Nuclear magnetic resonance (NMR) spectroscopy has evolved into an important technique in support of structure-based drug design because of its long tradition in the study of molecular interactions (1,2). Several NMR experiments have been used in generic binding assays to identify weak, but specific, binding between small molecules and a target protein. The advantage of these NMR screening methods is that they can be applied as soon as a target

From: Methods in Molecular Biology, vol. 300: Protein Nanotechnology, Protocols, instrumentation, and Applications Edited by: T. Vo-Dinh © Humana Press Inc., Totowa, NJ

Fig. 1. Principle of the trNOESY experiment. The trNOE is only operative when the ligand is bound, because the ligand experiences a long correlation time (xc) only in the bound state. The NOE is transferred and measured on the resonances of the ligand in the free state.

protein is available without the need for extensive assay development. NMR screening methods have been reviewed extensively by others (3-5) and are not described in detail here, with the exception of the transferred nuclear Overhauser effect spectroscopy (trNOESY) experiment.

trNOESY experiments (6-8) are routinely used to detect ligand binding to a target protein under conditions of fast exchange (ligands that bind with micromolar to millimolar dissociation constants). The advantages of the trNOESY method are that it does not require large amounts of pure, labeled protein; it is not limited by the size of the protein; and it can provide information about the structure of the bound form of the ligand. In the experiment, the intensity of each intraligand nuclear Overhauser effect (NOE) crosspeak is governed by the population-weighted cross-relaxation rate (9) (Fig. 1). Thus, the binding event is relatively straightforward to detect and does not require time-consuming chemical shift assignments. A strong negative NOE crosspeak is observed for binders, as opposed to weakly positive or zero NOE crosspeaks for the same compounds in the absence of the target receptor, as shown for MP-biocytin in Fig. 2. Thus, the sign flip of the NOE crosspeak between the free vs bound states acts as a simple binary filter to distinguish binders from nonbinders (10,11) (see Note 1).

An important parameter in selecting a reagent for use in a sensor is its relative binding affinity. Very recently, several laboratories, including our own, have begun to address the issue of whether NMR screening methods can simultaneously and rapidly provide this information (12-14). For the preparation of multivalent ligands that consist of two or more ligands that are weak

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