Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

Summary

We present a protocol for immobilizing single macromolecules in microfluidic devices and quantifying changes in their conformations under shear flow. This protocol is useful for characterizing the biomechanical and functional properties of biomolecules such as proteins and DNA in a flow environment.

Abstract

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as atomic force microscopy have limited temporal resolution, while Förster resonance energy transfer (FRET) only allow conformations to be inferred. Fluorescence microscopy, on the other hand, allows real-time in situ visualization of single molecules in various flow conditions. Our protocol describes the steps to capture conformational changes of single biomolecules under different shear flow environments using fluorescence microscopy. The shear flow is created inside microfluidic channels and controlled by a syringe pump. As demonstrations of the method, von Willebrand factor (VWF) and lambda DNA are labeled with biotin and fluorophore and then immobilized on the channel surface. Their conformations are continuously monitored under variable shear flow using total internal reflection (TIRF) and confocal fluorescence microscopy. The reversible unraveling dynamics of VWF are useful for understanding how its function is regulated in human blood, while the conformation of lambda DNA offers insights into the biophysics of macromolecules. The protocol can also be widely applied to study the behavior of polymers, especially biopolymers, in varying flow conditions and to investigate the rheology of complex fluids.

Introduction

Mechanisms of how biomolecules respond to environmental stimuli have been studied widely. In a flow environment in particular, shear and elongational forces regulate the conformational changes and potentially the function of biomolecules. Typical examples include shear-induced unraveling of lambda DNA and von Willebrand factor (VWF). Lambda DNA has been used as a tool to understand conformational dynamics of individual, flexible polymer chains and the rheology of polymer solutions^1,2,3,4. VWF is a natural flow sensor that aggregates platelet....

Protocol

1. Preparing VWF

1. Reconstitute human plasma VWF to prepare it for the labeling reactions. Add 100 µL of deionized (DI) water to 100 µg of lyophilized VWF to create a 1 mg/mL VWF stock solution.
2. Dialyze VWF stock solution in order to remove excess glycine, thereby increasing the biotin and fluorophore labeling efficiency.
   1. Transfer 50 µL of VWF stock solution into a 0.1 mL dialysis unit with a 10,000 molecular weight cut-off and seal with a cap. Store the remaining stock solution at -20 °C. VWF stock will be stable for up to 1 year at -20 °C.
   2. Run dialysis in 500 mL of 1x sterile phosphate-buffered saline (PBS) (0.01 M disodium....

Results

Observing the dynamic behavior of biomolecules such as VWF and lambda DNA is highly dependent on optimizing their binding to the device surface. Incubating surface treatments for the recommended times in the microfluidic device is crucial to obtaining binding with a few anchorage points, so that molecules can freely extend and relax upon changing flow. If the proteins or DNA are bound too strongly with multiple linkages, they will either extend to limited lengths or not extend at all. This occurs particularly with VWF wh.......

Discussion

To obtain high quality data of single-molecule conformational changes using fluorescence microscopy as described in this method, it is critical to incubate the molecule for the appropriate amount of time, minimize its nonspecific interactions with the surface and establish microscope settings that reduce photobleaching. The ability of the molecule to freely change conformation is related to the number of biotin-streptavidin interactions formed between the molecule and the surface. As mentioned previously, this must be co.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa Fluor 488 Labeling Kit	Invitrogen	A30006
Bio-Spin P-6 Gel Columns	Bio-Rad	7326221
Biotin	Sigma-Aldrich	B4501	Use as free biotin in Step 5.6
Biotin-14-dCTP	AAT Bioquest	17019
BSA-Biotin	Sigma-Aldrich	A8549
Coverslips	VWR	48393-195	No. 1 ½, 22 x 50 mm
dNTP Set	Invitrogen	10297018
Float Buoys for Mini Dialysis Device	Thermo Scientific	69588
Klenow Fragment (3'→5' exo-)	New England BioLabs	M0212S	Use for 10X reaction buffer in Step 2.1.1 and 1X reaction buffer in Step 2.2.2
Lambda DNA	New England BioLabs	N3011S
Mini Dialysis Device	Thermo Scientific	69570	10K MWCO, 0.1 mL volume
NEBuffer 4	New England BioLabs	B7004S
NHS-PEG4-Biotin	Thermo Scientific	21330
Protocatechuate 3,4-Dioxygenase	Sigma-Aldrich	P8279
Protocatechuic acid	Santa Cruz Biotechnology	sc-205818
Silicone Elastomer Kit for PDMS Fabrication	The Dow Chemical Company	4019862
Streptavidin	Sigma-Aldrich	85878
The Blocking Solution	CANDOR Bioscience	110 050	Use as casein blocking solution throughout protocol
Vinyl Cleanroom Tape	Fisher Scientific	19-120-3217
von Willebrand Factor, Human Plasma	Millipore Sigma	681300
YOYO-1 Dye	AAT Bioquest	17580
0.25 mm Inner Diameter Tubing	Cole-Parmer	EW-06419-00
25 Gauge Needle	Thomas Scientific	JG2505X