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The study of how the intracellular medium influences protein structural dynamics and protein-protein interactions is a captivating area of research for scientists aiming to comprehend biomolecules in their native environment. As the cellular environment can hardly be reproduced in vitro , direct investigation of biomolecules within cells has attracted growing interest in the past two decades. Among magnetic resonances, site-directed spin labeling coupled to electron paramagnetic resonance spectroscopy (SDSL-EPR) has emerged as a powerful tool for studying the structural properties of biomolecules directly in cells. Since the first in-cell EPR experiment was reported in 2010, substantial progress has been made, and this Review provides a detailed overview of the developments and applications of this spectroscopic technique. The strategies available for preparing a cellular sample and the EPR methods that can be applied to cells will be discussed. The array of spin labels available, along with their strengths and weaknesses in cellular contexts, will also be described. Several examples will illustrate how in-cell EPR can be applied to different biological systems and how the cellular environment affects the structural and dynamic properties of different proteins. Lastly, the Review will focus on the future developments expected to expand the capabilities of this promising technique.

The combined effects of the cellular environment on proteins led to the definition of a fifth level of protein structural organization termed quinary structure. To explore the implication of potential quinary structure for globular proteins, we studied the dynamics and conformations of Escherichia coli (E. coli) peptidyl-prolyl cis/trans isomerase B (PpiB) in E. coli cells. PpiB plays a major role in maturation and regulation of folded proteins by catalyzing the cis/trans isomerization of the proline imidic peptide bond. We applied electron paramagnetic resonance (EPR) techniques, utilizing both Gadolinium (Gd(III)) and nitroxide spin labels. In addition to using standard spin labeling approaches with genetically engineered cysteines, we incorporated an unnatural amino acid to achieve Gd(III)-nitroxide orthogonal labeling. We probed PpiB's residue-specific dynamics by X-band continuous wave EPR at ambient temperatures and its structure by double electron-electron resonance (DEER) on frozen samples. PpiB was delivered to E. coli cells by electroporation. We report a significant decrease in the dynamics induced by the cellular environment for two chosen labeling positions. These changes could not be reproduced by adding crowding agents and cell extracts. Concomitantly, we report a broadening of the distance distribution in E. coli, determined by Gd(III)-Gd(III) DEER measurements, as compared with solution and human HeLa cells. This suggests an increase in the number of PpiB conformations present in E. coli cells, possibly due to interactions with other cell components, which also contributes to the reduction in mobility and suggests the presence of a quinary structure., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

UreG is a cytosolic GTPase involved in the maturation network of urease, an Ni-containing bacterial enzyme. Previous investigations in vitro showed that UreG features a flexible tertiary organization, making this protein the first enzyme discovered to be intrinsically disordered. To determine whether this heterogeneous behavior is maintained in the protein natural environment, UreG structural dynamics was investigated directly in intact bacteria by in-cell EPR. This approach, based on site-directed spin labeling coupled to electron paramagnetic resonance (SDSL-EPR) spectroscopy, enables the study of proteins in their native environment. The results show that UreG maintains heterogeneous structural landscape in-cell , existing in a conformational ensemble of two major conformers, showing either random coil-like or compact properties. These data support the physiological relevance of the intrinsically disordered nature of UreG and indicates a role of protein flexibility for this specific enzyme, possibly related to the regulation of promiscuous protein interactions for metal ion delivery., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Purpose: Surgical treatment of primary lung T4 tumors is controversial especially when the cancer invades the mediastinal structures or the descending thoracic aorta. Conventional surgical treatment is associated with a high perioperative mortality and morbidity rate. Thoracic EndoVascular Aortic Repair has emerged as a valid off-label alternative to conventional surgery. We aimed to assess perioperative and midterm aortic-related outcome of patients who have undergone aortic stent-graft implantation, followed by en bloc surgical treatment of the involved aorta and lung cancer resection., Materials and Methods: From July 2017 to May 2020, we treated 5 patients diagnosed with a T4 lung cancer by the involvement of the descending thoracic aorta. When only the descending thoracic aorta is involved, a 2-stage procedure was considered, with aortic stent-graft implantation performed before tumor resection. One-stage strategy, with stent-graft implantation carried out before thoracotomy, was preferred for patients with the involvement of cardiac and/or other vascular mediastinal structures., Results: The mean age was 58.4 ± 6.2 years. All patients were affected by non-small cell lung cancer. All 5 patients required a single stent-graft to completely cover the involved segment of aorta. Four patients underwent a 2-stage procedure. One patient, with the involvement of the left inferior pulmonary vein, required a 1-stage en bloc resection of the left lower lobe, aortic wall adventitia, left inferior pulmonary vein, and reconstruction of the left atrial wall. Primary procedural success was achieved in all. At follow-up, no patient developed aortic-related complications. One patient died 2 years after surgery, due to local recurrence of the tumor., Conclusion: T4 lung resection combined with aortic stent-graft implantation can be safely performed. Endovascular surgery, by avoiding the use of cardiopulmonary bypass, aortic cross-clamping, and graft replacement, can reduce significant morbidity and mortality rate. Postoperative and long-term outcome of these patients treated with endovascular surgery is mainly related to pulmonary disease, not to aortic treatment.

Site-directed spin labeling (SDSL) combined with continuous wave electron paramagnetic resonance (cw EPR) spectroscopy is a powerful technique to reveal, at the local level, the dynamics of structural transitions in proteins. Here, we consider SDSL-EPR based on the selective grafting of a nitroxide on the protein under study, followed by X-band cw EPR analysis. To extract valuable quantitative information from SDSL-EPR spectra and thus give a reliable interpretation on biological system dynamics, a numerical simulation of the spectra is required. However, regardless of the numerical tool chosen to perform such simulations, the number of parameters is often too high to provide unambiguous results. In this study, we have chosen SimLabel to perform such simulations. SimLabel is a graphical user interface (GUI) of Matlab , using some functions of Easyspin . An exhaustive review of the parameters used in this GUI has enabled to define the adjustable parameters during the simulation fitting and to fix the others prior to the simulation fitting. Among them, some are set once and for all (g y , g z ) and others are determined (A z , g x ) thanks to a supplementary X-band spectrum recorded on a frozen solution. Finally, we propose guidelines to perform the simulation of X-band cw-EPR spectra of nitroxide labeled proteins at room temperature, with no need of uncommon higher frequency spectrometry and with the minimal number of variable parameters.

One of the greatest current challenges in structural biology is to study protein dynamics over a wide range of timescales in complex environments, such as the cell. Among magnetic resonances suitable for this approach, electron paramagnetic resonance spectroscopy coupled to site-directed spin labeling (SDSL-EPR) has emerged as a promising tool to study protein local dynamics and conformational ensembles. In this work, we exploit the sensitivity of nitroxide labels to report protein local dynamics at room temperature. We demonstrate that such studies can be performed while preserving both the integrity of the cells and the activity of the protein under investigation. Using this approach, we studied the structural dynamics of the chaperone NarJ in its natural host, Escherichia coli. We established that spin-labeled NarJ is active inside the cell. We showed that the cellular medium affects NarJ structural dynamics in a site-specific way, while the structural flexibility of the protein is maintained. Finally, we present and discuss data on the time-resolved dynamics of NarJ in cellular context., (© 2022 Wiley-VCH GmbH.)

A 70-year-old male patient was admitted to the emergency room in cardiac arrest. The patient was resuscitated and then referred to our cardiac surgery department, where he was diagnosed with suspected effusive constrictive pericarditis. A failed trial of TEE-guided pericardiocentesis led to the decision of surgical intervention. Sternotomy was performed and revealed pericardial thickening and very dense adhesions involving the pericardium and both pleurae, suggesting a neoplastic disease. An extensive pericardiectomy and bilateral pleural decortication were performed. After surgery, the patient improved significantly and was discharged from the intensive care unit 24 h later. Pericardial thickening, dense adhesions, the amount and color of pericardial fluid and the aspect of epicardial tissue increased our suspicion of neoplastic disease. Histological samples were sent to be analyzed immediately; a few days later, they were unexpectedly negative for any neoplastic disease but showed a group-B-hemolytic Streptococcus agalactiae infection, which causes pericarditis in extremely rare cases. Postoperatively, the patient, under intravenous antibiotic and anti-inflammatory therapy, remained asymptomatic and was discharged ten days after the surgery. At the three-month follow-up, transthoracic echocardiography showed a normal right and left ventricular function with no pericardial effusion.