Name: utilizing time-resolved protein-induced fluorescence enhancement to identify stable local conformations one &#945;-synuclein monomer at a time
Uploaded: 2021-05-30T14:00:00
Description: Watch this Scientific Journal Video about Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One ÃŽÂ±-Synuclein Monomer at a Time at JoVE.com

The pT-t7 plasmid encoding A56C &#945;-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).

1. Plasmid transformation
<ol>
	<li>Preparation of 0.5 L of SOC medium&#160;
	<ol>
		<li>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).</li>
		<li>Add double-distilled water (DDW) until total volume of 0.5 L.</li>
		<li>Adjust to pH 7 by adding sodium hydroxide (NaOH).</li>
		<li>Prepare stock aliquots of 100 mL and autoclave.</li>
		<li>Before using, add 0.5 mL of sterile magnesium chloride (MgCl2) and 1.8 mL of sterile glucose to 100 mL ...

Extensive biochemical and biophysical studies were performed to study the structural characteristics of &#945;-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 &#945;-Syn monomer free of binding. These works reported ...

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&#160;measurements 6,7, fluorescence anisotropy8,9, fluorescence quantum yields and lifetimes10,11, as a function of different fluorescence quenching12 or enhancement13 mechanisms. One of these mechanisms, better known as protein-induced fluorescence enhancement (PIFE)14 introduces the enhancement of fluorescence quantum yield and lifetime as a function of steric obstruction to the free isomerization of the fluorophore when in excited-state, caused by protein surfaces in the vicinity of the dye14,15,16,17,18,19. Both FRET and PIFE are considered spectroscopic rulers or proximity sensors since their measured parameter is directly linked to a spatial measure within the labeled biomolecule under measurement. While the FRET efficiency is related to the distance between a pair of dyes within a range of 3-10 nm20, PIFE tracks increases in fluorescence quantum yields or lifetimes related to the distance between the dye and a surface of a nearby protein in the range of 0-3 nm19.
Single-molecule FRET has been widely used for providing structural insights into many different protein systems, including intrinsically disordered proteins (IDPs)21, such as &#945;-Synuclein (&#945;-Syn)22. &#945;-Syn can form ordered structures following binding to different biomolecules and under different conditions23,24,25,26,27,28,29,30. However, when unbound, the &#945;-Syn monomer is characterized by high conformational heterogeneity with rapidly interconverting conformations31,32.
The conformations of &#945;-Syn have been studied previously using various different techniques that help in identifying conformational dynamics of such highly heterogeneous and dynamic protein systems33,34,35,36,37,38,39. Interestingly, single-molecule FRET (smFRET) measurements of &#945;-Syn in buffer reported a single FRET population39,40&#160;that is an outcome of time-averaging of conformations dynamically interconverting at times much faster than the typical diffusion time of &#945;-Syn through the confocal spot (times as fast as few microseconds and even faster than that, relative to typical millisecond diffusion times)40,41. However, using a FRET spectroscopic ruler with the 3-10 nm distance sensitivity sometimes reports only on overall structural changes in a small protein such as &#945;-Syn. Single-molecule measurements utilizing spectroscopic proximity sensors with shorter distance sensitivities have the potential to report on dynamics of local structures. Herein we perform single-molecule PIFE measurements of &#945;-Syn and identify different sub-populations of fluorescence lifetimes mapping to different local structures with transitions between them as slow as 100 ms. This work summarizes time-resolved smPIFE measurements of freely-diffusing &#945;-Syn molecules one at a time, in buffer and when bound to SDS-based membranes as a short-range single-molecule spectroscopic proximity sensor.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter Units</td>
			<td>Merc</td>
			<td>C7715</td>
			<td>cutoff: 100 kDa</td>
		</tr>
		<tr>
			<td>ammonium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>A4418</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr>
			<td>cysteamine</td>
			<td>Sigma-Aldrich</td>
			<td>30070</td>
			<td></td>
		</tr>
		<tr>
			<td>dialysis bags - MEGA GeBaFlex-tube</td>
			<td>Gene Bio-Application</td>
			<td>MEGA320</td>
			<td></td>
		</tr>
		<tr>
			<td>dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>43815</td>
			<td></td>
		</tr>
		<tr>
			<td>ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast SeeBand staining solution</td>
			<td>Gene Bio-Application</td>
			<td>SB050</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>50046</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>54457</td>
			<td></td>
		</tr>
		<tr>
			<td>HiTrap Desalting 5 mL</td>
			<td>Sigma-Aldrich</td>
			<td>GE17-1408</td>
			<td></td>
		</tr>
		<tr>
			<td>6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (TROLOX)</td>
			<td>Sigma-Aldrich</td>
			<td>238813</td>
			<td></td>
		</tr>
		<tr>
			<td>isopropyl &#946;-d-1-thiogalactopyranoside (IPTG)</td>
			<td>Sigma-Aldrich</td>
			<td>I5502</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Sigma-Aldrich</td>
			<td>L3152</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>63068</td>
			<td></td>
		</tr>
		<tr>
			<td>MonoQ column</td>
			<td>Sigma-Aldrich</td>
			<td>54807</td>
			<td></td>
		</tr>
		<tr>
			<td>protein LoBind tube</td>
			<td>Sigma-Aldrich</td>
			<td>EP0030108094</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td>Rinse a &#181;-slide 18</td>
			<td>Ibidi</td>
			<td>81816</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma-Aldrich</td>
			<td>75746</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>71507</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Cell spreaders, Drigalski spatulas</td>
			<td>mini-plast</td>
			<td>815-004-05-001</td>
			<td></td>
		</tr>
		<tr>
			<td>streptomycin sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>S9137</td>
			<td></td>
		</tr>
		<tr>
			<td>sulfo-Cy3&#160;maleimide</td>
			<td>abcam</td>
			<td>ab146493</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)</td>
			<td>Sigma-Aldrich</td>
			<td>75259</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>93363</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Sigma-Aldrich</td>
			<td>T7293</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Sigma-Aldrich</td>
			<td>Y1625</td>
			<td></td>
		</tr>
	</tbody>
</table>

utilizing time-resolved protein-induced fluorescence enhancement to identify stable local conformations one &#945;-synuclein monomer at a time

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 &#945;-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 &#945;-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled &#945;-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while &#945;-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.

As an IDP, when it is not bound to another biomolecule, &#945;-Syn exhibits structural dynamics between multiple conformations, with transitions at few microseconds40&#160;and even at hundreds of nanoseconds41. When &#945;-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...

Watch this Scientific Journal Video about Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One ÃŽÂ±-Synuclein Monomer at a Time at JoVE.com

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One ÃƒÅ½Ã‚Â±-Synuclein Monomer at a Time

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 &#945;-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural ...

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