Ligand-Protein Affinity Studies Using Long-Lived States of Fluorine-19 Nuclei

The lifetimes T-LLS of long-lived states or T-LLC of long-lived coherences can be used for the accurate determination of dissociation constants of weak protein ligand complexes. The remarkable contrast between signals derived from LLS or LLC in free and bound ligands can be exploited to search for weak binders with large dissociation constants K-D > 1 mM that are important for fragment-based drug discovery but may escape detection by other screening techniques. Alternatively, the high sensitivity of the proposed method can be exploited to work with large ligand-to-protein ratios, with an evident advantage of reduced consumption of precious proteins. The detection of F-19-F-19 long-lived states in suitably designed fluorinated spy molecules allows one to perform competition binding experiments with high sensitivity while avoiding signal overlap that tends to hamper the interpretation of proton spectra of mixtures.

Exploring Ligand Affinities for Proteins by NMR of Long-Lived States

Roberto Buratto

The detection of molecules that can bind to active sites of protein targets and the measurement of their affinities is a promising application of NMR. Nowadays, the screening of drug candidates is routinely done by NMR in pharmaceutical industry. We have proposed to use the relaxation of Long-Lived States (LLS) for drug screening by NMR. Long-lived states are nuclear spin states whose decay time constant Tlls can be much longer than the longitudinal relaxation time T1. LLS can be used to screen and determine the dissociation constant KD of molecular fragments that bind weakly to protein targets. The use of LLS for fragment screening leads to a spectacular increase in contrast between free and bound ligands, and thus allows one to characterize binding of fragments with very weak affinities, with KD in the millimolar range, which is difficult to achieve by other methods such as ITC. By exploiting the LLS behavior of a spy molecule, we experimentally demonstrate that it is possible to measure dissociation constants KD as large as 12 mM, corresponding to very weak binding, where most other biophysical techniques fail, including other NMR methods based on the observation of ligands. Furthermore, we have combined LLS for screening for improved contrast with 1H dissolution-DNP to enhance the sensitivity. DNP-enhanced screening for measuring LLS signals of a weak ligand allows one to use very low concentrations of ligands and proteins. We observed dramatic differences between the spectra of the ligand in the presence or absence of a protein, or in the presence of the protein combined with a stronger ligand. Moreover, we have explored LLS involving pairs of 19F nuclei to study binding phenomena. Indeed, fluorine detection is quite interesting because it offers the possibility to perform screening experiments without any problems due to overlapping signals. In a customdesigned fluorinated ligand that binds trypsin, we have observed a promising ratio Tlls / T1 > 4. This fluorinated ligand has been used as spy molecule in competition experiments, which allowed us to rank the affinities and estimate dissociation constants of arbitrary ligands that do not contain any fluorine.

EPFL2015

Probing the structure and function of the tachykinin neurokinin-2 receptor through biosynthetic incorporation of fluorescent amino acids at specific sites

Jonathan Knowles, Gerardo Turcatti, Horst Vogel

A general method for understanding the mechanisms of ligand recognition and activation of G protein-coupled receptors has been developed. A study of ligand-receptor interactions in the prototypic seven-transmembrane neurokinin-2 receptor (NK2) using this fluorescence-based approach is presented. A fluorescent unnatural amino acid was introduced at known sites into NK2 by suppression of UAG nonsense codons with the aid of a chem. misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during heterologous expression in Xenopus oocytes. Fluorescence-labeled NK2 mutants contg. an unique 3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3-diaminopropionic acid (NBD-Dap) residue at either site 103, in the first extracellular loop, or 248, in the third cytoplasmic loop, were functionally active. The fluorescent NK2 mutants were investigated by microspectrofluorimetry in a native membrane environment. Intermol. distances were detd. by measuring the fluorescence resonance energy transfer (FRET) between the fluorescent unnatural amino acid and a fluorescently labeled NK2 heptapeptide antagonist. These distances, calcd. by the theory of Forster, permit to fix the ligand in space and define the structure of the receptor in a mol. model for NK2 ligand-receptor interactions. Our data are the first report of the incorporation of a fluorescent unnatural amino acid into a membrane protein in intact cells by the method of nonsense codon suppression, as well as the first measurement of exptl. distances between a G protein-coupled receptor and its ligand by FRET. The method presented here can be generally applied to the anal. of spatial relationships in integral membrane proteins such as receptors or channels. [on SciFinder (R)]

1996

Exploring Ligand Affinities for Proteins by NMR of Long-Lived States

Roberto Buratto

EPFL2015