positively charged (protonated) over a wide pH range and interacts directly with the Schiff base counterion Asp-97; and (ii) photoisomerization-induced transfer of the Schiff base proton to the Asp-97 counterion disrupts its interaction with His-75 and triggers a histidine deprotonation.

We examine the structural changes during the primary photoreaction in blue-absorbing proteorhodopsin (BPR), a light-driven retinylidene proton pump, using low-temperature FTIR difference spectroscopy. Comparison of the light-induced BPR difference spectrum recorded at 80 K to that of green-absorbing proteorhodopsin (GPR) reveals that there are several differences in the BPR and GPR primary photoreactions despite the similar structure of the retinal chromophore and all-trans --> 13-cis isomerization. Strong bands near 1700 cm(-1) assigned previously to a change in hydrogen bonding of Asn230 in GPR are still present in BPR. However, additional bands in the same region are assigned on the basis of site-directed mutagenesis to changes occurring in Gln105. In the amide II region, bands are assigned on the basis of total (15)N labeling to structural changes of the protein backbone, although no such bands were previously observed for GPR. A band at 3642 cm(-1) in BPR, assigned to the OH stretching mode of a water molecule on the basis of H2(18)O substitution, appears at a different frequency than a band at 3626 cm(-1) previously assigned to a water molecule in GPR. However, the substitution of Gln105 for Leu105 in BPR leads to the appearance of both bands at 3642 and 3626 cm(-1), indicating the waters assigned in BPR and GPR exist in separate distinct locations and can coexist in the GPR-like Q105L mutant of BPR. These results indicate that there exist significant differences in the conformational changes occurring in these two types proteorhodopsin during the initial photoreaction despite their similar chromophore structures, which might reflect a different arrangement of water in the active site as well as substitution of a hydrophilic for hydrophobic residue at residue 105.

Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Delta1-49 apolipoprotein A-I fragment (Delta49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR --> M transition. Importantly the functional bR was solubilized in discoidal bR.NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Delta49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies.

Proteorhodopsins are an extensive family of photoactive membrane proteins found in proteobacteria distributed throughout the world's oceans which are often classified as green- or blue-absorbing (GPR and BPR, respectively) on the basis of their visible absorption maxima. GPR and BPR have significantly different properties including photocycle lifetimes and wavelength dependence on pH. Previous studies revealed that these different properties are correlated with a single residue, Leu105 in GPR and Gln105 in BPR, although the molecular basis for the different properties of GPR and BPR has not yet been elucidated. We have studied the unexcited states of GPR and BPR using resonance Raman spectroscopy which enhances almost exclusively chromophore vibrations. We find that both spectra are remarkably similar, indicating that the retinylidene structure of GPR and BPR are almost identical. However, the frequency of a band assigned to the retinal C13-methyl-rock vibration is shifted from 1006 cm (-1) in GPR to 1012 cm (-1) in BPR. A similar shift is observed in the GPR mutant L105Q indicating Leu and Gln residues interact differently with the retinal C13-methyl group. The environment of the Schiff base of GPR and BPR differ as indicated by differences in the H/D induced down-shift of the Schiff base vibration. Residues located in transmembrane helices (D-G) do not contribute to the observed differences in the protein-chromophore interaction between BPR and GPR based on the Raman spectra of chimeras. These results support a model whereby the substitution of the hydrophilic Gln105 in BPR with the smaller hydrophobic Leu105 in GPR directly alters the environment of both the retinal C13 group and the Schiff base.

Proteorhodopsins are a recently discovered class of microbial rhodopsins, ubiquitous in marine bacteria. Over 4000 variants have thus far been discovered, distributed throughout the oceans of the world. Most variants fall into one of two major groups, green- or blue-absorbing proteorhodopsin (GPR and BPR, respectively), on the basis of both the visible absorption maxima (530 versus 490 nm) and photocycle kinetics ( approximately 20 versus approximately 200 ms). For a well-studied pair, these differences appear to be largely determined by the identity of a single residue at position 105 (leucine/GPR and glutamine/BPR). We find using a combination of visible and infrared spectroscopy that a second difference is the protonation state of a glutamic acid residue located at position 142 on the extracellular side of helix D. In BPR, Glu142 (the GPR numbering system is used) is deprotonated and can act as an alternate proton acceptor, thus explaining the earlier observations that neutralization of the Schiff base counterion, Asp97, does not block the formation of the M intermediate. In contrast, Glu142 in GPR is protonated and cannot act in this state as an alternate proton acceptor for the Schiff base. On the basis of these findings, a mechanism is proposed for proton pumping in BPR. Because the pKa of Glu142 is near the pH of its native marine environment, changes in pH may act to modulate its function in the cell.

Recent studies demonstrate that photoactive proteins can react within several picoseconds to photon absorption by their chromophores. Faster subpicosecond protein responses have been suggested to occur in rhodopsin-like proteins where retinal photoisomerization may impulsively drive structural changes in nearby protein groups. Here, we test this possibility by investigating the earliest protein structural changes occurring in proteorhodopsin (PR) using ultrafast transient infrared (TIR) spectroscopy with approximately 200 fs time resolution combined with nonperturbing isotope labeling. PR is a recently discovered microbial rhodopsin similar to bacteriorhodopsin (BR) found in marine proteobacteria and functions as a proton pump. Vibrational bands in the retinal fingerprint (1175-1215 cm(-1)) and ethylenic stretching (1500-1570 cm(-1)) regions characteristic of all-trans to 13-cis chromophore isomerization and formation of a red-shifted photointermediate appear with a 500-700 fs time constant after photoexcitation. Bands characteristic of partial return to the ground state evolve with a 2.0-3.5 ps time constant. In addition, a negative band appears at 1548 cm(-1) with a time constant of 500-700 fs, which on the basis of total-15N and retinal C15D (retinal with a deuterium on carbon 15) isotope labeling is assigned to an amide II peptide backbone mode that shifts to near 1538 cm(-1) concomitantly with chromophore isomerization. Our results demonstrate that one or more peptide backbone groups in PR respond with a time constant of 500-700 fs, almost coincident with the light-driven retinylidene chromophore isomerization. The protein changes we observe on a subpicosecond time scale may be involved in storage of the absorbed photon energy subsequently utilized for proton transport.

Anabaena sensory rhodopsin (ASR) is a novel microbial rhodopsin recently discovered in the freshwater cyanobacterium Anabaena sp. PCC7120. This protein most likely functions as a photosensory receptor as do the related haloarchaeal sensory rhodopsins. However, unlike the archaeal pigments, which are tightly bound to their cognate membrane-embedded transducers, ASR interacts with a soluble cytoplasmic protein analogous to transducers of animal vertebrate rhodopsins. In this study, infrared spectroscopy was used to examine the molecular mechanism of photoactivation in ASR. Light adaptation of the pigment leads to a phototransformation of an all-trans/15-anti to 13-cis/15-syn retinylidene-containing species very similar in chromophore structural changes to those caused by dark adaptation in bacteriorhodopsin. Following 532 nm laser-pulsed excitation, the protein exhibits predominantly an all-trans retinylidene photocycle containing a deprotonated Schiff base species similar to those of other microbial rhodopsins such as bacteriorhodopsin, sensory rhodopsin II, and Neurospora rhodopsin. However, no changes are observed in the Schiff base counterion Asp-75, which remains unprotonated throughout the photocycle. This result along with other evidence indicates that the Schiff base proton release mechanism differs significantly from that of other known microbial rhodopsins, possibly because of the absence of a second carboxylate group at the ASR photoactive site. Several conformational changes are detected during the ASR photocycle including in the transmembrane helices E and G as indicated by hydrogen-bonding alterations of their native cysteine residues. In addition, similarly to animal vertebrate rhodopsin, perturbations of the polar head groups of lipid molecules are detected.