Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

Summary

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

Abstract

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

Introduction

Advances in single molecule force spectroscopy (SMFS) by AFM have enabled mechanical manipulation and precise characterization of single protein molecules. This characterization has produced novel insights about protein mechanics^1,2, protein folding³, protein-ligand interactions⁴, protein-protein interactions⁵, and protein-based engineered materials^6,7,8. SMFS is especially useful for studying protein unfolding, as stretching by AFM allows the che....

Protocol

1. Protein Preparation

1. DNA cloning.
   1. Synthesize a DNA sequence of interest, for example, the DNA sequence of NI₁₀C¹⁰, or isolate via PCR from the host organism using standard molecular biology techniques¹¹. Flank the gene of interest with restriction sites during synthesis or by placing sites in the 5’- end of the PCR primers to correspond to a module in the plasmid pEMI91 (Addgene #74888)¹².
   2. Separately digest both the plasmid pEMI91¹² and the DNA sequence of interest with a pair of restriction sites so that the sequence....

Results

Representative results from this protocol are shown in Figure 2. Both panels show representative force-extension curves from proteins. The top shows results from a I91 polyprotein, while the bottom shows the I91 protein flanking a protein-of-interest, the NI₁₀C molecule. These recordings show the characteristic force of I91 (200 pN) and contour length increment (28 nm) which indicates that the alignment and calibration of the AFM was successful. Th.......

Discussion

A critical step in the protocol is the use of a polyprotein, described in step 1.1.2, which serves as a positive control to "fingerprint" single-molecule events. Generally, there must be unfolding events of the polyprotein proteins (for I91, this means an unfolding force of about 200 pN and contour length increment of about 28 nm) to unambiguously conclude that the protein of interest has been unfolded. For example, when the protein of interest is flanked by three I91 domains from either side, then there must be .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AFM Specimen Discs, 15mm diameter	Ted Pella, Inc.	16218	Serve as base for glass substrate
Round Glass Coverslips, 15mm diamiter No.1 Thick	Ted Pella, Inc.	26024	serve as glass substrate and base for gold coating
Adhesive Tabs	Ted Pella, Inc.	16079	Paste on AFM Specimen Discs to provide a sticky face for attaching glass coverslips
STD Multimode head assembly	Bruker Nano Inc.	1B75C	AFM head
Glass probe holder	Bruker Nano Inc.	MTFML-V2	Glass probe holder for scanning in fluid with the MultiMode AFM.
Microlever AFM probes	Bruker Nano Inc.	MLCT	Silicon Nitride cantilevers with Silicon Nitride tips, ideal for contact imaging modes
AFM probes with Au coated tips	Bruker Nano Inc.	OBL-10	Cantilevers for pulling on proteins with low unfolding force
Multifunction Data Acquisition (DAQ) Card,16-Bit, 1 MS/s (Multichannel), 1.25 MS/s (1-Channel), 32 Analog Inputs	National Instruments	PCI-6259	Data Acquisition for signals from AFM head and Piezo Actuators
LISA Linear Piezo Stage Actuators	Physik Instrumente LP	P-753.11C	Piezo Actuator to control the position of substrate and perform pulling measurements
XY Piezo Stage	Physik Instrumente LP	P-541.2CD	Piezo Actuator to control the position of substrate and scan on substrate surface