Step size of the rotary proton motor in single FoF1-ATP synthase from a thermoalkaliphilic bacterium by DCO-ALEX FRET

Thermophilic enzymes can operate at higher temperatures but show reduced activities at room temperature. They are in general more stable during preparation and, accordingly, are considered to be more rigid in structure. Crystallization is often easier compared to proteins from bacteria growing at ambient temperatures, especially for membrane proteins. The ATP-producing enzyme FoF1-ATP synthase from thermoalkaliphilic Caldalkalibacillus thermarum strain TA2.A1 is driven by a Fo motor consisting of a ring of 13 c-subunits. We applied a single-molecule F\"orster resonance energy transfer (FRET) approach using duty cycle-optimized alternating laser excitation (DCO-ALEX) to monitor the expected 13-stepped rotary Fo motor at work. New FRET transition histograms were developed to identify the smaller step sizes compared to the 10-stepped Fo motor of the Escherichia coli enzyme. Dwell time analysis revealed the temperature and the LDAO dependence of the Fo motor activity on the single molecule level. Back-and-forth stepping of the Fo motor occurs fast indicating a high flexibility in the membrane part of this thermophilic enzyme.Comment: 14 pages, 7 figure

Molecular quantum spin network controlled by a single qubit

Scalable quantum technologies will require an unprecedented combination of precision and complexity for designing stable structures of well-controllable quantum systems. It is a challenging task to find a suitable elementary building block, of which a quantum network can be comprised in a scalable way. Here we present the working principle of such a basic unit, engineered using molecular chemistry, whose control and readout are executed using a nitrogen vacancy (NV) center in diamond. The basic unit we investigate is a synthetic polyproline with electron spins localized on attached molecular sidegroups separated by a few nanometers. We demonstrate the readout and coherent manipulation of very few ($\leq 6$) of these $S=1/2$ electronic spin systems and access their direct dipolar coupling tensor. Our results show, that it is feasible to use spin-labeled peptides as a resource for a molecular-qubit based network, while at the same time providing simple optical readout of single quantum states through NV-magnetometry. This work lays the foundation for building arbitrary quantum networks using well-established chemistry methods, which has many applications ranging from mapping distances in single molecules to quantum information processing.Comment: Author name typ

Diverse functions of Pmr1

Freies cytosolisches Ca2+ erfüllt in Eukaryonten für viele zelluläre Prozesse die Funktion eines "second messengers" und unterliegt daher einer exakten Kontrolle, um die normale Physiologie der Zelle aufrecht zu erhalten. Auf einen raschen Anstieg der cytosolischen Ca2+- Konzentration erfolgt ein aktives Entfernen des Ca2+ aus dem Cytosol. In der Hefe Saccharomyces cerevisiae geschieht dies durch die Ca2+- ATPasen Pmc1 und Pmr1, sowie durch den Ca2+/H+ Antiporter Vcx1. Der Weg auf dem überschüssiges Ca2+ wieder aus der Zelle ausgeschieden werden kann, ist bisher unbekannt. Eine Plasmamembran Ca2+- ATPase, wie von Säugerzellen bekannt, scheint in der Hefe Saccharomyces cerevisiae nicht zu existieren. Da Ca2+ in allen Subkompartimenten des Sekretionswegs eine wichtige Rolle spielt und zu dem dort im Vergleich zum Cytosol in wesentlich erhöhten Konzentrationen nachgewiesen wurde, liegt es nahe zu vermuten, dass Ca2+- verpackt in sekretorische Vesikel - die Zelle durch Exocytose verlassen könnte. Diese Hypothese wurde an Hand von Ca2+- Ausstrommessungen an verschiedenen Hefestämmen untersucht. Anhand von Stämmen, die aufgrund einer sec- Mutation ein temperatursensitives Sekretionsverhalten aufweisen, konnte gezeigt werden, dass Ca2+ , verpackt in sekretorische Vesikel, entlang des Sekretionswegs zur Plasmamembran transportiert und bei der Fusion der post- Golgi Vesikel mit der Plasmamembran aus der Hefezelle freigesetzt wird. Der Ca2+- Ausstrom erfolgt also über Exocytose. In einem weiteren Projekt wurde der Aminosäuretransport durch die Aminosäurepermease Gap1 in delta- pmr1- Zellen untersucht. Kinetische Transportmessungen mit radioaktiv markierten Aminosäuren ergaben, dass die Transportaktivität von Gap1 in delta- pmr1- Zellen drastisch reduziert ist und dass vermutlich eine Form von unkompetitiver Inhibierung von Gap1 in delta- pmr1-Zellen vorliegt. In einem dritten Projekt wurde nach genetischen Interaktionspartnern von Pmr1 gesucht. Dabei wurde festgestellt, dass delta- pmr1 delta- bst1, delta- pmr1 delta- sac2, delta- pmr1 delta- vps24 Doppelmutanten auf YPD- Platten einen starken Wachstumsdefekt aufweisen.The cytosolic free Ca2+- concentration of the yeast Saccharomyces cerevisiae is maintained at extremely low levels by the activity of the P- Type Ca2+- ATPases Pmc1 and Pmr1, and the antiport Vcx1. This is necessary because a transient increase in the cytosolic Ca2+ concentration regulates a wide variety of cellular processes. Animal cells exert tight control of free Ca2+ in the cytosol by balancing Ca2+ sequestration into internal stores with Ca2+ extrusion through well- studied Ca2+ pumps located at the plasma membrane. However a plasma membrane Ca2+ pump seems not to be existing in Saccharomyces cerevisiae. Very high Ca2+ concentrations are found in the organelles of the secretory pathway: therefore, it seems possible that Ca2+ is transported along the secretory pathway wrapped into secretory vesicles, which fuse with the plasma membrane to free their content by exocytosis. Here, we studied Ca2+ efflux out of different sec mutant strains at permessive and restrictive temperature. The result of the experiments show that the Ca2+ is extruded out of the cell by exocytosis. In a second project the amino acid transport by the general amino acid permease Gap1 was measured in delta- pmr1- cells. The transport activity of Gap1 in delta- pmr1 cells is drastically reduced, maybe by some sort of uncompetitive inhibition

Nanoscale nuclear magnetic resonance with chemical resolution

Nuclear magnetic resonance (NMR) spectroscopy is a key analytical technique in chemistry, biology and medicine. However, conventional NMR spectroscopy requires at least nanoliter sized sample volume to achieve sufficient signal. Here we combine the use of a quantum memory and high magnetic fields with a dedicated quantum sensor based on nitrogen vacancy centers in diamond to achieve chemical shift resolution in 1H and 19F NMR spectroscopy of 20 zeptoliter sample volumes. We demonstrate the application of NMR pulse sequences to achieve homonuclear decoupling and spin diffusion measurements. The best measured NMR linewidth of a liquid sample was ∼1 part per million, mainly limited by molecular diffusion. To mitigate the influence of diffusion we performed high resolution solid state NMR by applying homonuclear decoupling and achieved a 20-fold narrowing of the NMR linewidth

Relaxometry and Dephasing Imaging of Superparamagnetic Magnetite Nanoparticles Using a Single Qubit

To study the magnetic dynamics of superparamagnetic nanoparticles, we use scanning probe relaxometry and dephasing of the nitrogen vacancy (NV) center in diamond, characterizing the spin noise of a single 10 nm magnetite particle. Additionally, we show the anisotropy of the NV sensitivity’s dependence on the applied decoherence measurement method. By comparing the change in relaxation (<i>T</i>₁) and dephasing (<i>T</i>₂) time in the NV center when scanning a nanoparticle over it, we are able to extract the nanoparticle’s diameter and distance from the NV center using an Ornstein–Uhlenbeck model for the nanoparticle’s fluctuations. This scanning probe technique can be used in the future to characterize different spin label substitutes for both medical applications and basic magnetic nanoparticle behavior

Study protocol: the ear-nose-throat (ENT) prospective international cohort of patients with primary ciliary dyskinesia (EPIC-PCD).

INTRODUCTION Primary ciliary dyskinesia (PCD) is a rare, genetic, multiorgan disease with an estimated prevalence of 1 in 10 000. It affects mainly the upper and lower airways due to impaired mucociliary clearance. Almost all patients have sinonasal or otologic (ear-nose-throat, ENT) problems, although the ENT clinical phenotype may present great variability. Despite that, data on PCD ENT manifestations are scarce and based on small single-centre studies. To date, we know little about the spectrum and severity of PCD ENT disease, its association with lung disease, its course over life and its determinants of prognosis.This study protocol describes the aims and methods of the first prospective, observational, multinational cohort study focusing on ENT disease in patients with PCD. METHODS AND ANALYSIS The ENT prospective international cohort of patients with PCD (EPIC-PCD) is a prospective standardised observational clinical cohort set up as a multinational multicentre study, embedded into routine patient care. It aims to longitudinally characterise ENT disease in patients with PCD and its association with lung disease, and to identify determinants of its prognosis. Patients of all ages, diagnosed with PCD who undergo an ENT clinical assessment at least once a year at one of the participating centres will be invited to participate. Collected data include diagnostic test results, results of ENT examinations, lung function measurements, information on management of ENT disease and patient-reported data on clinical symptoms and health-related quality of life (QoL). Data are collected using the standardised PCD-specific FOLLOW-PCD form and the validated QoL-PCD questionnaire. ETHICS AND DISSEMINATION The study has been reviewed and approved by the Human Research Ethics Committees at all participating centres, based on local legislation. The results of the study will be published in scientific journals, presented at scientific conferences and disseminated to participants and national patient organisations. TRIAL REGISTRATION NCT04611516