Abstract
Physicochemical characterization of protein aggregates is important on one hand, due to its large impact in understanding many diseases for which formation of protein aggregates is one of the pathological hallmarks. On the other hand, recently it has been observed that bacterial inclusion bodies (IBs) are also highly pure proteinaceous aggregates of a few hundred nanometers produced by recombinant bacteria supporting the biological activities of the embedded polypeptides. From this fact arises a wide spectrum of uses of IBs as functional and biocompatible materials upon convenient engineering but very few is known about their physicochemical properties.
In this chapter we present methods for the characterization of protein aggregates as particulate materials relevant to their physicochemical and nanoscale properties.
Specifically, we describe the use of infrared spectroscopy (IR) for the determination of the secondary structure, dynamic light scattering (DLS) for sizing, nanosight for sizing and counting, and Z-potential measurements for the determination of colloidal stability. To study their morphology we present the use of atomic force microscopy (AFM). Cryo-transmission electron microscopy will be used for the determination of the internal structuration. Moreover, wettability and nanomechanical characterization can be performed using contact angle (CA) and force spectroscopic AFM measurements of the proteinaceous nanoparticles, respectively.
The physical principles of the methods are briefly described and examples of data for real samples and how that data is interpreted are given to help clarify capabilities of each technique.
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Carrio M, Gonzalez-Montalban N, Vera A et al (2005) Amyloid-like properties of bacterial inclusion bodies. J Mol Biol 347:1025–1037
García-Fruitós E, Gonzàlez-Montalbán N, Morell M et al (2005) Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins. Microb Cell Factories 4:27
García-Fruitós E, Rodriguez-Carmona E, Díez-Gil C et al (2009) Surface cell growth engineering assisted by a novel bacterial nanomaterial. Adv Mater 21:4249–4253
Avidan-Shpalter C, Gazit E (2006) The early stages of amyloid formation: biophysical and structural characterization of human calcitonin pre-fibrillar assemblies. Amyloid 13:216–225
Kumar S, Mohanty SK, Udgaonkar JB (2007) Mechanism of formation of amyloid protofibrils of barstar from soluble oligomers: evidence for multiple steps and lateral association coupled to conformational conversion. J Mol Biol 367:1186–1204
Li H, Rahimi F, Sinha S et al (2009) Amyloids and protein aggregation–analytical methods. In: Meyers RA (ed) Encyclopedia of analytical chemistry. John Wiley & Sons, New York
Stine WB, Snyder SW, Ladror US et al (1996) The nanometer-scale structure of amyloid-i3 visualized by atomic force microscopy. J Protein Chem 15(2):193–203
Rubin N, Perugia E, Goldschmidt M et al (2008) Chirality of amyloid suprastructures. J Am Chem Soc 130:4602–4603
Apetri MM, Maiti NC, Zagorski MG et al (2006) Secondary structure of a-synuclein oligomers: characterization by raman and atomic force microscopy. J Mol Biol 355:63–71
Mauro M, Craparo EF, Podestà A et al (2007) Kinetics of different processes in human insulin amyloid formation. J Mol Biol 366:258–274
Jansen R, Dzwolak W, Winter R (2005) Amyloidogenic self-assembly of insulin aggregates probed by high resolution atomic force microscopy. Biophys J 88:1344–1353
Ortega-Vinuesa JL, Tengvall P, Lundstrom I (1998) Aggregation of HSA, IgG, and fibrinogen on methylated silicon surfaces. J Colloid Interface Sci 207:228–239
Liu R, McAllister C, Lyubchenko Y et al (2004) Residues 17–20 and 30–35 of beta-amyloid play critical roles in aggregation. J Neurosci Res 75:162–171
Hoyer W, Cherny D, Subramaniam V et al (2004) Rapid self-assembly of a-synuclein observed by in situ atomic force microscopy. J Mol Biol 340:127–139
Goldsbury C, Green J (2005) Time-lapse atomic force microscopy in the characterization of amyloid-like fibril assembly and oligomeric intermediates. Methods Mol Biol 299:103–128
Lashuel HA, Lansbury PT (2006) Are amyloid diseases caused by protein aggregates that mimic bacterial pore-forming toxins? Q Rev Biophys 39:167–201
Cano-Garrido O, Rodríguez-Carmona E, Díez-Gil C et al (2013) Supramolecular organization of protein-releasing functional amyloids solved in bacterial inclusion bodies. Acta Biomater 9:6134–6142
Díez-Gil C, Krabbenborg S, García-Fruitós E et al (2010) The nanoscale properties of bacterial inclusion bodies and their effect on mammalian cell proliferation. Biomaterials 31:5805–5812
Tatkiewicz WI, Seras-Franzoso J, Garcıa-Fruitós E et al (2013) Two-dimensional microscale engineering of protein-based nanoparticles for cell guidance. ACS Nano 7:4774–4784
Ami D, Natalello A, Gatti-Lafranconi P et al (2005) Kinetics of inclusion body formation studied in intact cells by FT-IR spectroscopy. FEBS Lett 579:3433–3436
Dong AD, Huang P, Caughey WS (1990) Protein secondary structures in water from second-derivative amide I infrared spectra. Biochemistry 29:3303–3308
Parra A, Casero E, Lorenzo E et al (2007) Nanomechanical properties of globular proteins: lactate oxidase. Langmuir 23:2747–2754
Acknowledgement
The authors are indebted to the Cell Culture Unit of the “Servei de Cultius Cellulars, Producció d’Anticossos i Citometria” (SCAC), and to the “Servei de Microscòpia,” both at the UAB. We are also indebted to the Protein Production Platform and Biomaterial, Processing and Nanostructuring Unit (CIBER-BBN) for helpful technical assistance (http://bbn.ciber-bbn.es/programas/plataformas/equipamiento). This work was supported by the DGI Grant POMAs (CTQ2010-19501), AGAUR (Grants SGR2009-516), and the Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN). W.T. is grateful to the Consejo Superior de Investigaciones Científicas (CSIC) for a “JAE-pre” fellowship.
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Tatkiewicz, W. et al. (2015). Methods for Characterization of Protein Aggregates. In: García-Fruitós, E. (eds) Insoluble Proteins. Methods in Molecular Biology, vol 1258. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2205-5_22
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DOI: https://doi.org/10.1007/978-1-4939-2205-5_22
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