Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping

Summary

A method for investigating the structure of a protein photoreceptor using atomic force microscopy (AFM) is described in this paper. PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) reveals intact protein dimers on a mica surface.

Abstract

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be measured to ~10 pN by a laser and sectored detector, which can be converted to image topography. Amplitude modulation or “tapping mode” AFM involves the probe making intermittent contact with the surface while oscillating at its resonant frequency to produce an image. Used in conjunction with a fluid cell, tapping-mode AFM enables the imaging of biological macromolecules such as proteins in physiologically relevant conditions. Tapping-mode AFM requires manual tuning of the probe and frequent adjustments of a multitude of scanning parameters which can be challenging for inexperienced users. To obtain high-quality images, these adjustments are the most time consuming.

PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) produces an image by measuring a force response curve for every point of contact with the sample. With ScanAsyst software, PF-QNM can be automated. This software adjusts the set-point, drive frequency, scan rate, gains, and other important scanning parameters automatically for a given sample. Not only does this process protect both fragile probes and samples, it significantly reduces the time required to obtain high resolution images. PF-QNM is compatible for AFM imaging in fluid; therefore, it has extensive application for imaging biologically relevant materials.

The method presented in this paper describes the application of PF-QNM to obtain images of a bacterial red-light photoreceptor, RpBphP3 (P3), from photosynthetic R. palustris in its light-adapted state. Using this method, individual protein dimers of P3 and aggregates of dimers have been observed on a mica surface in the presence of an imaging buffer. With appropriate adjustments to surface and/or solution concentration, this method may be generally applied to other biologically relevant macromolecules and soft materials.

Introduction

Atomic force microscopy (AFM) has become a very important tool for investigating the structural and mechanical properties of surfaces, thin films, and single molecules since its invention in 1986 (Figure 1).^1-3 Using a liquid-cell, the method has become particularly useful in studies of biological macromolecules and even living cells in a physiologically relevant environment.^4-10 Tapping-mode AFM has traditionally been used for imaging soft materials or loosely bound molecules to the surface, since contact-mode AFM is typically unsuitable due to the damage caused by the lateral forces exerted on the sample by the cantilever.^....

Protocol

1. Computer and Microscope Set Up

1. Open the valve of the N₂ cylinder and adjust the knobs to ensure the air table is floating and level.
2. Turn on the computer, controller, and fiber optic light in this order.
3. Set the scanner to AFM/LFM mode and center the camera over the AFM head.
4. Open software. Select experiment category “Nanomechanical Properties”, “Quantitative Nanomechanical Mapping” under experiment group, and “PeakForce QNM in Fluid” under experiment. Click Load Experiment and then Setup.
5. Focus the camera using the Z objective to see the surface of the stage. Using t....

Results

Representative AFM images of a photoreceptor protein, P3, in its light-adapted state are presented in Figures 3 and 4. A freshly-cleaved mica substrate (Figure 3A) is a suitable, flat surface for protein adsorption. Collecting an image of clean mica as a negative control is important for several reasons. First, it insures the liquid cell is clean and no residual materials from previous experiments will contaminate the surface. Second, it tests the quality of the probe. I.......

Discussion

AFM is a scanning probe microscopy method fully capable of imaging proteins and other biological macromolecules in physiologically relevant conditions. In comparison to X-ray crystallography and NMR, one limitation of AFM is its inability to achieve the same resolution, particularly lateral resolution. When using AFM to analyze a molecule on any surface, the impact of the surface and the probe on the image of the molecule must also be considered when data are analyzed. Deconvoluting of the probe’s impact on the acq.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tris-HCl	Fisher Scientific	O4997-100
NaCl	Acros Organics	7647-14-5
MgCl2	Acros Organics	7791-18-6
Multimode 8 AFM	Bruker-Nano	492-008-011	equipped with Nanoscope V controller and J scanner
Probe	Bruker-Nano	SNL-10, ScanAsyst-Fluid+
Tapping Mode Fluid Cell	Bruker-Nano	MTFML
Mica V-4 Grade	SPI supplies	1150503	25 x 25 x .26 mm
Sample support disk	nanoSurf	BT02236
Petri dish	Plasta-Medic, Inc.		100 mm x 15 mm
micropipettors	Denville Scientific	XL 3000i
RpBphP3			Prepared according to cited references
Nanoscope software	Bruker-Nano
Fiber Optic Light	Digital Instruments Inc.	F0-50
Pelco AFM Disc Gripper	Ted Pella Inc	1668	12 mm
1 ml syringe	McKesson	102-ST1C
Eppendorf tubes	Denville Scientific	C2171
The Pymol Molecular Graphic System v.1.5.0.1	Schrodinger, LLC