Dependence of Effective Molarity on Linker Length for an Intramolecular Protein−Ligand System

Abstract

This paper reports dissociation constants and "effective molarities" (M eff ) for the intramolecular binding of a ligand covalently attached to the surface of a protein by oligo(ethylene glycol) (EG n ) linkers of different lengths (n = 0, 2, 5, 10, and 20), and compares these experimental values with theoretical estimates from polymer theory. As expected, the value of M eff is lowest when the linker is too short (n = 0) to allow the ligand to bind noncovalently at the active site of the protein without strain, is highest when the linker (n = 2) is the optimal length to allow such binding to occur, and decreases monotonically as the length increases past this optimal value (but, only by a factor of approximately eight from n = 2 to n = 20). These experimental results are not compatible with a model in which the single bonds of the linker are completely restricted when the ligand has bound non-covalently to the active site of the protein, but are quantitatively compatible with a model that treats the linker as a random-coil polymer. Calorimetry revealed that enthalpic interactions between the linker and the protein are not important in determining the thermodynamics of the system. Taken together, these results suggest that the manifestation of the linker in the thermodynamics of binding is exclusively entropic. The values of M eff are, theoretically, intrinsic properties of the EG n linkers, and can be used to predict the avidities of multivalent ligands with these linkers for multivalent proteins. The weak dependence of M eff on linker length suggests that multivalent ligands containing flexible linkers that are longer than the spacing between the binding sites of a multivalent protein will be effective in binding, and that the use of flexible linkers with length somewhat greater than the optimal distance between binding sites is a justifiable strategy for use in the design of multivalent ligands.

High-Affinity Peptide-Based Collagen Targeting Using Synthetic Phage Mimics: From Phage Display to Dendrimer Display

“…The enhanced affinity can be understood by assuming that binding of one peptide ligand increases the effective molarity (EM) to 0.34 ( 0.07 mM for binding to a second receptor site. 18 This number is consistent with the length of the flexible linker used here and an average distance of ∼30 Å between two peptide binding sites. 19 This analysis also indicates that multivalent binding is only effective when EM > K inter and that statistical factors contribute to enhance the effect of multivalent interactions.…”

Isolating Contributions from Intersegmental Transfer to DNA Searching by Alkyladenine DNA Glycosylase*

“…The lack of a length dependence for the different length PEG tethers is not surprising because PEG is extremely flexible and has a short persistence length (31). Previous studies of the properties of PEG tethers provide estimates for the effective concentration of tethered duplex that a protein bound to one duplex would experience (32,33). For the PEG-10 substrate (tethered by 60 ethylene glycol units), the effective concentration is estimated to be ϳ3 mM, which appears to be sufficiently high to support rapid transfer.…”

Cucurbit[8]uril templated supramolecular ring structure formation and protein assembly modulation

“…12 The binding process for ring formation can be described by a two-step process with binding constant K 1 representing the intermolecular binding of the first FGGG-motif of 1 in the cavity of Q8 and K 2 the intramolecular binding of the second FGGG of 1 (Fig. 1b).…”