NMR‐Based Screening of Membrane Protein Ligands

Abstract

Membrane proteins pose problems for the application of NMR-based ligand-screening methods because of the need to maintain the proteins in a membrane mimetic environment such as detergent micelles: they add to the molecular weight of the protein, increase the viscosity of the solution, interact with ligands non-specifically, overlap with protein signals, modulate protein dynamics and conformational exchange and compromise sensitivity by adding highly intense background signals. In this article, we discuss the special considerations arising from these problems when conducting NMR-based ligand-binding studies with membrane proteins. While the use of (13)C and (15)N isotopes is becoming increasingly feasible, (19)F and (1)H NMR-based approaches are currently the most widely explored. By using suitable NMR parameter selection schemes independent of or exploiting the presence of detergent, (1)H-based approaches require least effort in sample preparation because of the high sensitivity and natural abundance of (1)H in both, ligand and protein. On the other hand, the (19)F nucleus provides an ideal NMR probe because of its similarly high sensitivity to that of (1)H and the lack of natural (19)F background in biologic systems. Despite its potential, the use of NMR spectroscopy is highly underdeveloped in the area of drug discovery for membrane proteins.

Exhaustive Fluorine Scanning toward Potent p53–Mdm2 Antagonists

“…[13, 14] Last but not least, 19 F is very useful in NMR of complex biological systems due to its high sensitivity and zero natural background. [15] Herein, we report the systematic F-scanning of a scaffold derived from the Ugi four-component reaction (Ugi-4CR) for the synthesis, optimization and biophysical characterization of potent p53-Mdm2 antagonists.…”

NMR of liquid crystals and micellar solutions

“…92 Various aggregates, such as spherical and elongated micelles, tubular and rodlike aggregates, as well as vesicles, are induced by adding sodium 2-naphthalenesulfonate (SNphs) or sodium benzenesulfonate (SBzs). However, SNphs and SBzs cannot induce similar aggregate transitions for the systems of C12CsC12(Me) (s = 6,8,12). The possible mechanism of these phenomena is proposed based on the results of UV spectra and 1 H NMR measurements.…”

“…While the use of 13 C and 15 N isotopes is becoming increasingly feasible, 19 F and 1 H NMR-based approaches are currently the most widely explored. 12 Solution NMR has become the tool of choice for detecting structures of the small proteins or peptides and to characterize their interactions with biomimetic micelles. This approach was used to characterize the structure and orientation of cyclotides, mini-proteins characterized by a circular backbone and a knotted disulfide core, bound to dodecylphosphocholine micelles.…”

Applications of In-Cell NMR in Structural Biology and Drug Discovery

“…In addition, NMR active nuclei such as 19 F and 31 P can be incorporated to a protein, making 19 F and 31 P NMR possible in determining conformational changes of proteins induced by ligand binding or post-translational modifications [44,45,46,47,48]. In fragment-based drug discovery (FBDD), NMR is frequently used in identifying fragments with different binding affinities [49,50]. Proton-based NMR spectroscopies have been successfully used in this field.…”