Colocalization of dots in the upright microscopy images of VSNEA, as evidenced by a comparison between the bright field and the corresponding fluorescence images, strongly suggests a selective binding of the peptides to the Au tips of VSNEA without any nonspecific binding to other areas of the substrate (Figure 3a,b).
A non-repetitive (XP n sequence like identified in RgCrtC (10×) and TrCrtC (9×) can have different functions as for instance stabilizing the enzyme by binding noncovalently to other proteins, binding to other hydrophobic structures like hydrophobic substrates or function as a "molecular trigger" passing signals to the inner membrane.
NCAM is a membrane glycoprotein receptor of the immunoglobulin supergene family that mediates cell-to-cell adhesion via homophilic binding to other NCAM molecules and cell-to-substrate adhesion via heterophilic binding (NCAM binding to another ligand or counter-receptor) [ 30].
The phosphorylated sequence, designated here as the pS9 peptide, is thought to auto-inhibit GSK-3 by acting as a pseudo-substrate that blocks binding of other substrates (Dajani et al., 2001; Frame et al., 2001).
Although enzymes are simply catalysts in the process, and can therefore theoretically be re-used, in practice this may be difficult due to denaturing, inhibition, or irreversible binding to the substrate or other non-targeted materials.
The proposal that different SS classes would compete with each other for binding to potential substrate chains is consistent with the observation that SSII from pea embryos is a distributive enzyme that dissociates from the glucan substrate after each glucose unit addition [ 48, 50].
For Pro425, mutation to Arg resulted in loss of binding for MTX and other substrates, but substantial preservation of PMX binding, presumably reflecting a conformation change induced by the Arg substitution.
In subsequent crossing-binding analysis, candidate 9.1 showed broad substrate binding affinity to other PCB compounds, while candidate 9.2 showed a high specificity for the two PCBs with hydroxyl functional groups.
These observations, as well as comparison to structures of other kinases bound to substrates, indicate that the inhibitory peptides act as true pseudo-substrates that mimic binding to substrate, rather than stabilizing a distinct catalytically inactive conformation of GSK-3.
Thus, the binding of the side chain of L-arginine via interaction with both domains of the protein is likely crucial for triggering conformational change required to generate the bindings sites of the other substrates.
