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With our strong expertise and extensive experience in affinity and kinetics measurement for dimers/polymers. Creative Biolabs provide label-free and high-throughput Surface Plasmon Resonance (SPR) and Bio-Layer Interferometry (BLI) technologies based services, which is capable of satisfying any of your specific demand.
Monomer is the simplest form of a carbohydrate, nucleic acid, lipid, or protein (such as glucose, nucleotide and amino acid). Dimer is a pairing of structurally similiar monomers (such as sucrose and phospholipid). Polymer is a set of many indentical monomers associated together through polymerization (such as dynamic actin filaments and microtubules). Dimers or polymers are joined by either covalent bonds or hydrogen bonds, while we focus on the affinity analysis of the latter form. Y. R. Kim et al. performed a process named enzymatic surface initiated polymerization (ESIP) with SPR system to work out affinity analysis for polyhydroxybutyrate (PHB) (Figure 1). ESIP has been extensively studied due to its unique ability which allows the robust and dense polymers to grow directly from the gold surface using an immobilized initiator.
Figure 1. Different concentration of PHA synthase binding to the mixed SAMs and subsequent polymerization (left panel). SPR sensorgram showing the binding of His-tag PHA synthase to a Ni-NTA functionalized gold surface and the subsequent dissociation of the same protein from the surface by free imidazole (right panel). PHA, polyhydroxyalkanoate; SAMs, Self-assembled mono- layers. (Macromol. Biosci., 2006)
Take XMAP215 polymerase as another example, which regulates microtubule growth via its repeat domains locate at the N termini (TOG domains), which binds tubulin dimers, therefore the affinity for tubulin can be observed through the polymerization kinetics.
Figure 2. Model of TOG12+++ on the plus end of a microtubule. TOG12+++, the TOG12 fragment with the strong microtubule-binding domain. (PNAS, 2011)
As shown in Fiugre 2, the following reaction scheme of the microtubule polymerization is:
Where microtubule end (Tn) represents the enzyme, free tubulin dimer (T) is the substrate, XMAP215 (X) is the activator. The relative affinity of the activator for the intermediate state is given by α, while the association rate change of the intermediate state due to the activator is given by α’, thus, α and α’ are defined as :
From the formula above, we can confirm the tubulin-binding affinity correlates with the activity of XMAP215, which localizes to the growing microtubule ends primarily through microtubule lattice diffusion. Finally, the flux of XMAP215 molecules to the microtubule end is given by:
Where D indicates the diffusion coefficient of XMAP215 on the microtubule lattice, Kon and Koff are the association and dissociation rate constants, respectively, for the binding of XMAP215 to microtubule. From the above formula, we notice that the activity of XMAP215 polymerase increases with the microtubule-binding affinity.
Figure 3. The affinity analysis of XMAP215 polymerization
Mutation or removal in XMAP215 leads to a higher KD of the polymerization without the change of νmax. Once XMAP215 targets to the microtubule end, the tubulin affinity can determine the maximal growth rate (νmax) at any fixed tubulin concentration (Figure 3, A). The graph shows theoretical dose response of a protein with a constant νmax and an altered Kon during microtubule-lattice binding: 4 x reduced in red, 2 x reduced in green, not reduced in blue. We can note that the KD value is changed (Figure 3, B).
M. Vidal, et al. (2004). Design of peptoid analogue dimers and measure of their affinity for Grb2 SH3 domains. Biochemistry. 43 (23): 7336-7344
P. O. Widlund, et al. (2011). XMAP215 polymerase activity is built by combining multiple tubulin-binding TOG domains and a basic lattice-binding region. PNAS. 108 (7): 2754-2746.
S. Elbaum-Garfinkle et al. (2014). Tau mutants bind tubulin heterodimers with enhanced affinity. PNAS. 111 (17): 6311-6316.
Y. R. Kim, et al. (2006). Real-Time Analysis of Enzymatic Surface-Initiated Polymerization Using Surface Plasmon Resonance (SPR). 6 (2): 145-152.
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