Gangbildveränderungen im Rahmen von Dual-Tasking bei Parkinson-Patienten und Gesunden

Das idiopathische Parkinson-Syndrom (IPS) ist eine chronische progrediente Erkrankung, dessen Prävalenz mit zunehmendem Lebensalter steigt und dass daher in unserer älter werdenden Gesellschaft eine zunehmende Bedeutung bekommt. Gesundheitspolitisch spielt dabei die Morbidität aufgrund von Stürzen im Rahmen der Gangbildveränderungen eine zentrale Rolle. Es wurden für diese Arbeit die Daten von zwei Patientenkollektiven mit unterschiedlicher Krankheitsdauer und eine Kontrollgruppe ausgewertet. Dabei wurden mit Hilfe eines tragbaren Sensors die Hauptkomponenten des Gehens, sowie die unter Dual Task Bedingungen auftretenden Gangbildveränderungen untersucht. Bei diesen Dual Task Untersuchungen mussten die Probanden neben dem Gehen jeweils eine zusätzliche motorisch und eine kognitiv betonte Aufgabe ausführen (non-walking Task). Bei den Dual Task Untersuchungen scheint der non-walking Task mit motorischer Komponente (in dieser Arbeit Kreuze auf ein Blatt Papier setzten) gegenüber einem parallel durchgeführten rein kognitiven Test (Subtrahieren in 7er Schritten) die sensitivere Methode zu sein, um Leistungsverluste bei den IPS-Patienten zu erfassen. Die Kreuzgeschwindigkeit war signifikant unterschiedlich zwischen IPS und Kontrollen, nicht jedoch die Subtraktionsgeschwindigkeit. Bei der Berechnung der Dual Task Kosten, die anzeigen in welchem Ausmaß die Ausführung einer zuvor einzeln erfüllten Aufgabe an Qualität abgenommen hat, wenn noch eine zusätzliche Aufgabe bearbeitet werden muss, zeigten sich bei den Gang-Parametern, entgegen unserer ursprünglichen Annahme, weder beim gleichzeitigen Kreuze setzen noch beim gleichzeitigen Subtrahieren signifikante Unterschiede zwischen IPS-Patienten und Kontrollen. In unseren Untersuchungen wurden die Abstriche beim Dual Tasking bei der Ausführung des non-walking Tasks gemacht, was bedeuten würde, dass IPS-Patienten im Sinne einer „posture first“ Strategie sinnvollerweise der Stabilisierung des Gangbildes den Vorzug geben

Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET

Pulsed electron-electron double resonance spectroscopy (PELDOR/DEER) and single-molecule Förster resonance energy transfer spectroscopy (smFRET) are frequently used to determine conformational changes, structural heterogeneity, and inter probe distances in biological macromolecules. They provide qualitative information that facilitates mechanistic understanding of biochemical processes and quantitative data for structural modelling. To provide a comprehensive comparison of the accuracy of PELDOR/DEER and smFRET, we use a library of double cysteine variants of four proteins that undergo large-scale conformational changes upon ligand binding. With either method, we use established standard experimental protocols and data analysis routines to determine inter-probe distances in the presence and absence of ligands. The results are compared to distance predictions from structural models. Despite an overall satisfying and similar distance accuracy, some inconsistencies are identified, which we attribute to the use of cryoprotectants for PELDOR/DEER and label-protein interactions for smFRET. This large-scale cross-validation of PELDOR/DEER and smFRET highlights the strengths, weaknesses, and synergies of these two important and complementary tools in integrative structural biology

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology

An integrated transport mechanism of the maltose ABC importer

ATP-binding cassette (ABC) transporters use the energy of ATP hydrolysis to transport a large diversity of molecules actively across biological membranes. A combination of biochemical, biophysical, and structural studies has established the maltose transporter MalFGK2 as one of the best characterized proteins of the ABC family. MalF and MalG are the transmembrane domains, and two MalKs form a homodimer of nucleotide-binding domains. A periplasmic maltose-binding protein (MalE) delivers maltose and other maltodextrins to the transporter, and triggers its ATPase activity. Substrate import occurs in a unidirectional manner by ATP-driven conformational changes in MalK2 that allow alternating access of the substrate-binding site in MalF to each side of the membrane. In this review, we present an integrated molecular mechanism of the transport process considering all currently available information. Furthermore, we summarize remaining inconsistencies and outline possible future routes to decipher the full mechanistic details of transport by MalEFGK2 complex and that of related importer systems

Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET

Pulsed electron-electron double resonance spectroscopy (PELDOR/DEER) and single-molecule Förster resonance energy transfer spectroscopy (smFRET) are frequently used to determine conformational changes, structural heterogeneity and inter probe distances in biological macromolecules. They provide qualitative information that facilitates mechanistic understanding of biochemical processes and quantitative data for structural modeling. To provide a comprehensive comparison of the accuracy of PELDOR/DEER and smFRET, we use a library of double cysteine variants of four proteins that undergo large-scale conformational changes upon ligand binding. With either method, we use established standard experimental protocols and data analysis routines to determine inter-probe distances in the presence and absence of ligands. The results are compared to distance predictions from structural models. Despite an overall satisfying and similar distance accuracy, some inconsistencies are identified, which we attribute to the use of cryoprotectants for PELDOR/DEER and label-protein interactions for smFRET. This large-scale cross-validation of PELDOR/DEER and smFRET highlights the strengths, weaknesses, and synergies of these two important and complementary tools in integrative structural biology.Paper is available as a pre-print: https://doi.org/10.1101/2020.11.23.394080 and will be published in Nature Communications under manuscript number NCOMMS-20-47944C