The relationship between effective molarity and affinity governs rate enhancements in tethered kinase-substrate reactions

Scaffold proteins are thought to accelerate protein phosphorylation reactions by tethering kinases and substrates together, but there is little quantitative data on their functional effects. To assess the contribution of tethering to kinase reactivity, we compared intramolecular and intermolecular kinase reactions in a minimal model system. We find that tethering can enhance reaction rates in a flexible tethered kinase system, and the magnitude of the effect is sensitive to the structure of the tether. The largest effective molarity we obtained was ∼0.08 µM, which is much lower than the effects observed in small molecule model systems and tethered protein-ligand interactions. We further demonstrate that the tethered, intramolecular reaction only makes a significant contribution to observed rates when the scaffolded complex assembles at concentrations below the effective molarity. These findings provide a quantitative framework that can be applied to understand endogenous protein scaffolds and to engineer synthetic networks.

Quantification of cooperativity in the self-assembly of H-bonded rosettes

The effective molarity associated with the macrocyclisation step in the self-assembly of triaminopyrimidines with barbiturates and with cyanates was measured as 2 M.

The canonical behavior of the entropic component of thermodynamic effective molarity. An attempt at unifying covalent and noncovalent cyclizations

The statistically corrected entropic component of effective molarity (EMS*) complies with the “canonical” values expressed by the log plot of EMS* vs. the number n of single bonds in the ring product.

Immunochemical engineering of cell surfaces to generate virus resistance

Modern immunochemical engineering allows the creation of cells that either secrete antibodies or incorporate them into various cellular compartments, including the plasma membrane. Because the receptors for most viruses are known, if one can achieve the proper stoichiometry and geometry, plasma membrane-associated antibodies to these receptors should block viral infection. In this report, we test this concept for two different viruses, human rhinovirus and HIV. Plasma membrane-tethered antibodies efficiently rendered cells permanently nonpermissive for infection by both these viruses. Membrane-bound antibodies were much more efficient than free antibody in preventing infection, likely because of the effective molarity of membrane bound antibodies. Such resistant cells may restore immune-competence to otherwise compromised HIV patients.