Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One α-Synuclein Monomer at a Time

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 biomolecules^1,2, probing how different biomolecular parameters distribute as well as how they dynamically interconvert between different sub-populations of these parameters at sub-millisecond resolution^3,4,5. 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 of SOC.
2. Thaw BL21 (DE3) competent Escherichia coli cells on ice.
3. Mix the competent cells gently.
....

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 used^39,40. 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 nature^{33,34,35,36,37,38}. 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 .......

Materials

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