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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in α-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

Abstract

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural dynamics and its contributions to biology. While the FRET-based ruler reports on inter-dye distances in the 3-10 nm range, other spectroscopic techniques, such as protein-induced fluorescence enhancement (PIFE), report on the proximity between a dye and a protein surface in the shorter 0-3 nm range. Regardless of the method of choice, its use in measuring freely-diffusing biomolecules one at a time retrieves histograms of the experimental parameter yielding separate centrally-distributed sub-populations of biomolecules, where each sub-population represents either a single conformation that stayed unchanged within milliseconds, or multiple conformations that interconvert much faster than milliseconds, and hence an averaged-out sub-population. In single-molecule FRET, where the reported parameter in histograms is the inter-dye FRET efficiency, an intrinsically disordered protein, such as the α-Synuclein monomer in buffer, was previously reported as exhibiting a single averaged-out sub-population of multiple conformations interconverting rapidly. While these past findings depend on the 3-10 nm range of the FRET-based ruler, we sought to put this protein to the test using single-molecule PIFE, where we track the fluorescence lifetime of site-specific sCy3-labeled α-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled α-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while α-Synuclein might be disordered globally, it nonetheless attains stable local structures. In summary, in this work we highlight the advantage of using different spectroscopic proximity sensors that track local or global structural changes one biomolecule at a time.

Introduction

Over the past two decades, single-molecule fluorescence-based methods have become a powerful tool for measuring biomolecules1,2, probing how different biomolecular parameters distribute as well as how they dynamically interconvert between different sub-populations of these parameters at sub-millisecond resolution3,4,5. The parameters in these techniques include the energy transfer efficiency in FRET measurements 6,7, fluorescence anisotropy8<....

Protocol

1. Plasmid transformation

  1. Preparation of 0.5 L of SOC medium 
    1. Weigh 10 g of tryptone, 2.5 g of Yeast extract, 0.25 g of sodium chloride (NaCl), 0.1 g of potassium chloride (KCl).
    2. Add double-distilled water (DDW) until total volume of 0.5 L.
    3. Adjust to pH 7 by adding sodium hydroxide (NaOH).
    4. Prepare stock aliquots of 100 mL and autoclave.
    5. Before using, add 0.5 mL of sterile magnesium chloride (MgCl2) and 1.8 mL of sterile glucose to 100 mL .......

Representative Results

As an IDP, when it is not bound to another biomolecule, α-Syn exhibits structural dynamics between multiple conformations, with transitions at few microseconds40 and even at hundreds of nanoseconds41. When α-Syn crosses the confocal spot, it may undergo thousands of transitions between conformations. Indeed, this was the case when smFRET was used39,40. Here we perform smPIFE measurements in order to p.......

Discussion

Extensive biochemical and biophysical studies were performed to study the structural characteristics of α-Syn and its disordered nature33,34,35,36,37,38. Several works have already utilized freely-diffusing smFRET to investigate the intra-molecular dynamics of the α-Syn monomer free of binding. These works reported .......

Acknowledgements

The pT-t7 plasmid encoding A56C α-Syn mutant was given to us as a present from Dr. Asaf Grupi, Dr. Dan Amir and Dr. Elisha Haas. This paper was supported by the National Institutes of Health (NIH, grant R01 GM130942 to E.L. as a subaward), the Israel Science Foundation (grant 3565/20 within the KillCorona - Curbing Coronavirus Research Program), the Milner Fund and the Hebrew University of Jerusalem (startup funds).

....

Materials

NameCompanyCatalog NumberComments
Amicon Ultra-15 Centrifugal Filter UnitsMercC7715cutoff: 100 kDa
ammonium sulfateSigma-AldrichA4418
BSASigma-AldrichA9647
cysteamineSigma-Aldrich30070
dialysis bags - MEGA GeBaFlex-tubeGene Bio-ApplicationMEGA320
dithiothreitol (DTT)Sigma-Aldrich43815
ethylenediaminetetraacetic acid (EDTA)Sigma-AldrichE5134
Fast SeeBand staining solutionGene Bio-ApplicationSB050
GlycineSigma-Aldrich50046
D-GlucoseSigma-AldrichG7021
HEPESSigma-Aldrich54457
HiTrap Desalting 5 mLSigma-AldrichGE17-1408
6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (TROLOX)Sigma-Aldrich238813
isopropyl β-d-1-thiogalactopyranoside (IPTG)Sigma-AldrichI5502
LB brothSigma-AldrichL3152
Magnesium chlorideSigma-Aldrich63068
MonoQ columnSigma-Aldrich54807
protein LoBind tubeSigma-AldrichEP00301080940.5 mL
Rinse a µ-slide 18Ibidi81816
SDSSigma-Aldrich75746
Sodium acetateSigma-AldrichS2889
Sodium hydroxideSigma-AldrichS8045
Sodium phosphate monobasic monohydrateSigma-Aldrich71507
Sterile Cell spreaders, Drigalski spatulasmini-plast815-004-05-001
streptomycin sulfateSigma-AldrichS9137
sulfo-Cy3 maleimideabcamab146493
Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)Sigma-Aldrich75259
Tris-HClSigma-Aldrich93363
TryptoneSigma-AldrichT7293
Yeast ExtractSigma-AldrichY1625

References

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Time resolvedProtein induced Fluorescence EnhancementSingle moleculeAlpha synucleinStructural SubpopulationsLocal ConformationsSulfo Cy3BSAWater Immersion ObjectiveLaser FocusPhoton DetectionFRETBurstsPhoton HDF5

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