smFRET and DEER Distance Measurements as Applied to Disordered and Structured Proteins
| Autor: | Ruoyi Qiu, Erkang Ou, Tatyana I. Smirnova, Keith Weninger, Hugo Sanabria, Sergey Milikisiyants |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
Synaptobrevin
Chemistry 010401 analytical chemistry Biophysics 02 engineering and technology 021001 nanoscience & nanotechnology 01 natural sciences Fluorescence Acceptor 0104 chemical sciences law.invention Crystallography Protein structure Förster resonance energy transfer law Molecule 0210 nano-technology SNARE complex Electron paramagnetic resonance |
| Zdroj: | Biophysical Journal. 110(3) |
| ISSN: | 0006-3495 |
| DOI: | 10.1016/j.bpj.2015.11.2987 |
| Popis: | FRET and DEER are two spectroscopic methods that are widely applied for biophysical studies of protein structure. Both methods are based on measuring dipolar interactions - electrical dipoles in case of FRET and magnetic dipoles in case of DEER - between specifically labeled protein sites. The experimental data are then analyzed to derive the distance between the interacting dipoles and relate this distance to the structure of biomacromolecule(s). Molecular volume of EPR labels is generally smaller vs. that of the fluorescent probes and DEER experiments can be carried out by labeling cysteines with identical molecular tags whereas FRET typically relies on orthogonal labeling with distinct donor and acceptor fluorophores. Another essential difference is that FRET can be performed under physiological conditions, but DEER typically requires cryogenic or near cryogenic temperatures because of short phase memory time for nitroxides at ambient conditions. Finally, single molecule (sm) FRET reports on conformation of individual protein molecules whereas DEER provides information on ensemble average. While the distance ranges of these two methods overlaps the direct comparison of FRET and DEER data is rarely found in the literature. Here we report on the distance measurements and conformational states using both smFRET and DEER on three protein systems. We attached probes to a unique pair of cysteines in the neuronal SNARE protein SNAP-25. SNAP-25 is highly disordered in isolation, but it folds into a stable alpha-helix bundle upon forming SNARE complex with syntaxin and synaptobrevin. We also labeled intrinsically disordered Glutamate Receptor Cytoplasmic Domain N2B. Results of smFRET and DEER distances and distance distribution are compared for disordered SNAP-25 and folded SNAP-25 within the SNARE complex and for disordered N2B. |
| Databáze: | OpenAIRE |
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