Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy

Channelrhodopsin-2 from Chlamydomonas reinhardtii is a light-gated ion channel. Over recent years, this ion channel has attracted considerable interest because of its unparalleled role in optogenetic applications. However, despite considerable efforts, an understanding of how molecular events during the photocycle, including the retinal trans-cis isomerization and the deprotonation/reprotonation of the Schiff base, are coupled to the channel-opening mechanism remains elusive. To elucidate this question, changes of conformation and configuration of several photocycle and conducting/nonconducting states need to be determined at atomic resolution. Here, we show that such data can be obtained by solid-state NMR enhanced by dynamic nuclear polarization applied to ¹⁵N-labeled channelrhodopsin-2 carrying 14,15-¹³C₂ retinal reconstituted into lipid bilayers. In its dark state, a pure all-trans retinal conformation with a stretched C14-C15 bond and a significant out-of-plane twist of the H-C14-C15-H dihedral angle could be observed. Using a combination of illumination, freezing, and thermal relaxation procedures, a number of intermediate states was generated and analyzed by DNP-enhanced solid-state NMR. Three distinct intermediates could be analyzed with high structural resolution: the early P1500 K-like state, the slowly decaying late intermediate P4480, and a third intermediate populated only under continuous illumination conditions. Our data provide novel insight into the photoactive site of channelrhodopsin-2 during the photocycle. They further show that DNP-enhanced solid-state NMR fills the gap for challenging membrane proteins between functional studies and X-ray–based structure analysis, which is required for resolving molecular mechanisms.Since their discovery (1), channelrhodopsins (ChRs) have generated enormous interest because of the rapid development of their applications in optogenetics (2–7). Commonly, ChR2 from Chlamydomonas reinhardtii (8) and its variants are used thanks to their favorable expression levels. They are the only proteins known today functioning as light-gated ion channels (Fig. 1A). Like other microbial retinal proteins, they undergo a periodic photocycle. In ChRs, this photocycle is coupled to channel opening and closing as revealed in electrophysiological recordings (8). A chimera of ChR1 and ChR2 has been crystallized to yield a structure at 2.3-Å resolution (9). However, little is known on how this coupling functions on a molecular level, and a large number of studies based on visible (10–13), IR (11, 14–19), resonance Raman spectroscopy (20, 21), and EPR spectroscopy (22, 23) has been performed to address this question.Open in a separate windowFig. 1.(A) Visualization of dimeric ChR2 reconstituted into the lipid bilayer as used in this study [cartoon based on the crystal structure of the ChR1/2 chimera (data from ref. 9)]. Blue light illumination activates ChR2. (B) Single turnover (black arrows) and continuous illumination photocycle (blue arrows) (14, 40). (C) Schematic view of the experimental setup for generating and measuring different photointermediates. (D) The DNP enhancement is generated by magnetization transfer from the biradical AMUPOL to ChR2.The photocycles of microbial rhodopsins are usually compared with bacteriorhodopsin, the first discovered and most studied light-driven proton pump (24). Without any illumination, microbial retinal proteins thermally equilibrate into a dark state (25). In the case of bacteriorhodopsin, for example, this state contains a mixture of two species termed bacteriorhodopsin₅₆₈ (all-trans,15-anti retinal Schiff base) and bacteriorhodopsin₅₄₈ (13-cis,15-syn conformation) (26, 27). On illumination, light adaption occurs from the dark state to the ground state, which contains only the all-trans,15-anti conformer as the photocycle starting point (28). A similar light–dark adaption has been found in halorhodopsin from Halobacterium salinarium (29). However, such a light/dark adaption in conjunction with a conformer mixture does not seem to be a general property of microbial membrane proteins. Other systems have been described where the ground state contains only an all-trans,15-anti retinal Schiff base chromophore [e.g., green proteorhodopsin (30), Anabaena sensory rhodopsin (31), Oxyrrhis marina proteorhodopsin (32), sensory rhodopsin I from H. salinarum (33) and Salinibacter ruber (34), and sensory rhodopsin II from Natronobacterium pharaonis (35, 36) and H. salinarum (37)].In ChR2, the retinal is covalently bound to the lysine residue 257 conserved in all retinal proteins through a Schiff base linkage (38). The X-ray structure of the ChR chimera shows the retinal in an all-trans configuration (9), although other conformations cannot be excluded at the obtained resolution. Results of retinal extraction in conjunction with resonance Raman studies were interpreted as an isomer mixture containing 30% of a 13-cis retinal in dark- and light-adapted ChR2 (20). In addition, nanosecond IR spectroscopy on the E123T mutant of ChR2 indicated the presence of some 13-cis retinal in the dark state using a similar spectroscopic assignment as in the resonance Raman study (39). In contrast to bacteriorhodopsin, no light adaption was observed using resonance Raman techniques (20) or visual spectroscopy (12). The occurrence of a conformer mixture in the ground state without light adaption would make ChR2 unique among the microbial retinal proteins, but additional data are needed to confirm these observations more directly at improved atomic resolution.The current model of the ChR2 photocycle is shown in Fig. 1B (14, 40). According to this model, blue light excitation leads to a retinal all-trans to 13-cis isomerization, resulting in a red-shifted first intermediate P1500 (12) resembling a K-like state, which most likely contains a 13-cis,15-anti retinal Schiff base chromophore similar to Bacteriorhodopsin (27). To our knowledge, such red-shifted K-like intermediates occur in all microbial retinal proteins (38). Schiff base deprotonation leads to the M-like state P2390 (10, 11). This state is followed by the red-shifted intermediate P3520, which has previously been correlated with the open state (10). However, later data confirmed that channel opening occurs before P3520 formation and might happen during a spectroscopically silent transition between P2a390 and P2b390 states (41). The last photocycle intermediate is the long-lived intermediate P4480 state (τ = 24 s), which is referred to as the desensitized state with a spectral characteristic similar to the ground state (11, 42). In addition, time-resolved FTIR spectroscopy indicated that P3520 could partially convert directly to the ground state (14).The situation becomes more complicated under continuous light illumination (40, 43). Under these conditions, a high transient current is observed first that is quickly reduced to a much lower steady-state current. After turning off the irradiation, the steady-state current decays biexponentially. This observation can only be explained by a branching of the photocycle. Two open states and two closed states are required to quantitatively describe the observed behavior under continuous light conditions. The two closed states are most likely the ground state and the desensitized state P4480 that accumulates under continuous illumination and is identical to the same intermediate from a single turnover (18). One of the open states is probably the open state P3520 observed in single-turnover experiments. However, little is known about the identity of the second open state, which only occurs under continuous light conditions. It might be an M-like P2390 state, another P3520 state, or another unknown state. Light excitation of probably P4480 creates this additional state. This state or group of states here is referred to as P_x containing at least one open state (Fig. 1B). It is also likely that the open states P3520 and P_x to some extent can convert directly to the ground state, which is indicated by dashed lines in Fig. 1B.All of the above-described states were detected by visible and FTIR spectroscopy, and assignments of spectroscopic signatures to conformational and configurational states of the retinal were based on analogous data previously studied. However, detailed information on bond lengths or torsion angles that would also link to quantum chemical calculation is still missing. To fill this gap between static crystallographic data on the one hand and kinetic and functional data based on optical spectroscopy and electrophysiology on the other hand, we applied solid-state magic angle spinning NMR on isotope-labeled ChR2 and retinal to obtain site-resolved structural data directly in a membrane environment under various experimental conditions. In this way, fine details of the chromophore conformation during the photocycle could be resolved, which will be important to understand the link between channel and photocycle activity in ChR2. A limitation using proteoliposomes is the amount of sample that can be studied, because the protein-to-lipid ratio cannot be increased too much without compromising protein integrity. In addition, trapping photointermediates works best using samples with low optical density, which reduces further the usable amount of protein, resulting in a poor NMR signal-to-noise ratio. Therefore, cross-effect dynamic nuclear polarization (DNP) enhanced magic angle spinning (MAS) NMR [review in the work by Maly et al. (44)] was indispensable in overcoming these sensitivity problems (Fig. 1C). This technique requires temperatures around 100 K that are also compatible with trapping of photointermediates as outlined below. DNP-enhanced MAS NMR is not yet a routine method but is applied increasingly to complex, mechanistic studies on retinal proteins (45–48) and other membrane proteins (49–51).Here, DNP-enhanced solid-state NMR spectroscopy has been applied to ¹⁵N-labeled ChR2 carrying 14,15-¹³C₂ retinal reconstituted into lipid bilayers and incubated with the DNP polarizing agent AMUPOL (52) in a glycerol–water mixture. The labeling scheme adopted here is shown in Fig. 2A. The ¹³C14 chemical shift is sensitive to the configuration of the C13-C14 bond. Together with the neighboring ¹³C15 atom, the two ¹³C-labeled spins can be used for double quantum filtering of this spin pair against the natural abundance background and at the same time, offer the possibility to study the length and the dihedral angle of the C14-C15 bond. Furthermore, the chemical shift of the Schiff base nitrogen is also sensitive to the chromophore conformation, reports on the protonation state of the Schiff base, and reflects counterion interactions. Using this approach, we were able to provide a first analysis, to our knowledge, of the retinal–Schiff base chromophore in ChR2 in its ground state as well as three different photointermediate states at atomic resolution.Open in a separate windowFig. 2.(A) DNP-enhanced MAS NMR has been applied to U-¹⁵N-ChR2 containing 14,15-¹³C all-trans–retinal. (B) A 62-fold signal enhancement is achieved for ¹³C cross-polarization (CP; CP vs. CP + DNP). The ¹³C natural abundance background can be efficiently suppressed by a double quantum filter (DQF; DQF + DNP), resulting in a spectrum with only the resonances of C14 and C15. As a control, one additional CP spectrum was acquired at 850 MHz close to room temperature (CP at RT). The gray bar indicates where the ¹³C14 signal would be expected for a chromophore in the 13-cis,15-syn conformation. (C) ¹³C14-¹³C15 double-quantum (DQ) build-up curve. (D) HCCH dephasing curves for the C14-C15 spin system in ChR2 during two rotor periods reporting on the HCCH dihedral angle.     

MYC family amplification and clinical risk-factors interact to predict an extremely poor prognosis in childhood medulloblastoma

The MYC oncogenes are the most commonly amplified loci in medulloblastoma, and have previously been proposed as biomarkers of adverse disease prognosis by us and others. Here, we report focussed and comprehensive investigations of MYCC, MYCN and MYCL in an extensive medulloblastoma cohort (n?=?292), aimed to define more precisely their biological significance and optimal clinical application to direct improved disease risk-stratification and individualisation of therapy. MYCC and MYCN expression elevations were multifactorial, associated with high-risk (gene amplification, large-cell/anaplastic pathology (LCA)) and favourable-risk (WNT/SHH molecular subgroups) disease features. Highly variable cellular gene amplification patterns underlay overall MYC copy number elevations observed in tumour biopsies; we used these alternative measures together to define quantitative methodologies and thresholds for amplification detection in routinely collected tumour material. MYCC and MYCN amplification, but not gain, each had independent prognostic significance in non-infants (≥3.0-16.0?years), but MYCC conferred a greater hazard to survival than MYCN when considered across this treatment group. MYCN's weaker group-wide survival relationship may be explained by its pleiotropic behaviour between clinical disease-risk groups; MYCN predicted poor prognosis in clinical high-risk (metastatic (M+) or LCA), but not standard-risk, patients. Extending these findings, survival decreased in proportion to the total number of independently significant high-risk features present (LCA, M+ or MYCC/MYCN amplification). This cumulative-risk model defines a patient group characterised by ≥2 independent risk-factors and an extremely poor prognosis (<15% survival), which can be identified straightforwardly using the reported MYC amplification detection methodologies alongside clinical assessments, enabling targeting for novel/intensified therapies in future clinical studies.     

Quinols as novel therapeutic agents. 2.(1) 4-(1-Arylsulfonylindol-2-yl)-4-hydroxycyclohexa-2,5-dien-1-ones and related agents as potent and selective antitumor agents

A series of substituted 4-(1-arylsulfonylindol-2-yl)-4-hydroxycyclohexa-2,5-dien-1-ones (indolylquinols) has been synthesized on the basis of the discovery of lead compound 1a and screened for antitumor activity. Synthesis of this novel series was accomplished via the "one-pot" addition of lithiated (arylsulfonyl)indoles to 4,4-dimethoxycyclohexa-2,5-dienone followed by deprotection under acidic conditions. Similar methodology gave rise to the related naphtho-, 1H-indole-, and benzimidazole-substituted quinols. A number of compounds in this new series were found to possess in vitro human tumor cell line activity substantially more potent than the recently reported antitumor 4-substituted 4-hydroxycyclohexa-2,5-dien-1-ones(1) with similar patterns of selectivity against colon, renal, and breast cell lines. The most potent compound in the series in vitro, 4-(1-benzenesulfonyl-6-fluoro-1H-indol-2-yl)-4-hydroxycyclohexa-2,5-dienone (1h), exhibits a mean GI(50) value of 16 nM and a mean LC(50) value of 2.24 muM in the NCI 60-cell-line screen, with LC(50) activity in the HCT 116 human colon cancer cell line below 10 nM. The crystal structure of the unsubstituted indolylquinol 1a exhibits two independent molecules, both participating in intermolecular hydrogen bonds from quinol OH to carbonyl O, but one OH group also interacts intramolecularly with a sulfonyl O atom. This interaction, which strengthens upon ab initio optimization, may influence the chemical environment of the bioactive quinol moiety. In vivo, significant antitumor activity was recorded (day 28) in mice bearing subcutaneously implanted MDA-MB-435 xenografts, following intraperitoneal treatment of mice with compound 1a at 50 mg/kg.     

Postmarketing evaluation of the safety and effectiveness of varicella vaccine

BACKGROUND: The Oka strain of live attenuated varicella virus was licensed for use in healthy children in the United States in March, 1995. We report a postmarketing evaluation of the short term safety of this vaccine within Kaiser Permanente. METHODS: After licensure varicella vaccination was introduced into the preventive care program of the Northern California Kaiser Permanente Medical Care Program. Potential adverse events after vaccination with varicella vaccine were identified from automated clinical databases of hospitalizations, emergency room visits and clinic visits. Deaths were identified from automated clinical databases at Kaiser as well as from the State death records for California. To evaluate safety, rates of diagnosis-specific events in the risk periods were compared with the rates of such diagnosis-specific events in two self control and one historical control period. RESULTS: During the study period of April 1, 1995, to December 31, 1996, a total of 89753 adults and children received varicella vaccine. A total of 3200 relative risks were calculated, and of these 5 hospital diagnostic categories, 9 emergency visit diagnostic categories and 30 outpatient diagnostic categories demonstrated at least 1 relative risk with a P value of <0.05 in 1 or more age groups and in comparisons with 1 control period or more. The p value for these tests was not adjusted for multiple comparisons. Of these categories 14 demonstrated an increased risk either in more than 1 age group or against more than 1 comparison group. These categories included elective procedures, febrile seizure, febrile illness, well child, acute gastroenteritis, varicella, congenital anomaly, "rule out sepsis," trauma, viral syndrome, apnea, back pain, congenital valvular heart disease and vision evaluation for glasses. Of these the outcomes of elective procedure, congenital anomaly, congenital valvular heart disease, well child and vision evaluation for glasses were judged not to have a biologically plausible association with vaccination. A second diagnostic grouping included febrile illness, viral illness, febrile seizure and "rule out sepsis." In an analysis of these events which adjusted for the concomitant administration of M-M-R(II) vaccine, none of the associations was statistically associated with receipt of varicella vaccine. The diagnostic category of "rule out sepsis" still had a relative risk of 1.95 with P = 0.02. None of the children in the "rule out sepsis" category had positive bacteriologic cultures from any other normally sterile site. Because of the large number of gastroenteritis cases, we reviewed a random sample of 100 exposed and 100 unexposed cases. From this review no consistent time association or clustering of any of these events was seen in the exposed follow-up time interval. Only gastroenteritis and negative evaluations for sepsis were thought to be possibly associated with receipt of varicella vaccine. Although there was a statistically significant increased risk over the entire 30 day-period, there was no clustering of these events within the 30-day window. CONCLUSION: In this study population of 89753 children and adults, the varicella vaccine (Oka strain, Merck) appeared to have a favorable safety profile. In addition rates of varicella-like rash and of breakthrough cases were both low and consistent with the rates observed in prelicensure studies.     

Health in south-eastern Europe: a troubled past, an uncertain future

The political and economic turmoil that occurred in south-eastern Europe in the last decade of the twentieth century left a legacy of physical damage. This aspect of the conflict has received considerable coverage in the media. However, surprisingly less has been reported about the effects of that turmoil on the health of the people living in the region. In an attempt to identify and synthesize data on these effects, we carried out a systematic review and used the results to put together a searchable online database of documents, reports, and published material, the majority of which have not previously been easily accessible (http:// www.lshtm.ac.uk/ecohost/see/index.php). The database covers the period from the early 1990s to 2003 and will be of considerable interest to policy-makers. It contains 762 items, many of them annotated and available for downloading. This paper synthesizes the main findings obtained from the material in the database and emphasizes the need for concerted action to improve the health of people in south-eastern Europe. Furthermore, we also recommend that agencies working in post-conflict situations should invest in developing and maintaining online databases that would be useful to others working in the area.     

Species-specific profiles of mycotoxins produced in cultures and associated with conidia of airborne fungi derived from biowaste

The potential to produce mycotoxins and non-volatile secondary metabolites was investigated for approximately 250 freshly isolated fungal strains. Among the eleven most relevant species, viz. Aspergillus flavus, A. fumigatus, A. niger, A. parasiticus, A. versicolor, Emericella nidulans, Paecilomyces variotii, Penicillium brevicompactum, P. clavigerum, P. crustosum, and P. polonicum, a wide range of metabolites partly of toxicological relevance was identified. Several unknown metabolites were found for the less frequent species, which were primarily investigated for chemotaxonomic delimitation from closely related species. The spectra of metabolites in conidial extracts and culture extracts (containing also mycelium and medium) were compared for a limited number of relevant fungi. Some mycotoxins, such as sterigmatocystin in Emericella nidulans, were not present in the conidial extracts, though produced by most strains. Fumigaclavine C, tryptoquivaline, and trypacidin, characteristic for A. fumigatus, were found in conidial extracts, but highly toxic compounds such as gliotoxin and fumitremorgens were not present. Finally, compounds such as cyclopenol, cyclopenin, and penitrem A being characteristic for certain penicillia, were found in conidial extracts and are therefore assumed to occur in native bioaerosols.     

Patho-anatomical features of so-called Ph1− chronic myeloid leukemia

Summary Chronic myeloid leukemia without the Philadelphia chromosome (Ph^1– CML) is described and distinguished from chronic myeloid leukemia with the Philadelphia chromosome (Ph¹⁺ CML) on the basis of clinical and autopsy findings of four cases. Ph^1– CML showed clinical, hematological, and patho-anatomical features which could be regarded as typical.Patho-anatomically Ph^1– CML differed from Ph¹⁺ CML in the variable maturation of the leukemic proliferation in the bone marrow and extramedullary infiltrates. Up to the terminal phase Ph^1– CML can be of an extremely mature cell type. However, it can also show myeloblastic transformation after an initially mature cell stage. Ph^1– CML infiltrates are found in tissues and organs which Ph¹⁺ CML usually does not infiltrate or only to a low degree until a blastie crisis.On the basis of its course and clinical and patho-anatomical features Ph^1– CML looks like an atypical chronic myeloid leukemia. However, it is better called an acute myeloid leukemia of the mature cell type.The authors are grateful to Mr. K.-H. Tedsen for his excellent technical assistance and to Mrs. M. Soehring for translation and secretarial help.     

Histopathology of N-methyl-N-nitrosourea-induced mesenchymal tumours of the rat urinary bladder.

The present study reports the induction, histopathology, immunocytochemistry, growth pattern and proliferative behaviour of mesenchymal tumours of the urinary bladder of rats induced by a single intravesical dose (5 mg/kg/body weight) of N-methyl-N-nitrosourea (MNU). In 14 of 283 female Wistar rats (incidence: 4.9%). 16 non-epithelial tumours had developed after an experimental period of 14 months. The most common histological type induced was of fibrohistiocytic origin (eight benign-appearing and three malignant fibrous histiocytomas). Furthermore, two pure histiocytomas (one benign and one malignant) and three capillary and cavernous haemangiomas were produced. Since no metastases occurred and no clear-cut distinction between a merely expansive and a truly invasive growth was possible, the diagnosis of malignancy was based on the degree of cellular atypia and the mitotic activity. The benign-appearing fibrous histiocytomas showed a mean mitotic index of 0.06% and the malignant fibrous histiocytomas of 0.34%. The mitotic activity of the malignant histiocytoma was threefold (0.78%) as high as in the benign-appearing histiocytoma (0.25%). There exist close morphological similarities between the induced mesenchymal tumours in the rat and those occurring in the human bladder. Although the spectrum of histological types of mesenchymal tumours seen in the rat bladder was narrower than that encountered in man, MNU seems suitable for further studying the histogenesis, histopathology and biology of experimentally induced non-epithelial bladder neoplasms to gain information for a better understanding of the pathogenesis of human disease.