We describe a strategy for experimentally-constraining computational simulations of intrinsically disordered proteins (IDPs), using α-synuclein, an IDP with a central role in Parkinson's disease pathology, as an example. Previously, data from single-molecule Förster Resonance Energy Transfer (FRET) experiments have been effectively utilized to generate experimentally constrained computational models of IDPs. However, the fluorophores required for single-molecule FRET experiments are not amenable to the study of short-range (<30 Å) interactions. Using ensemble FRET measurements allows one to acquire data from probes with multiple distance ranges, which can be used to constrain Monte Carlo simulations in PyRosetta. To appropriately employ ensemble FRET data as constraints, we optimized the shape and weight of constraining potentials to afford ensembles of structures that are consistent with experimental data. We also used this approach to examine the structure of α-synuclein in the presence of the compacting osmolyte trimethylamine-N-oxide. Despite significant compaction imparted by 2 M trimethylamine-N-oxide, the underlying ensemble of α-synuclein remains largely disordered and capable of aggregation, also in agreement with experimental data. These proof-of-concept experiments demonstrate that our modeling protocol enables one to efficiently generate experimentally constrained models of IDPs that incorporate atomic-scale detail, allowing one to study an IDP under a variety of conditions.
Background: Low back pain is a very common disease. Many patients with chronic low back pain (CLBP) have been treated by complementary and alternative medicine such as acupuncture (AT) treatment. A type of AT, thread embedding acupuncture (TEA), consists of a thread that can continually stimulate at the AT points and has mechanical and chemical effects. Although TEA was widely used in clinical practice, there was little evidence of its efficacy and safety for CLBP. Methods: This clinical trial was randomized, controlled, assessor-blinded, two-armed, parallel, and conducted in multiple centers. Four Korean medical institutions recruited 38 outpatients with CLBP. The participants were randomly allocated to a treatment group (TEA combined with AT) or a control group (only AT) in a 1:1 ratio. All participants received conventional AT twice a week for 8 weeks (16 sessions) at 15 AT points (GV3 and bilateral BL23, BL24, BL25, BL26, BL40, BL60, and EX-B5) and the treatment group participants additionally received TEA once a week for 8 weeks (8 sessions) on 10 AT points in the multifidus, spinal erector, and lumbar quadrate muscles. The primary outcome measure of this study was the change of visual analog scale (VAS) from baseline (0 week) to the end of intervention (8 weeks). Secondary outcome measures included clinically relevant improvement (minimal clinically important difference) and 3% to 50% decrease on VAS, disability level (Korean version of Roland and Morris disability questionnaire), quality of life (Korean version of European quality of life 5dimension), global assessment (patient global impression of change), economic analysis, credibility test, and safety assessment. Results: The treatment group showed a significant reduction in VAS scores when compared with the control group (–33.7 ± 25.1 vs –15.6 ± 17.0, P = .013). As for the secondary outcome measures, the treatment group showed significant difference in 50% decrease on VAS and patient global impression of change. There was no serious adverse event associated with TEA and AT. Conclusion: This clinical trial documents the efficacy and safety of TEA combined with AT for the management of CLBP.
Site-specific fluorescence probes can be used to measure distances within proteins when used as part of a Förster resonance energy transfer (FRET) pair. Here we report the synthesis of a coumarin maleimide (Mcm-Mal) that is fluorogenic upon reaction with cysteine. We demonstrate that cysteine, acridonylalanine (Acd) double mutant proteins can be produced by unnatural amino acid mutagenesis and reacted with Mcm-Mal to generate Mcm/Acd labeled proteins for FRET studies. The Mcm/Acd FRET pair is minimally-perturbing, easy to install, and well-suited to studying protein distances in the 15-40 Å range. Furthermore, Mcm/Acd labeling can be combined with tryptophan fluorescence in three color FRET to monitor multiple interactions in one experiment.
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