Name: atomic force microscopy of red-light photoreceptors using peakforce quantitative nanomechanical property mapping
Uploaded: 2014-10-24T14:00:00
Description: Watch this Scientific Journal Video about Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping at JoVE.com

The NSF-MRI program (CHE: 1229103) is acknowledged for funding the purchase of new control electronics, software, liquid cells, and other equipment needed to assemble a dual AFM/STM. We acknowledge the shared facilities at The University of Chicago NSF-MRSEC program (DMR-0820054) for assistance with AFM instrumentation, training, and imaging, and&#160;for instrument time made available by the Materials Research Facilities Network (DMR-0820054). We particularly thank Dr. Qiti Guo, Dr. Justin Jureller, and Prof. Ka Yee Lee for welcoming our students before the funding of the NSF-MRI proposal that brought the necessary instrumentation to our campus. We acknowledge funding from a Title III STEM grant (ID: P031C110157) awarded to Northeastern Illinois University that provided summer research stipends for students and faculty as well as support for consumable supplies. Finally, we acknowledge Bruker-Nano, Inc. for continued instrumental support and for permission to reproduce a plot showing the mechanism of PeakForce QNM and to use the word ScanAsyst to describe the automated software.

1. Computer and Microscope Set Up
<ol>
	<li>Open the valve of the N2 cylinder and adjust the knobs to ensure the air table is floating and level.</li>
	<li>Turn on the computer, controller, and fiber optic light in this order.</li>
	<li>Set the scanner to AFM/LFM mode and center the camera over the AFM head.</li>
	<li>Open software. Select experiment category &#8220;Nanomechanical Properties&#8221;, &#8220;Quantitative Nanomechanical Mapping&#8221; under experiment group, and &#8220;PeakForce QNM in Fluid&#8...

<ol>
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Vis. Exp. 71, (2013).</li><li>Bahatyrova, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Native+Architechure+of+a+Photosynthetic+Membrane.">The Native Architechure of a Photosynthetic Membrane.</a> Nature. 430, 1058-1061 (2004).</li><li>Sturgis, J. N., Tucker, J. D., Olsen, J. D., Hunter, C. N., Niederman, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+Force+Microscopy+Studies+of+Native+Photosynthetic+Membranes.">Atomic Force Microscopy Studies of Native Photosynthetic Membranes.</a> Biochem. 48, 3679-3698 (2009).</li><li>Pelling, A. E., Li, Y., Shi, W., Gimzewski, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domain+Structure+of+a+Unique+Bacterial+Red+Light+Photoreceptor+as+Revealed+by+Atomic+Force+Microscopy.">Domain Structure of a Unique Bacterial Red Light Photoreceptor as Revealed by Atomic Force Microscopy.</a> Mater. Res. Soc. Symp. Proc. 1652, (2013).</li><li>Rockwell, N. C., Lagarias, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+phytochrome:+a+picture+is+worth+a+thousand+spectra.">The structure of phytochrome: a picture is worth a thousand spectra.</a> Plant Cell. 18, 4-14 (2006).</li><li>Rockwell, N. C., Shang, L., Martin, S. S., Lagarias, J. 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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&#8217;s impact on the acq...

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.11 Tapping-mode AFM substantially reduces these forces by having the tip intermittently touch the surface rather than being in constant contact. In this mode, the cantilever is oscillated at or near its resonant frequency normal to the surface. Similarly to contact-mode AFM, topography is analyzed by plotting the movement of the z-piezo as a function of xy (distance).
The cantilever dynamics can be quite unstable at or near resonance; therefore, they are very challenging to automate outside of a &#8220;steady-state&#8221; situation. Specifically, these dynamics depend on both the sample properties and scanning environment. For a soft molecule adsorbed to a hard(er) surface, a well-tuned feedback loop for the molecule may lead to feedback oscillation for the surface. Operation in fluid further complicates the tuning of the cantilever. Changes in temperature or fluid levels require constant readjustment of set point, gains, and other imaging parameters. These adjustments tend to be very time-consuming and challenging for users.
Peak Force Quantitative Nanomechanical Property Mapping (PF-QNM), like tapping mode AFM, avoids lateral interactions by intermittently contacting the sample (Figure 2).12-15 However, PF-QNM operates in non-resonant mode and frequencies much lower than tapping-mode AFM. This eliminates the tuning challenges of tapping-mode AFM, particularly those exacerbated by the presence of fluid. With PF-QNM, images are collected by taking a force response curve at every point of contact. With the addition of ScanAsyst software,15 adjustment of the scanning parameters can be automated and a high-resolution image obtained in a matter of minutes by even inexperienced users. Once the user becomes more familiar with the AFM, any or all of the automated parameters may be disabled at any time which permits the experimentalist to fine tune the image quality manually. Since its inception, PF-QNM has been applied to map bacteriorhodopsin, a membrane protein, and other native proteins at the submolecular level.16-18 For bacteriorhodopsin, there is a direct correlation between protein flexibility and X-ray crystallographic structures.12 PF-QNM has been utilized to investigate living cells with high resolution.19,20 Furthermore, PF-QNM data has elucidated important connections between structure and mechanics within the erythrocyte membrane that are critical for cell integrity and function.21
We have employed scanning probe microscopy (SPM) methods,22 including AFM,23 to study the structure of red-light photoreceptors called bacteriophytochromes (BphPs).24,25 They consist of a light sensing module covalently linked to a signaling-effector module such as histidine kinase (HK).26 The light-sensing module typically contains a bilin chromophore which undergoes structural transformation upon absorption of a photon, with a series of structural changes reaching the signaling-effector module and leading to a global transformation of the entire protein.24,27-29 Based on this transformation, there are two distinct light absorbing states of BphPs, a red and far-red light absorbing state, denoted as Pr and Pfr. Pr is thermally stable, dark-adapted state for most BphPs.28 The molecular basis of Pr/Pfr photoconversion is not entirely understood due to limited structural knowledge of these proteins. With the exception of one structure from D. radiodurans,30 all published X-ray crystallographic structures of these proteins are in the dark-adapted state and lack effector domain. The intact BphPs are too large to be effectively studied by Nuclear Magnetic Resonance (NMR) and are notoriously difficult to crystallize in their intact form (particularly in the light-adapted state) for X-ray crystallography. BphPs have recently been engineered as infrared fluorescent protein markers (IFP&#8217;s).31 Structural characterization of these proteins can further aid in effective IFP design.32-36
The focus of this article is to present a procedure for imaging of BphPs using liquid-cell AFM via PF-QNM. The method is demonstrated by studies of the light-adapted state of the bacteriophytochrome RpBphP3 (P3) from the photosynthetic bacterium R. palustris. The AFM procedure presented here is convenient and straightforward approach for imaging of proteins as well as other biological macromolecules. With this method, structural details of individual molecules can be collected in a short period of time, similar to an upper-level science course laboratory session. Through measuring cross-sections and completing further dimensional analyses, experimental data can be compared to useful computational models.37-42

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog #</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Fisher Scientific</td>
			<td>O4997-100</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Acros Organics</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Acros Organics</td>
			<td>7791-18-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Multimode 8 AFM</td>
			<td>Bruker-Nano</td>
			<td>492-008-011</td>
			<td>equipped with Nanoscope V controller and J scanner</td>
		</tr>
		<tr>
			<td>Probe</td>
			<td>Bruker-Nano</td>
			<td>SNL-10, ScanAsyst-Fluid+</td>
			<td></td>
		</tr>
		<tr>
			<td>Tapping Mode Fluid Cell</td>
			<td>Bruker-Nano</td>
			<td>MTFML</td>
			<td></td>
		</tr>
		<tr>
			<td>Mica V-4 Grade</td>
			<td>SPI supplies</td>
			<td>1150503</td>
			<td>25 x 25 x .26 mm</td>
		</tr>
		<tr>
			<td>Sample support disk</td>
			<td>nanoSurf</td>
			<td>BT02236</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Plasta-Medic, Inc.</td>
			<td></td>
			<td>100 mm x 15 mm&#160;</td>
		</tr>
		<tr>
			<td>micropipettors</td>
			<td>Denville Scientific</td>
			<td>XL 3000i</td>
			<td></td>
		</tr>
		<tr>
			<td>RpBphP3</td>
			<td></td>
			<td></td>
			<td>Prepared according to cited references</td>
		</tr>
		<tr>
			<td>Nanoscope software</td>
			<td>Bruker-Nano</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber Optic Light</td>
			<td>Digital Instruments Inc.</td>
			<td>F0-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Pelco AFM Disc Gripper</td>
			<td>Ted Pella Inc</td>
			<td>1668</td>
			<td>12 mm</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>McKesson</td>
			<td>102-ST1C</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td>Denville Scientific</td>
			<td>C2171</td>
			<td></td>
		</tr>
		<tr>
			<td>The Pymol Molecular Graphic System v.1.5.0.1</td>
			<td>Schrodinger, LLC</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

atomic force microscopy of red-light photoreceptors using peakforce quantitative nanomechanical property mapping

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 &#8220;tapping mode&#8221; 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.

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...

Watch this Scientific Journal Video about Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping at JoVE.com

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

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.

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 ...

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