Abstract
To understand cellular processes such as biochemical pathways and signaling networks, we need to understand binding and reaction rates of often competing reactions, their dependence on cellular concentrations of participating molecules, and the regulation of these rates through allostery, posttranslational modifications, or other mechanisms. To do so, we break these systems down into their elementary steps, which are almost invariably either unimolecular or bimolecular reactions that frequently occur on sub-second, often sub-millisecond, time scales. Rapid mixing techniques, which generally achieve mixing in less than 2 ms, are generally suitable for the study of such reactions. The application of these techniques to the study of enzyme mechanisms is described in several excellent texts (Cornish-Bowden, Fundamentals of enzyme kinetics, 1995; Gutfreund, Kinetics for the life sciences. Receptors, transmitters and catalysis, 1995); flow techniques are used to study individual steps by monitoring the approach to equilibrium (the pre-steady state) under single turnover conditions.
The individual steps in complex biochemical reaction schemes determine how fast systems can respond to incoming signals and adapt to changed conditions [1, 2]. This chapter is concerned with in vitro techniques that have been developed to study fast reactions in solution, and we present the study of various interactions of calmodulin as an example. The kinetic information obtained with these techniques is indispensable for understanding the dynamics of biochemical processes and complements the static structural and thermodynamic information available from X-ray crystallography, NMR, and equilibrium binding studies.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Cornish-Bowden A (1995) Fundamentals of enzyme kinetics. Portland Press, London
Gutfreund H (1995) Kinetics for the life sciences. Receptors, transmitters and catalysis. Cambridge University Press, Cambridge
Berridge MJ, Bootman MD, Lipp P (1998) Calcium-a life and death signal. Nature 395:645–648
Soderling TR, Stull JT (2001) Structure and regulation of calcium/calmodulin-dependent protein kinases. Chem Rev 101:2341–2352
Chattopadhyaya R, Meador WE, Means AR et al (1992) Calmodulin structure refined at 1.7 Å resolution. J Mol Biol 228:1177–1192
Heidorn DB, Trewhella J (1988) Comparison of the crystal and solution structures of calmodulin and troponin C. Biochemistry 27:909–915
Finn BE, Evenas J, Drakenberg T et al (1995) Calcium-induced structural changes and domain autonomy in calmodulin. Nat Struct Biol 2:777–783
Barman TE, Bellamy SR, Gutfreund H et al (2006) The identification of chemical intermediates in enzyme catalysis by the rapid quench-flow technique. Cell Mol Life Sci 63:2571–2583
Shastry MCR, Luck SD, Roder H (1998) A continuous-flow capillary mixer to monitor reactions on the microsecond time scale. Biophys J 74:2714–2721
Martin SR, Schilstra MJ (2013) Rapid mixing kinetic techniques. In: Williams M, Daviter T (eds) Protein-ligand interactions. Methods in molecular biology (methods and protocols), vol 1008. Humana Press, Totowa, NJ, pp p119–p138
Eccleston JF, Martin SR, Schilstra MJ (2008) Rapid kinetic techniques. Methods Cell Biol 84:445–477
Browne JP, Strom M, Martin SR et al (1997) The role of β-sheet interactions in domain stability, folding, and target recognition reactions of calmodulin. Biochemistry 36:9550–9561
Clapperton JA, Martin SR, Smerdon SJ et al (2002) Structure of the complex of calmodulin with the target sequence of calmodulin-dependent protein kinase I: studies of the kinase activation mechanism. Biochemistry 41:14669–14479
Martin SR, Andersson-Teleman A, Bayley PM et al (1985) Kinetics of calcium dissociation from calmodulin and its tryptic fragments. A Quin 2 stopped‑flow fluorescence study reveals a two‑domain structure. Eur J Biochem 151:543–550
Halford SE (1971) Escherichia coli alkaline phosphatase. An analysis of transient kinetics. Biochem J 125:319–327
De La Cruz EM, Ostap EM, Sweeney HL (2001) Kinetic mechanism and regulation of myosin VI. J Biol Chem 276:32373–32381
Eccleston JF, Hutchinson JP, White HD (2001) Stopped-flow techniques. In: Harding SE, Chowdhry BZ (eds) Protein-ligand interactions: structure and spectroscopy. Oxford University Press, Oxford
De La Cruz EM, Wells AL, Rosenfeld SS et al (1999) The kinetic mechanism of myosin V. PNAS 96:13726–13731
Eccleston JF, Petrovic A, Davis CT et al (2006) The kinetic mechanism of the SufC ATPase: the cleavage step is accelerated by SufB. J Biol Chem 281:8371–8378
Kuzmic P (1996) Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase. Anal Biochem 237:260–273
Schilstra MJ, Martin SR, Keating SM (2008) Methods for simulating the dynamics of complex biological processes. Methods Cell Biol 84:807–842
Woodward SKA, Eccleston JF, Geeves MA (1991) Kinetics of the interaction of 2′(3′)-O-(N-methylanthraniloyl)-ATP with myosin subfragment 1 and actomyosin subfragment 1: characterization of two acto.S1.ADP complexes. Biochemistry 30:422–430
Johnson ML (2008) Nonlinear least-squares fitting methods. Methods Cell Biol 84:781–805
Press WH, Teukolsky BP, Vetterling WT et al (1990) Numerical recipes. The art of scientific computing. Cambridge University Press, Cambridge
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Martin, S.R., Schilstra, M.J. (2021). Interactions of a Signal Transduction Protein Investigated by Fluorescence Stopped-Flow Kinetics. In: Daviter, T., Johnson, C.M., McLaughlin, S.H., Williams, M.A. (eds) Protein-Ligand Interactions. Methods in Molecular Biology, vol 2263. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1197-5_3
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1197-5_3
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1196-8
Online ISBN: 978-1-0716-1197-5
eBook Packages: Springer Protocols