Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

Summary

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

Abstract

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis¹, atherogenesis² and wound healing³. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance^4-7.

Single molecule techniques such as optical trapping⁸, atomic force microscopy⁹, and magnetic tweezers^10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level¹². Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

Protocol

1. Flow Cell Preparation

1. Coverslips (#1.5, 22x22 mm and 22x40 mm, VWR) are cleaned using sonication.
   1. Add the coverslips to a small glass container capable of holding coverslips and fitting in the sonicator (see step 2).
   2. Fill the container with isopropanol and sonicate in a bath sonicator for 20 minutes.
   3. Discard the isopropanol and rinse the coverslips with copious quantities of deionized water produced by a Barnsted MilliQ apparatus or similar device. Fill the container with water and sonicate for 20 minutes.
   4. After sonication, dry the coverslips in a stream of filtered, dust-free air. Use only coverslips that ap....

Discussion

This protocol describes a new use for a classical single molecule technique. Magnetic tweezers allow medium to high-throughput single molecule assays in a cost-efficient manner. However, like all experimental techniques there are challenges and potential pitfalls.

Limitations of magnetic tweezers

Compared to an optical trap the spatial and temporal resolution of a MT apparatus is low. Moreover, the forces generated by the simple MT described here are 30 pN or less, sig.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Micro Cover Glass #1.5 (22x22)	VWR	48366-067
Micro Cover Glass #1.5 (22x40)	VWR	48393-048
Lambda DNA	Invitrogen	25250-010
T4 DNA Ligase	Invitrogen	15224-041
Microcon Ultracel YM-100	Millipore	42413
Anti-Digoxigenin	Roche Diagnostics	11-333-089-001
Tween 20	Sigma	P9416-100ML
Anti-myc Antibody	Invitrogen	46-0603
Bovine Serum Albumin	Sigma	B4287-5G
Dynabeads M-280 Streptavidin	Invitrogen	658.01D
Dynabeads MyOne T1 Streptavidin	Invitrogen	658.01D
p-Aminophenylmercuric Acetate	Calbiochem	164610
Biotin-Maleimide	Sigma Aldrich	B1267
Biotin labeled oligo	IDT DNA	Custom synthesis
Digoxigenin labeled oligo	IDT DNA	Custom synthesis
Collagen peptide gene	DNA 2.0	Custom synthesis
MMP-1 cDNA	Harvard Plasmid Database
z-translator	Thorlabs	MTS50
Servo controller for translator	Thorlabs	TDC001