Abstract
The physicochemical characterization of protein aggregates yields an important contribution to further our understanding on many diseases for which the formation of protein aggregates is one of the pathological hallmarks. On the other hand, bacterial inclusion bodies (IBs) have recently been shown to be highly pure proteinaceous aggregates of a few hundred nanometers, produced by recombinant bacteria supporting the biological activities of the embedded polypeptides. Despite the wide spectrum of uses of IBs as functional and biocompatible materials upon convenient engineering, 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 dynamic light scattering (DLS) for sizing, nanoparticle tracking analysis for sizing and counting, and zeta potential measurements for the determination of colloidal stability. To study the morphology of protein aggregates we present the use of atomic force microscopy (AFM) and scanning electron microscopy (SEM). Cryo-transmission electron microscopy (cryo-TEM) 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 (FS-AFM) measurements of the proteinaceous nanoparticles, respectively. Finally, the 4′4-dithiodipyridine (DTDP) method is presented as a way of relatively quantifying accessible sulfhydryl groups in the structure of the nanoparticle .
The physical principles of the methods are briefly described and examples are given to help clarify capabilities of each technique.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
García-Fruitós E, Rodríguez-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 (2006) Amyloids and protein aggregation—analytical methods. Encycl Anal Chem Appl Theory Instrum
Teimouri A, Azami SJ, Keshavarz H et al (2018) Anti-toxoplasma activity of various molecular weights and concentrations of chitosan nanoparticles on tachyzoites of RH strain. Int J Nanomedicine 13:1341
Stine WB, Snyder SW, Ladror US et al (1996) The nanometer-scale structure of amyloid-β visualized by atomic force microscopy. J Protein Chem 15:193–203
Sanagavarapu K, Nüske E, Nasir I et al (2019) A method of predicting the in vitro fibril formation propensity of A$β$40 mutants based on their inclusion body levels in E. coli. Sci Rep 9:1–14
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 α-synuclein oligomers: characterization by raman and atomic force microscopy. J Mol Biol 355:63–71
Kumar S, Tepper K, Kaniyappan S et al (2014) Stages and conformations of the Tau repeat domain during aggregation and its effect on neuronal toxicity. J Biol Chem 289:20318–20332
Manno 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, Lundström 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 α-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. In: Amyloid proteins. Springer, pp 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
Chaibva M, Gao X, Jain P et al (2018) Sphingomyelin and GM1 influence huntingtin binding to, disruption of, and aggregation on lipid membranes. Acs Omega 3:273–285
Cano-Garrido O, Sánchez-Chardi A, Parés S et al (2016) Functional protein-based nanomaterial produced in microorganisms recognized as safe: a new platform for biotechnology. Acta Biomater 43:230–239
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, Garcia-Fruitos E et al (2013) Two-dimensional microscale engineering of protein-based nanoparticles for cell guidance. ACS Nano 7:4774–4784
Ruggeri FS, Habchi J, Cerreta A et al (2016) AFM-based single molecule techniques: unraveling the amyloid pathogenic species. Curr Pharm Des 22:3950–3970
Riener CK, Kada G, Gruber HJ (2002) Quick measurement of protein sulfhydryls with Ellman’s reagent and with 4,4′-dithiodipyridine. Anal Bioanal Chem 373:266–276
Martínez-Miguel M, Kyvik AR, Martínez-Moreno A et al (2020) Stable anchoring of bacteria-based protein nanoparticles for surface enhanced cell guidance. J Mater Chem B 8:5080–5088
Delgado AV, González-Caballero F, Hunter RJ et al (2005) Measurement and interpretation of electrokinetic phenomena (IUPAC technical report). Pure Appl Chem 77:1753–1805
Parra A, Casero E, Lorenzo E et al (2007) Nanomechanical properties of globular proteins: lactate oxidase. Langmuir 23:2747–2754
Acknowledgments
The authors are grateful for the financial support received from MOTHER and Mol4Bio (MAT2016-80826-R and PID2019- 105622RBI00) granted by the DGI (Spain), GenCat (SGR-918 and SGR-229) financed by DGR (Catalunya), the SpanishMinistry of Economy and Competitiveness (MINECO) through the “Severo Ochoardquo; Programme for Centres of Excellence in R&D (SEV-2015-0496 and CEX2019-000917-S), the COST Action CA15126 Between Atom and Cell, Fundació La Marató de TV3 (Nr. 201812). This study has been also supported by the Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), an initiative funded by the VI National R&D&I Plan, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. The NTA, DLS and zeta potential measurements have been performed by the Biomaterial Processing and Nanostructuring Unit (U6) of the ICTS “NANBIOSISrdquo;, a unit of the CIBER network in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN) located at the Institute of Materials Science of Barcelona (ICMAB-CSIC). J.G. is also grateful to MINECO for a “Ramon y Cajalrdquo; fellowship (Nr. RYC-2017-22614), the Max Planck Society through the Max Planck Partner Group “Dynamic Biomimetics for Cancer Immunotherapyrdquo; in collaboration with the Max Planck Institute for Medical Research (Heidelberg, Germany).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Martínez-Miguel, M. et al. (2022). Methods for the Characterization of Protein Aggregates. In: Garcia Fruitós, E., Arís Giralt, A. (eds) Insoluble Proteins. Methods in Molecular Biology, vol 2406. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1859-2_29
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1859-2_29
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1858-5
Online ISBN: 978-1-0716-1859-2
eBook Packages: Springer Protocols