Abstract
Surface plasmon resonance (SPR) can be used to analyze both binding affinities and kinetic parameters between a ligand and an analyte. SPR can be performed by either cross-linking a given ligand to a sensor chip covalently or utilizing high-affinity non-covalent interactions to secure a ligand in a particular conformation to a chip, both of which have their potential advantages. SPR measurements are mass based and reflect the proportional amount of analyte bound to a given ligand at a given concentration when flowed at a set rate in order to determine the binding parameters of a given biochemical interaction. The resultant sensorgram can indicate different types of binding events as well as provide both ka and kd, which can be used to determine an equilibrium dissociation constant KD. SPR can be used to measure binding affinity of proteins involved in fusion such as between SNAREs, SNAREs, and proteins that interact with them such as Sec18 (NSF) or Sec17 (alpha-SNAP), or to measure the binding of any fusion-related protein to a specific lipid or other small molecules; however, KDs are determined by SPR using a titration of concentrations of analyte and a maximum point on the sensorgram signifying saturation of the protein in order to determine a steady-state KD.
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Bakhtiar R (2012) Surface Plasmon resonance spectroscopy: a versatile technique in a Biochemist’s toolbox. J Chem Ed 90:203–209
Karlsson R, Michaelsson A, Mattsson L (1991) Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system. J Immunol Methods 145:229–240
Neumann T, Junker HD, Schmidt K, Sekul R (2007) SPR-based fragment screening: advantages and applications. Curr Top Med Chem 7:1630–1642
Sasser T, Qiu QS, Karunakaran S, Padolina M, Reyes A, Flood B et al (2012) Yeast lipin 1 orthologue pah1p regulates vacuole homeostasis and membrane fusion. J Biol Chem 287:2221–2236
Starr ML, Hurst LR, Fratti RA (2016) Phosphatidic acid sequesters Sec18p from cis-SNARE complexes to inhibit priming. Traffic 17:1091–1109
Bayburt TH, Sligar SG (2002) Single-molecule height measurements on microsomal cytochrome P450 in nanometer-scale phospholipid bilayer disks. Proc Natl Acad Sci U S A 99:6725–6730
Denisov IG, Grinkova YV, Lazarides AA, Sligar SG (2004) Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size. J Am Chem Soc 126:3477–3487
Haas A, Wickner W (1996) Homotypic vacuole fusion requires Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF). EMBO J 15:3296–3305
de Mol NJ, Fischer MJ (2010) Surface plasmon resonance: a general introduction. Methods Mol Biol 627:1–14
Futamura M, Dhanasekaran P, Handa T, Phillips MC, Lund-Katz S, Saito H (2005) Two-step mechanism of binding of apolipoprotein E to heparin: implications for the kinetics of apolipoprotein E-heparan sulfate proteoglycan complex formation on cell surfaces. J Biol Chem 280:5414–5422
Acknowledgments
This work was supported in part by NIH grant GM101132 to RAF.
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Sparks, R.P., Jenkins, J.L., Fratti, R. (2019). Use of Surface Plasmon Resonance (SPR) to Determine Binding Affinities and Kinetic Parameters Between Components Important in Fusion Machinery. In: Fratti, R. (eds) SNAREs. Methods in Molecular Biology, vol 1860. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8760-3_12
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DOI: https://doi.org/10.1007/978-1-4939-8760-3_12
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