The two-photon reversible reaction of the bistable jumping spider rhodopsin-1

Bistable opsins are photopigments expressed in both invertebrates and vertebrates. These light-sensitive G-protein-coupled receptors undergo a reversible reaction upon illumination. A first photon initiates the cis to trans isomerization of the retinal chromophore—attached to the protein through a protonated Schiff base—and a series of transition states that eventually results in the formation of the thermally stable and active Meta state. Excitation by a second photon reverts this process to recover the original ground state. On the other hand, monostable opsins (e.g., bovine rhodopsin) lose their chromophore during the decay of the Meta II state (i.e., they bleach). Spectroscopic studies on the molecular details of the two-photon cycle in bistable opsins are limited. Here, we describe the successful expression and purification of recombinant rhodopsin-1 from the jumping spider Hasarius adansoni (JSR1). In its natural configuration, spectroscopic characterization of JSR1 is hampered by the similar absorption spectra in the visible spectrum of the inactive and active states. We solved this issue by separating their absorption spectra by replacing the endogenous 11-cis retinal chromophore with the blue-shifted 9-cis JSiR1. With this system, we used time-resolved ultraviolet-visible spectroscopy after pulsed laser excitation to obtain kinetic details of the rise and decay of the photocycle intermediates. We also used resonance Raman spectroscopy to elucidate structural changes of the retinal chromophore upon illumination. Our data clearly indicate that the protonated Schiff base is stable throughout the entire photoreaction. We additionally show that the accompanying conformational changes in the protein are different from those of monostable rhodopsin, as recorded by light-induced FTIR difference spectroscopy. Thus, we envisage JSR1 as becoming a model system for future studies on the reaction mechanisms of bistable opsins, e.g., by time-resolved x-ray crystallography

The structure of the core NuRD repression complex provides insights into its interaction with chromatin

The NuRD complex is a multi-protein transcriptional corepressor that couples histone deacetylase and ATP-dependent chromatin remodelling activities. The complex regulates the higher-order structure of chromatin, and has important roles in the regulation of gene expression, DNA damage repair and cell differentiation. HDACs 1 and 2 are recruited by the MTA1 corepressor to form the catalytic core of the complex. The histone chaperone protein RBBP4, has previously been shown to bind to the carboxy-terminal tail of MTA1. We show that MTA1 recruits a second copy of RBBP4. The crystal structure reveals an extensive interface between MTA1 and RBBP4. An EM structure, supported by SAXS and crosslinking, reveals the architecture of the dimeric HDAC1:MTA1:RBBP4 assembly which forms the core of the NuRD complex. We find evidence that in this complex RBBP4 mediates interaction with histone H3 tails, but not histone H4, suggesting a mechanism for recruitment of the NuRD complex to chromati

Ultrafast structural changes direct the first molecular events of vision

視覚に関わるタンパク質の超高速分子動画 --薄暗いところで光を感じる仕組み--. 京都大学プレスリリース. 2023-03-23.Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs). A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation

Role of HRCT in detection and characterization of pulmonary abnormalities in patients with febrile neutropenia

Background: Fever is of grave concern in the management of patients with neutropenia with early detection of a focus of infection being the major goal. As lungs are the most common focus, chest imaging is of vital importance. This Institute Review Board approved prospective study was undertaken to assess the usefulness of high resolution computed tomography (HRCT) in early detection and characterization of pulmonary abnormalities in febrile neutropenia. Materials and Methods: A total of 104 consecutive patients (M:F:75:29, age range 11-66 years) with fever of 38.2°C or more with an absolute neutrophil count 10 mm with or without surrounding GGO or cavitations was sensitive (95.23%) and specific (96.7%) for fungal infection, while small (1-4 mm) random or centrilobular nodules with tree-in-bud appearance was sensitive (90%) and highly specific (97.02%) for tuberculosis. Diagnosis of pyogenic infection based on presence of air-space consolidation, pleural effusion, GGO or centrilobular nodules showed a sensitivity of 84.78% and specificity of 93.84%, whereas patchy or diffuse GGO, interstitial thickening and/or air-space consolidation showed high sensitivity (86.7%) and specificity (96.8%) for Pneumocystis jiroveci pneumonia. Conclusion: HRCT chest is an excellent modality in the diagnostic work-up of patients with febrile neutropenia allowing early detection and characterization of pulmonary abnormalities

Outcomes of Management of Patients with Acute Mesenteric Ischemia: A Prospective Study

Introduction: Acute mesenteric ischemia (AMI) is the sudden onset of small intestinal hypoperfusion, which can be due to reduction or cessation of arterial inflow. It can be embolic or thrombotic. AMI is a potentially fatal vascular emergency, with overall mortality of 60%–80%. The present study was designed to study presentation, risk factors, and various outcomes of AMI patients, who were treated in the tertiary hospital. Methodology: All patients with features suggestive of AMI were taken in study. All patients underwent explorative laparotomy with exteriorization of bowel after initial resuscitation. Postoperatively, refeeding enteroclysis was done. Observation and Results: The total mortality rate was 62.50%. In patients with functional bowel length of 1 foot, 90.90% patients died. In patients with functional bowel length of 5–6 feet, only 12.50% died. It was observed that the mortality rate was more in the age group of 50–59 years with functional bowel length of 1 foot or <1 foot. Refeeding enteroclysis is an effective technique to use distal bowel. Contrast-enhanced computed tomography of the abdomen more suggestive of this pathology. Conclusions: A strong clinical suspicion and an aggressive approach should be considered in dealing with this condition because the outcome mainly depends on rapid diagnosis and treatment. With understanding of the pathogenesis of AMI and the better utilization of available radiological investigations, an improved outcome can be achieved. Mortality rates with refeeding enteroclysis are comparable with that of bowel anastomosis. This condition needs future research

Crystal structure of jumping spider rhodopsin-1 as a light sensitive GPCR

Light-sensitive G protein-coupled receptors (GPCRs)-rhodopsins-absorb photons to isomerize their covalently bound retinal, triggering conformational changes that result in downstream signaling cascades. Monostable rhodopsins release retinal upon isomerization as opposed to the retinal in bistable rhodopsins that "reisomerize" upon absorption of a second photon. Understanding the mechanistic differences between these light-sensitive GPCRs has been hindered by the scarcity of recombinant models of the latter. Here, we reveal the high-resolution crystal structure of a recombinant bistable rhodopsin, jumping spider rhodopsin-1, bound to the inverse agonist 9-cis retinal. We observe a water-mediated network around the ligand hinting toward the basis of their bistable nature. In contrast to bovine rhodopsin (monostable), the transmembrane bundle of jumping spider rhodopsin-1 as well that of the bistable squid rhodopsin adopts a more "activation-ready" conformation often observed in other nonphotosensitive class A GPCRs. These similarities suggest the role of jumping spider rhodopsin-1 as a potential model system in the study of the structure-function relationship of both photosensitive and nonphotosensitive class A GPCRs.ISSN:0027-8424ISSN:1091-649