Phototactic and Chemotactic Signal Transduction by Transmembrane Receptors and Transducers in Microorganisms

Microorganisms show attractant and repellent responses to survive in the various environments in which they live. Those phototaxic (to light) and chemotaxic (to chemicals) responses are regulated by membrane-embedded receptors and transducers. This article reviews the following: (1) the signal relay mechanisms by two photoreceptors, Sensory Rhodopsin I (SRI) and Sensory Rhodopsin II (SRII) and their transducers (HtrI and HtrII) responsible for phototaxis in microorganisms; and (2) the signal relay mechanism of a chemoreceptor/transducer protein, Tar, responsible for chemotaxis in E. coli. Based on results mainly obtained by our group together with other findings, the possible molecular mechanisms for phototaxis and chemotaxis are discussed

Regulation of environmental responses and its physiological significance in Vibrio species

研究成果の概要 (和文) : コレラ菌アミノ酸走性受容体を２つ同定し，うち一つは胆汁の主要成分タウリンの受容体としても働くことを見出した．タウリン応答およびタウリン受容体発現が37℃培養で顕著に増加したことから，タウリン走性がコレラ菌感染過程で重要な役割を果たす可能性が示唆された．一方，海洋ビブリオ菌においてもアミノ酸走性センサーを同定し，側毛発現時に発現誘導されることを示唆する結果を得た．さらに，走化性シグナル伝達系因子と相同な蛋白質群が，微好気条件で極局在することを見出した．これらのシグナル伝達系は宿主腸内などの微好気条件で働く可能性がある．研究成果の概要 (英文) : We have identified two amino acid receptors of Vibrio cholerae for chemotaxis, one of which also senses taurine, a major component of bile. The taurine response and the expression of the taurine chemoreceptor was enhanced at elevated temperature, raising an intriguing possibility that taurine taxis play a role in pathogenesis of the bacteria. We also identified an amino acid chemoreceptor in marine Vibrio sp., the expression of which may be induced coordinately with lateral flagella. We also found that chemotaxis-related signaling proteins localized to a cell pole under micro aerobic conditions, implying that these signaling systems function under specific conditions such as in the host intestine

Localization of transmembrane receptors and functional and structural compartentalization of the cytoplasmic membrane

研究成果の概要 (和文) : 大腸菌の走化性受容体および全ヒスチジンキナーゼ,コレラ菌の走化性様シグナル伝達系蛋白質の局在機構に関して解析し,膜貫通型受容体の局在とその制御の機構について新たな知見を得た.また,細胞膜中での膜貫通型受容体の動きを解析し,アクチン様細胞骨格またはそれに付随する構造により制限されることを見出した.さらに,FlAsHとHaloTagによる走化性受容体の標識を試み,局在観察可能な系を構築した.以上の知見は,細菌膜貫通型受容体の機能と細胞膜の構築に関して重要な知見をもたらすものである.研究成果の概要 (英文) : The chemoreceptors and the histidine kinases of Escherichia coli as well as protein components of chemotaxis-related signaling systems were analyzed for their localization to obtain several important results concerning the mechanisms underlying localization and its control of transmembrane sensors. Movements of the chemoreceptor through the membrane were analyzed to find that they are restricted by actin-like cytoskeleton or structures associated with it. The chemoreceptor was labeled with FlAsH and HaloTag to establish new systems for localization analyses. These results shed new light on the localization/function of transmembrane sensors and the architecture of the cytoplasmic membrane in prokaryotic cells

The retina-prosthesis interface in two rat models of retinitis pigmentosa and functional changes associated with photoreceptor loss.

With increasingly successful clinical trials, the use of prosthetic devices to replace the function of photoreceptors has now become a viable option to treat degenerative diseases of the retina, including age-related macular degeneration (AMD) and retinitis pigmentosa (RP). Using prosthetics, patients have demonstrated the ability to read large text, recognize small objects, and navigate. While promising, this represents coarse visual function, with the highest visual acuity equivalent to 20/560, still below the legal definition of blindness, 20/200. To investigate the benefit of more sophisticated designs, I implanted two types of photovoltaic prosthetic devices in two models of RP, Tg S334ter-3 and Tg P23H-1 rats. The performance of a new bipolar photovoltaic array (bPVA) design with local ground returns was compared to a monopolar photovoltaic array (mPVA) with a single ground return. Regardless of implant design, stimulation parameters at threshold were at least 1.5 orders of magnitude below safety limits. Age and duration of implantation had no significant effect on stimulation thresholds while threshold increased inversely to pixel size. In addition to treating the symptoms of these diseases, a better understanding of the functional consequences of degeneration will help guide future treatment strategies. I examined changes in visual function in the retina and the superior colliculus (SC). I found profound changes in visual function in both models. First, the ON pathway is preferentially lost due to a shift from equal numbers of ON and OFF ganglion cells in the retina and ON dominated responses in the SC to OFF dominated responses in both. In the Tg P23H-1 rat, this shift is progressive with age, while in the Tg S334ter-3 this shift is evident early in life. In late stages of the disease, there is little response from the ON pathway. In animals implanted with bPVA devices, there was a significant change in the response based on the location of the prosthesis, with a preservation of the ON pathway in areas directly under the influence of the prosthesis. Either the presence of the device or basal electrical activity may induce a trophic influence that attenuates this change in visual function

Phototactic and Chemotactic Signal Transduction by Transmembrane Receptors and Transducers in Microorganisms

Microorganisms show attractant and repellent responses to survive in the various environments in which they live. Those phototaxic (to light) and chemotaxic (to chemicals) responses are regulated by membrane-embedded receptors and transducers. This article reviews the following: (1) the signal relay mechanisms by two photoreceptors, Sensory Rhodopsin I (SRI) and Sensory Rhodopsin II (SRII) and their transducers (HtrI and HtrII) responsible for phototaxis in microorganisms; and (2) the signal relay mechanism of a chemoreceptor/transducer protein, Tar, responsible for chemotaxis in E. coli. Based on results mainly obtained by our group together with other findings, the possible molecular mechanisms for phototaxis and chemotaxis are discussed

細菌ロドプシン類タンパク質の生物物理学的研究

Microbial rhodopsins are typical membrane proteins with seven transmembrane alpha-helices (helices A-G). This type of protein has a retinal molecule as the chromophore. The microbial rhodopsins use the retinal chromophore attached at a conserved lysine residue to absorb photons for light-energy conversion and light-signal transduction. The molecular mechanisms of microbial rhodopsins have been discussed at the individual amino acid level. In this study, we have prepared amino acid replaced mutants for sensory rhodopsin II from Halobacterium salinarum (HsSRII), and archaerhodopsin discovered by our group from salt lake in Inner Mongolia of China (HeAR). Their photochemical properties were studied by flash photolysis and the the function of amino acids which have been not reported until now was clarified. HsSRII is a negative phototaxis receptor, which makes bacterium avoid blue-green light. In this study. The amino acid replaced mutants of aspartic acid 103 (D103) of HsSRII were prepared and analyzed with flash photolysis. Aspartic acid 103 of HsSRII corresponds to D115 of bacteriorhodopsin (BR). This amino acid residue D115 is functionally important in BR. Aspartic acid D103 were replaced with asparagine (D103N) or glutamic acid (D103E). The work revealed that a substitution of D103 with asparagine (D103N) or glutamic acid (D103E) can resulted in large changes in HsSRII photocycle. These alterations include acceleration of the decay of the M intermediate, prolonging the lifetime of intermediates in the later part of photocycle, and appearance of a long-lived shorter-wavelength photoproduct. Thus, D103 of HsSRII may play an important role in regulation of the photocycle of HsSRII. HeAR is a light driven outward proton pump like to BR. All key functional amino acid residues for proton pump of BR are conserved in HeAR. In the present work, we payed attention to amino acids which are only conserved in ARs, prepared the mutants T164A, S165A and T164A/S165A (T164 and/or S165 were replaced with an alanine) and analyzed the photocycles of the mutants with flash photolysis. The amino acid replacements caused profound changes to the photocycle of HeAR including acceleration of the decay of M intermediate, prolonging lifetime of intermediates in the later part of photocycle, and appearance of additional two intermediates which were evident in the photocycles of T164 mutants. These results suggest that although T164 and S165 are located at the far end of the photoactive center, these two amino acid residues are important for regulation of the HeAR photocycle. Moreover, the thermal stability of three-dimensional structure of HeAR and its mutants were investigated by small angle x-ray scattering (SAXS). In addition, the photocycles of HeAR reconstituted into lipid and HeAR in the presence of detergent were compared and the influence of the different environment on HeAR photocycle was investigated.細菌ロドプシン類タンパク質は7本のα-へリックスから構成され、発色団としてビタミンAアルデヒド（レチナール）を持つ光受容膜タンパク質であり、レチナールに吸収された光エネルギーの生体エネルギーへの変換や、外界の光情報の受容に関わっている。細菌ロドプシン類タンパク質は、生体膜中での立体構造が明らかにされた最初の膜タンパク質であり、モデル膜タンパク質として、詳細に研究され、それらの機能について、個々のアミノ酸レベルで、その分子機構が議論されている。本研究では、センサリーロドプシンII（SRII）と、中国内モンゴルの塩湖から我々のグループによって発見されたHalorubrum sp. ejinoor の持つアーキロドプシン(HeAR)について、新たなアミノ酸変異体を作成し、その光化学的性質を調べ、これまで分かっていなかったアミノ酸の働きを明らかにした。SRIIは、細菌が青緑光から逃げる負の走光性の光受容体である。本研究では、バクテリオロドプシン（BR）のプロトンポンプ機能に重要なアミノ酸残基であるアスパラギン酸（D）115に対応するSRIIのD103の変異体を作成した。SRIIのD103をアスパラギン（D103N）やグルタミン酸（D103E）に置換した変異体を作成し、その光反応を調べた。その結果、変異体では、M中間体の崩壊は速くなるが、光反応サイクル後半の中間体の寿命が長くなること、また、長寿命の副産物の生成が観察され、SRIIのD103は、SRIIの光反応サイクルの回転速度の調節に重要な役割を果たすことが分かった。HeARは、BRと同様に光駆動外向きプロトンポンプとして働く。ARのアミノ酸配列はBRとよく似ており、これまで、プロトンポンプ機能に必須であることが明らかになっているアミノ酸は全て保存されていた。しかし、ARだけに共通して保存されているアミノ酸があることに注目し、これらのアミノ酸（N末より164番目のトレオニン；T164と165番目のセリン；S165）をアラニン（A）に置換した変異体、T164A、S165AとT164A/S165Aを作製し、それらの光化学的性質を調べた。その結果、変異体では、M中間体の崩壊は速くなるが、光反応サイクル後半の中間体の寿命が長くなり、また、T164の変異では光反応中間体の数が増加した。発色団レチナールから離れた場所にあるにもかかわらず、HeARのT164とS165は、HeARの光反応サイクルの回転速度の調節に重要な役割を果たすことが分かった。また、HeARの立体構造の熱安定性をX線小角散乱法（SAXS）によって調べた。脂質に再構成したHeARと界面活性剤存在下でのHeARの光反応を比較検討し、タンパク質周辺環境の違いの影響についても調べた。室蘭工業大学 (Muroran Institute of Technology)博士（工学

Vibrational spectroscopy of an optogenetic rhodopsin: a biophysical study of molecular mechanisms

In this dissertation,the membrane protein channelrhodopsin-1 from the green flagellate algae Chlamydomonas agustae (CaChR1) is studied using a variety of spectroscopic techniques developed in the Rothschild Molecular Biophysics Laboratory at Boston University. Over the last decade, channelrhodopsins have proven to be effective optogenetic tools due to their ability to function as light-gated ion channels when expressed in neurons. This ability allows neuroscientists to optically activate an inward directed photocurrent which depolarizes the neuronal membranes and triggers an action potential. Although a variety of channelrhodopsins with different properties have been used, the underlying mechanisms of channelrhodopsin functionality is not yet fully understood. The protein studied here has several advantageous properties compared to the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii including a red shifted visible absorption and slower light inactivation despite having a lower channel current. Elucidating the internal molecular mechanisms underlying the function of CaChR1 provides critical insight into the large class of channelrhodopsin proteins leading toward improved bioengineering for specific optogenetic applications. Here near-IR pre-resonance Raman spectroscopy of CaChR1 provides information on the structure of the unphotolyzed (P0) retinal chromophore, the Schiff base protonation state, and presence of carboxylic acid residues interacting with the Schiff base. Low-temperature FTIR difference spectroscopy combined with site-directed mutagenesis and isotope labeling provide information on changes occurring in the retinal chromophore and protein during the primary phototransition (P0 to P1). This includes information about changes involving protonation state of binding-pocket residues, protein backbone structure, and internal water molecules. Further experiments combining low-temperature and time-resolved FTIR-difference spectroscopy reveal additional information about structural changes during the transition from the unphotolyzed state to the active (open channel) state of the protein (P0 to P2). This work has resulted in an initial model that describes key proton transfer events which occur between the Schiff base and carboxylic acid residues inside the active site of CaChR1. The model raises the possibility that ion channel gating and ion specificity is regulated by the protonation changes of two key residues (Glu 169 and Asp299) located near the Schiff base

Signal Transduction in tandem HAMP domains

The incidence of HAMP tandems in bacterial signaling proteins is low and presently it is unknown what physiological advantage may be gained by using a tandem. Presently a simple general mechanism of HAMP signaling which satisfactorily accounts for all experimental data cannot be presented. To study signal transduction via HAMP domains we used an in vitro biochemical system in which the signal output is affected exclusively by the HAMP domain that is inserted between the Tsr receptor as the input and the Rv3645 adenylyl cyclase as output domain. Initially neither the HAMP tandem nor its respective monomers operated as signal transducers in our system. The introduction of five targeted mutations in the first α-helix of NpHAMP1 which adapted this sequence somewhat to the equivalent Tsr sequence was required to obtain a functional, i.e. signal-transducing HAMP tandem. Replacement of the entire α-helix NpAS11-mut5 by the equivalent sequence of HAMPTsr the chimeric HAMP monomer (AS1Tsr/NpAS2) was fully operational in that serine strongly inhibited AC activity. Furthermore, in combination with NpHAMP2 in tandem the sign of the output signal was inverted as predicted. This left us with two HAMP tandem constructs with opposite outputs of the serine signal as initiated by serine-binding to the periplasmic domain of Tsr. The differences between both constructs were confined to the first α-helix of the first HAMP domain in the tandem as all other segments remained unchanged. Both constructs received the same conformational signal from Tsr. One might then reasonably speculate that the first α-helix (α-helix-1) is ultimately responsible for formation of different ground states of the output domain which leads to differences in signal output. In a series of experiments, AS1 of NpHAMP1 was extensively mutated to decipher which residues actually might determine such different states. In NpAS11-Tsr and NpAS11-mut5, five amino acid residues in α-helix-1 were responsible for defining opposite ground states. Just manipulating the α-helix-1 in a HAMP tandem was sufficient to produce opposite signaling outputs. The data do not permit making a similar claim for signal transduction through a HAMP monomer. This finding is hard to explain with rotation as a major HAMP signaling mechanism. In N. pharaonis sensory rhodopsin-I and its cognate transducer complex, SRI-HtrI has a dual function by mediating attractant and repellant responses whereas SRII-HtrII mediates only repellant responses. Both transducers have a HAMP tandem. In such a system signal rapid changes in signal input may require a fast track system for adaptation which has been reported for SRI-HtrI complex signaling. Our data allow the speculation that HAMP tandems by virtue of their intrinsic sequences prime a signal transduction system for a distinct organismal response to peculiar environmental cues such as light in N. pharaonis. The results complicate predictions of HAMP mediated signaling based on our current structural knowledge base. According to the gearbox model of HAMP signal transduction one might consider that HAMP1 may rotate in both directions. However, such an interpretation would clash with the fact that the signal emanating from the Tsr membrane receptor is the same irrespective of the type of HAMP domain attached to its C-terminal membrane exit. It is similarly questionable whether other proposals for signal transduction such as the piston or the dynamic bundle models alone could plausibly explain the above results. Rotation as one structural parameter for HAMP signal transduction is not excluded, rather it ought to be seen in conjunction with other molecular movements which might control four helix bundle stability in essence by regulated unfolding. Stability in this context is not restricted to the HAMP module alone but includes adjacent regions with which the HAMP domain is in a continuous structural balance. The possibility to switch the sign of the output signal by a single amino acid mutation in a HAMP tandem context may be an evolutionary advantage in the multiplicity of HAMP-mediated signaling systems and may expand the versatility of such units.HAMP* vermittelte Signaltransduktion ist allgegenwärtig (> 28.000 HAMP Datenbank Einträge). In den meisten Fällen ist zwischen einem Sensor und einem Ausgabemodul eine HAMP Domäne (HAMP Monomer) eingesetzt. Sie dient wahrscheinlich als Adapter zwischen der Sensor- und Effektordomäne. Der vorgeschlagene Mechanismus der HAMP Signaltransduktion durch Drehung wurde durch eine Kristallstruktur einer seriellen dreifach-HAMP aus Pseudomonas aeruginosa gestärkt. Das Rotationsmodell würde vorhersagen, dass sich mit jeder zusätzlichen HAMP Domäne das Vorzeichen des Ausgangssignals umkehrt. Diese Vorhersage wurde durch biochemische Experimente überprüft, indem eine HAMP-Tandem Domäne des HtrII Photorezeptors aus N. pharaonis verwendet wurde. Das grundlegende Design unserer getesteten Konstrukte mit Tsr als Sensor und Rv3645 AC als Effektor wurde beibehalten. Das grundlegende Design unserer getesten Konstrukte war jeweils Tsr als Sensor und Rv3645 AC als Effecktor mit der zu untersuchenden HAMP Domäne dazwischen. Es war nicht verwunderlich, dass zunächst weder das HAMP-Tandem noch seine jeweiligen Monomere in den getesteten Konstrukten als Signalgeber fungierten, weil in NpHtrII das Lichtsignal zwischen sensorischem Rhodopsin II und der Chemotaxiseinheit HtrII innerhalb der Membran weiter gegeben wird. Die Einführung von fünf gezielten Mutationen in der ersten α-Helix von NpHAMP1, die deren Sequenz stärker an die von Tsr angleicht, war erforderlich, um eine funktionale, d.h. signaltransduzierende HAMP-Tandem Domäne zu erhalten. Beide HAMP Monomere allein waren inaktiv als Signalgeber. Der überraschende Befund war, dass das Vorzeichen des Ausgangssignals nicht wie vorhergesagt umgedreht wurde.Wenn die gesamte erste α-Helix (AS1) im NpHAMP Monomer durch die äquivalente α-Helix von HAMPTsr ersetzt wurde (AS11-Tsr/NpH1), wurde das Konstrukt gehemmt. Mit NpHAMP2 als HAMP-Tandem allerdings wurde das Vorzeichen des Ausgangssignals invertiert, d.h. das Tandem-Konstrukt wurde aktiviert. Die Unterschiede im Ausgangssignal zwischen beiden Konstrukten können der ersten α-Helix der ersten HAMP Domäne im Tandem zugerechnet werden, da alle anderen Segmente unverändert blieben. Man kann annehmen, dass die erste α-Helix letztlich verantwortlich ist für die Bildung von unterschiedlichen Grundzuständen der Effektordomäne.In einer Serie von Experimenten wurde durch zahlreiche Mutationen in AS1 von NpHAMP1 untersucht, welche Aminosäuren die verschiedenen Zustände bestimmen. Das Ergebnis von 48 Mutationen in NpAS11-mut5 und AS11-Tsr/NpH1 Tandem Konstrukten war, dass fünf Aminosäuren für die gegensätzlichen Grundzustände verantwortlich waren. Dies würde dafür sprechen, dass das Signal, das vom Tsr Membranrezeptor ausgeht, immer das gleiche ist, unabhängig von der Art der angeschlossenen HAMP Domäne. So ist es fraglich, ob andere Modelle der Signaltransduktion wie z.B. das Kolben-Modell oder das ''dynamic bundle model'' die obigen Ergebnisse plausibel erklären. Rotation als alleiniger struktureller Parameter für die HAMP Signaltransduktion wird kaum ausreichen, sondern sollte in Verbindung mit anderen molekularen Bewegungen gesehen werden, welche Einfluss auf die Stabilität des Vierhelixbündels der HAMP Domäne nehmen können. Hierzu lässt sich z.B. das „regulated unfolding’’ nennen. Bei dieser These ist die Stabilität nicht auf das HAMP Modul allein beschränkt, sondern umfasst auch benachbarte Bereiche, mit denen sich die HAMP Domäne in einem kontinuierlichen Strukturgleichgewicht befindet. Eine plausible Interpretation wäre, dass HAMP Domänen verschiedene Grundzustände eines sensorischen Systems definieren und entsprechend gegensätzliche physiologische Reaktionen auslösen können. In dem einen Grundzustand lagert sich das katalytische AC Homodimer richtig zusammen, sodass bei Stimulation des Tsr Rezeptors sich die Untereinheiten voneinander distanzieren, wodurch es zu einer Hemmung der Enzymaktivität kommt. Im Gegensatz dazu kommen im anderen Grundzustand die Untereinheiten durch ein Serinsignal zusammen, was zu einer erhöhten Enzymaktivität führt. *- Histidine kinases, Adenylyl cyclases, Methyl-accepting chemotaxis proteins and Phosphatases

Untersuchungen zur Signalweiterleitung in Chemo- und Photorezeptoren

Die gerichtete Bewegung auf einen Lockstoff zu oder von einem Schreckstoff fort ermöglicht es einem Organismus optimale Lebensbedingungen zu finden. Die Zelle bewegt sich mit Hilfe von Flagellen, die über eine Zwei-Komponenten Signalkaskade gesteuert werden. Der Eingang eines Signals wird von membranständigen Chemo- oder Photorezeptoren detektiert und über die Membran ins Zellinnere weitergeleitet. Der Aspartatrezeptor aus E. coli (Tar) und der Transducer HtrII im Komplex mit SRII aus H. salinarum und N. pharaonis wurden in dieser Arbeit näher untersucht. Es wurden Elektronenspinresonanz Messungen an Proteoliposomen durchgeführt. Zudem gaben Methylierungsexperimente Hinweise auf die Kooperation zwischen den Rezeptoren. FTIR Messungen sowie Festkörper NMR Messungen wurden zur Aufklärung des aktiven Signalzustandes von NpSRII herangezogen

FTIR difference and resonance raman spectroscopy of rhodopsins with applications to optogenetics

Thesis (Ph. D.)--Boston UniversityThe major aim of this thesis is to investigate the molecular basis for the function of several types of rhodopsins with special emphasis on their application to the new field of optogenetics. Rhodopsins are transmembrane biophotonic proteins with 7 a-helices and a retinal chromophore. Studies included Archaerhodopsin 3 (AR3), a light driven proton pump similar to the extensively studied bacteriorhodopsin (BR); channelrhodopsins 1 and 2, light-activated ion channels; sensory rhodopsin II (SRII), a light-sensing protein that modulates phototaxis used in archaebacteria; and squid rhodopsins (sRho), the major photopigment in squid vision and a model for human melanopsin, which controls circadian rythms. The primary techniques used in these studies were FTIR difference spectroscopy and resonance Raman spectroscopy. These techniques, in combination with site directed mutagenesis and other biochemical methodologies produced new knowledge regarding the structural changes of the retinal chromophore, the location and function of internal water molecules as well as specific amino acids and peptide backbone. Specialized techniques were developed that allowed rhodopsins to be studied in intact membrane environments and in some cases in vivo measurements were made on rhodopsin heterologously expressed in E. coli thus allowing the effects of interacting proteins and membrane potential to be investigated. Evidence was found that the local environment of one or more internal water molecules in SRII is altered by interaction with its cognate transducer, Htrii, and is also affected by the local lipid environment. In the case of AR3, many of the broad IR continuum absorption changes below 3000 cm-1, assigned to networks of water molecules involved in proton transport through cytoplasmic and extracellular portions in BR, were found to be very similar to BR. Bands assigned to water molecules near the Schiff base postulated to be involved in proton transport were, however, shifted or absent. Structural changes of internal water molecules and possible bands associated with the interaction with ,8-arrestins were also detected in photoactivated squid rhodopsin when transformed to the acid Meta intermediate. Near-IR confocal resonance Raman 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. On the basis of these measurements, a model is proposed which provides a possible explanation for the observed fluorescence dependence of AR3 and other microbial rhodopsins on transmembrane potential