Abstract
Dissecting the functional basis of pathogenicity and resistance in the context of plant innate immunity benefits greatly from detailed knowledge about biomolecular interactions, as both resistance and virulence depend on specific interactions between pathogen and host biomolecules. While in vivo systems provide biological context to host-pathogen interactions, these experiments typically cannot provide quantitative biochemical characterization of biomolecular interactions. However, in many cases, the biological function does not only depend on whether an interaction occurs at all, but rather on the “intensity” of the interaction, as quantified by affinity. Specifically, microbial effector proteins may bind more than one host target to exert virulence functions, and looking at these interactions more closely than would be possible in a purely black-and-white qualitative assay (as classically based on plant or yeast systems) can reveal new insights into the evolutionary arms race between host and pathogen. Recent advances in biomolecular interaction assays that can be performed in vitro allow quantification of binding events with ever greater fidelity and application range. Here, we describe assays based on microscale thermophoresis (MST) and surface plasmon resonance (SPR). Using these technologies allows affinity determination both in steady-state and in kinetic configurations, providing two conceptually independent pathways to arrive at quantitative affinity data describing the interactions of pathogen and host biomolecules.
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
References
von Mering C et al (2002) Comparative assessment of large-scale data sets of protein-protein interactions. Nature 417:399–403
Aloy P, Russell RB (2004) Ten thousand interactions for the molecular biologist. Nat Biotechnol 22:1317–1321
Berggård T, Linse S, James P (2007) Methods for the detection and analysis of protein-protein interactions. Proteomics 7:2833–2842
Van Der Merwe PA (2001) Surface plasmon resonance. In: Harding SE, Chowdhry BZ (eds) Protein–ligand interactions: hydrodynamics and calorimetry. Oxford University Press, Oxford, pp 137–170
Duhr S, Braun D (2006) Why molecules move along a temperature gradient. Proc Natl Acad Sci U S A 103:19678–19682
Jerabek-Willemsen M et al (2011) Molecular interaction studies using microscale thermophoresis. Assay Drug Dev Technol 9:342–353
Myszka DG (1997) Kinetic analysis of macromolecular interactions using surface plasmon resonance biosensors. Curr Opin Biotechnol 8:50–57
Stenberg E et al (1991) Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proteins. J Colloid Interface Sci 143:513–526
Mayo CS, Hallock RB (1989) Immunoassay based on surface plasmon oscillations. J Immunol Methods 120:105–114
Myszka DG et al (1998) Extending the range of rate constants available from BIACORE: interpreting mass transport-influenced binding data. Biophys J 75:583–594
Johnsson B et al (1991) Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors. Anal Biochem 198:268–277
Karlsson R, Fält A (1997) Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors. J Immunol Methods 200:121–133
Wofsy C, Goldstein B (2002) Effective rate models for receptors distributed in a layer above a surface: application to cells and biacore. Biophys J 82:1743–1755
Rich RL, Myszka DG (2000) Advances in surface plasmon resonance biosensor analysis. Curr Opin Biotechnol 11:54–61
Andersson K et al (1999) Identification and optimization of regeneration conditions for affinity-based biosensor assays. A multivariate cocktail approach. Anal Chem 71(13):2475–2481
Saerens D et al (2008) Single-domain antibodies as building blocks for novel therapeutics. Curr Opin Pharmacol 8:600–608
Rich RL, Myszka DG (2008) Survey of the year 2007 commercial optical biosensor literature. J Mol Recognit 21:355–400
Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 60:523–533
Sahdev et al (2008) Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol Cell Biochem 307:249–264
Young CL et al (2012) Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications. Biotechnol J 7:620–634
Shaner NC et al (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22:1567–1572
Prescott M et al (1999) The length of polypeptide linker affects the stability of green fluorescent protein fusion proteins. Anal Biochem 273:305–307
Rich RL, Myszka DG (2010) Grading the commercial optical biosensor literature-Class of 2008: ‘The Mighty Binders’. J Mol Recognit 23:1–64
Acknowledgments
We thank Deutsche Forschungsgemeinschaft (SFB1101) for funding.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Reinhard, A., Nürnberger, T. (2017). Steady-State and Kinetics-Based Affinity Determination in Effector-Effector Target Interactions. In: Shan, L., He, P. (eds) Plant Pattern Recognition Receptors. Methods in Molecular Biology, vol 1578. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6859-6_7
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6859-6_7
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6858-9
Online ISBN: 978-1-4939-6859-6
eBook Packages: Springer Protocols