Name: characterizing individual protein aggregates by infrared nanospectroscopy and atomic force microscopy
Uploaded: 2019-09-12T15:00:00
Description: Watch this Scientific Journal Video about Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy at JoVE.com

The authors thank Swiss National Foundation for Science (SNF) for the financial support (grant number P2ELP2_162116 and P300P2_171219), the Darwin College, Erasmus+ program for the financial support (grant number 2018-1-LT01-KA103-046719-15400-P3) and the research leading to these results has received funding from the European Research Council under the European Union&#39;s Seventh Framework Programme (FP7/2007-2013) through the ERC grant PhysProt (agreement number 337969), the Newman Foundation (T.P.J.K.) and The Cambridge Centre for Misfolding Diseases (C.G., M.V., and T.P.J.K.).

1. Aggregation assays on fluorescence plate readers
NOTE: The protocol described here is an example of how to study the aggregation of any protein or peptide by chemical kinetics. In particular, it describes an optimized protocol to study the aggregation of the A&#946;42 peptide, which is involved in the onset and progression of Alzheimer&#8217;s disease58,59. A similar protocol can be adjusted and adopted towards studying the aggregatio...

The first critical step in this protocol is the preparation of monomeric proteins, such as in the case of A&#946;42 solution described in steps 1.1 and 1.2. It is essential to initiate the aggregation process from a highly pure, monomeric solution, as the presence of oligomeric or aggregated species may result in poor reproducibility of the aggregation kinetics58, and induce artefacts in the AFM measurements (e.g., fibrillar species will be evident at the initial stages of the aggregation), which ...

Over 40 million people worldwide are currently affected by neurodegenerative disorders, such as Alzheimer&#8217;s (AD)1 and Parkinson&#8217;s (PD)2 diseases. More generally, more than fifty pathologies are associated at the molecular level with protein misfolding and aggregation, a process that leads to the proliferation of insoluble fibrillar protein aggregates, known as amyloid deposits3,4. The molecular origins of neurodegeneration and its links with protein conformational changes of proteins leading to amyloid formation, however, remain unclear, in large part because of the high level of heterogeneity, transient nature and nanoscale dimensions of the pathological aggregates4,5.
Highly successful investigations of protein structures in the last several decades have been based widely on the use of bulk methods, including X-ray crystallography, cryo-electron microscopy and nuclear magnetic resonance spectroscopy5,6,7,8,9. Within this class of techniques, infrared (IR) spectroscopy has emerged as a sensitive analytical tool to unravel the chemical properties of biological systems such as proteins8. IR methods allow the quantification of protein secondary and quaternary structural changes during their misfolding and aggregation. In addition, in order to further decipher at the microscopic level the mechanistic details involved in the complex free energy landscapes of protein during their aggregation, a major advance has been the development of chemical kinetics tools to extend to complex self-assembly pathways including amyloid fibrils formation5,6,7,10,11,12. However, bulk spectroscopic methods provide only average information on the heterogeneous ensemble of species present in solution or involved in specific microscopic steps, thus rendering the investigation of the biophysical properties of individual aggregated species challenging at the nanoscale level13,14.
Several microscopy techniques with the capability of operating on scales smaller than the diffraction limit of light have emerged in the last decades. This class of methods includes electron microscopy (EM) and atomic force microscopy (AFM). While scanning electron microscopy (SEM) and transmission electron microscopy (TEM) provide two-dimensional (2D) images of a specimen, AFM has emerged in the last decades as a powerful and versatile technique to study three-dimensional (3D) morphologies, as well as the nanomechanical properties of a sample with sub-nanometer resolution13,14,15,16,17,18,19,20,21,22,23,24,25,26,27. The rationale behind studying protein aggregation via AFM is that this approach enables the investigation of the morphology of individual species present in solution13,14,16,17,19,20,21,25,27,28,29,30,31,32,33,34,35,36,37. In particular, by monitoring the sample as a function of time, AFM allows the investigation of the evolution of the morphology of the species within the sample, which makes it possible to follow and visualize the pathways of amyloid formation23,25,38,39,40,41,42. Furthermore, AFM enables the quantification of structural parameters such as cross-sectional heights and lengths of the individual species present in solution13,19,30,31,32,33,34,35,36,37,40,43,44,45,46,47,48. However, the study of a single biophysical property, such as morphology, is often not sufficient when studying heterogeneous and complex biological systems. AFM, SEM or TEM imaging methods alone do not readily reveal the chemical properties of heterogeneous species of amyloid aggregates at the nanoscale.
A major advance for the analysis of heterogeneous biological samples at this scale has been made recently with the development and application to the field of protein aggregation of infrared nanospectroscopy (AFM-IR)24,26,38,42,49,50,51,52. This innovative method exploits the combination of the spatial resolution of AFM (~1&#8722;10 nm) with the chemical analysis power of IR. The AFM-IR technique is based on the measurement of the photothermal induced resonance effect driven by an IR laser, and on the measurement of the thermal expansion of the sample under investigation by the AFM tip. The sample can be illuminated by the IR laser directly from the top or from the bottom in total internal reflection, similarly as in conventional infrared spectroscopy24,42,52,53. The IR laser can be pulsed with typical frequencies in the order of hundreds of kilohertz (1&#8722;1000 kHz) and tuned over a wide spectral range, typically between 1000&#8722;3300 cm-1. Although the laser source covers an area of ~30 &#181;m diameter, the spatial resolution of the AFM-IR technique is determined nominally by the AFM tip diameter, which detects the local thermal expansion of the system. AFM-IR is well suited to study biological samples because the IR signal is proportional to their thickness up to 1&#8722;1.5 &#181;m, and the resulting IR spectra are generally in agreement with the corresponding FTIR transmission spectra13,54,55. For this reason, established methods of analysis in spectroscopy can be readily applied, such as the study of chemical shifts, band shape change and de-convolution by second derivatives analysis52. Overall, combining the spatial resolution of AFM with the chemical recognition power of IR spectroscopy, AFM-IR enables the simultaneous acquisition of a wide range of morphological, mechanical and chemical properties of a sample at the nanoscale.
Here, we illustrate a protocol for the characterization of the process of protein aggregation that exploits the combination of in vitro fluorescence assays, high-resolution AFM imaging and nanoscale AFM-IR. This combined approach has already excelled in providing detailed results in studying the chemical and structural properties of individual micro-droplets formed by protein aggregates, in the study of liquid-liquid protein phase separation, and in investigating the heterogeneity and biophysical properties of individual aggregated species at the nanoscale23,26,38,45,50,53,56,57.

<table>
	<tbody>
		<tr>
			<td>AFM-IR system</td>
			<td>Anasys Instruments</td>
			<td>nanoIR 2 or 3</td>
			<td>Systems to measure thermal expansion in contact and resonance mode</td>
		</tr>
		<tr>
			<td>Corning 96-well Half Area Black/Clear Bottom Polystyrene NBS Microplate</td>
			<td>Corning</td>
			<td>3881</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Microplate Aluminium Sealing Tape</td>
			<td>Corning</td>
			<td>6570</td>
			<td></td>
		</tr>
		<tr>
			<td>Double Sided Adhesive Discs</td>
			<td>AGAR Scientific</td>
			<td>AGG3347N</td>
			<td></td>
		</tr>
		<tr>
			<td>FLUOstar Omega</td>
			<td>BMG Labtech</td>
			<td>415-101</td>
			<td>Platereader</td>
		</tr>
		<tr>
			<td>Mica Disc 10mm V1</td>
			<td>AGAR Scientific</td>
			<td>AGF7013</td>
			<td></td>
		</tr>
		<tr>
			<td>Park NX10 AFM system</td>
			<td>Park Systems</td>
			<td>N/A</td>
			<td>Atomic Force Microscope</td>
		</tr>
		<tr>
			<td>Platypus Ultra-Flat Gold Chips</td>
			<td>Platypus Technologies</td>
			<td>AU.1000.SWTSG</td>
			<td></td>
		</tr>
		<tr>
			<td>PPP-NCHR-10 cantilevers</td>
			<td>Park Systems</td>
			<td>PPP-NCHR-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LowBind Tubes, 2.0mL</td>
			<td>Eppendorf</td>
			<td>30108132</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon gold coated cantilevers</td>
			<td>Anasys Instruments</td>
			<td>PR-EX-nIR2</td>
			<td></td>
		</tr>
		<tr>
			<td>SPM Specimen Discs 12mm</td>
			<td>AGAR Scientific</td>
			<td>AGF7001</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterizing individual protein aggregates by infrared nanospectroscopy and atomic force microscopy

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with neurodegenerative conditions such as Alzheimer&#8217;s and Parkinson&#8217;s diseases. In particular low molecular weight aggregates, amyloid oligomers, have been shown to possess generic cytotoxic properties and are implicated as neurotoxins in many forms of dementia. We illustrate the use of methods based on atomic force microscopy (AFM) to address the challenging task of characterizing the morphological, structural and chemical properties of these aggregates, which are difficult to study using conventional structural methods or bulk biophysical methods because of their heterogeneity and transient nature. Scanning probe microscopy approaches are now capable of investigating the morphology of amyloid aggregates with sub-nanometer resolution. We show here that infrared (IR) nanospectroscopy (AFM-IR), which simultaneously exploits the high resolution of AFM and the chemical recognition power of IR spectroscopy, can go further and enable the characterization of the structural properties of individual protein aggregates, and thus offer insights into the aggregation mechanisms. Since the approach that we describe can be applied also to the investigations of the interactions of protein assemblies with small molecules and antibodies, it can deliver fundamental information to develop new therapeutic compounds to diagnose or treat neurodegenerative disorders.

A representative time course of A&#946;42 aggregation, as measured by the ThT fluorescence assay, is shown in Figure 1. The aggregation process is commonly characterized by a sigmoidal curve, where a lag phase is initially observed, and is followed by a steep growth phase, before the curve reaches a plateau when an equilibrium steady state is reached6,7,58. It is essential to ensure that an optimize...

Watch this Scientific Journal Video about Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy at JoVE.com

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

We describe the application of infrared nanospectroscopy and high-resolution atomic force microscopy to visualize the process of protein self-assembly into oligomeric aggregates and amyloid fibrils, which is closely associated with the onset and development of a wide range of human neurodegenerative disorders.

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with ...

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