Label-free quantitative 1H NMR spectroscopy to study low-affinity ligand–protein interactions in solution: A contribution to the mechanism of polyphenol-mediated astringency

Abstract

Nuclear magnetic resonance (NMR) spectroscopy is well-established in assessing the binding affinity between low molecular weight ligands and proteins. However, conventional NMR-based binding assays are often limited to small proteins of high purity and may require elaborate isotopic labeling of one of the potential binding partners. As protein–polyphenol complexation is assumed to be a key event in polyphenol-mediated oral astringency, here we introduce a label-free, ligand-focused 1H NMR titration assay to estimate binding affinities and characterize soluble complex formation between proteins and low molecular weight polyphenols. The method makes use of the effects of NMR line broadening due to protein–ligand interactions and quantitation of the non-bound ligand at varying protein concentrations by quantitative 1H NMR spectroscopy (qHNMR) using electronic reference to access in vivo concentration (ERETIC 2). This technique is applied to assess the interaction kinetics of selected astringent tasting polyphenols and purified mucin, a major lubricating glycoprotein of human saliva, as well as human whole saliva. The protein affinity values (BC50) obtained are subsequently correlated with the intrinsic mouth-puckering, astringent oral sensation imparted by these compounds. The quantitative NMR method is further exploited to study the effect of carboxymethyl cellulose, a candidate “anti-astringent” protein binding antagonist, on the polyphenol–protein interaction. Consequently, the NMR approach presented here proves to be a versatile tool to study the interactions between proteins and low-affinity ligands in solution and may find promising applications in the discovery of bioactives.

NMR-Based Studies on Odorant–Melanoidin Interactions in Coffee Beverages

“…The signal shape as well as the signal width, measured as the full width at half maximum (FWHM), indicated high binding affinity to macromolecular coffee melanoidins (Figure 3). The FWHM of 2,3-diethyl-5-methylpyrazine changed from 2.02 Hz in the aqueous reference to 4.16 Hz in the sample with added melanoidins and therefore led to significant signal broadening of 2.14 Hz and was shifted to higher frequencies by 1.42 Hz, which is supported by literature, where it is reported that compounds exhibiting non-covalent π-π interactions studied by NMR spectroscopy often show line broadening and shift of resonances to higher frequencies [36,37,18]. In comparison, the aroma active compounds 4-hydroxy-2,5-dimethyl-3(2H)-furanone and 2,3-butanedione, representing furanones and diketones are mostly unaffected by the addition of the HMW fraction >10 kDa.…”

“…Interactions such as complex formation by e.g. non-covalent (π-π), dipole-dipole or van der Waals interactions were revealed to be disbanded during liquid chromatography because of the dynamic equilibrium between lightly associated complexation partners [18]. Thus, bound and unbound states of the ligands could not be differentiated, and state of the art GC or UHPLC analysis remains not suitable for observing dynamic non-covalent interactions.…”

“…In the literature non-covalent interactions were reported to influence proteins and ligands [19][20][21][22], the oral astringency of polyphenols [18], as well as the co-pigmentation of anthocyanins [23].…”

Capillary electrophoresis‐based approaches for the study of affinity interactions combined with various sensitive and nontraditional detection techniques

“…Each of the above‐mentioned biologically active molecules is characterized by its specific physicochemical properties and behavior that change after the formation of the complex with the corresponding interacting partner. Based on these alterations, many different approaches to studying the interactions have been used–equilibrium dialysis , nuclear magnetic resonance , circular dichroism , fluorescence‐based techniques , isothermal titration calorimetry , continuous ultrafiltration , surface plasmon resonance (SPR) , Fourier transform infrared spectroscopy , MS , Raman spectroscopy , molecular modeling , HPLC , and thermophoresis , including CE as well. Besides the substantial instrumental development, the necessity of measured data quality as high as possible is essential requirement of these studies.…”

“…Each of the above-mentioned biologically active molecules is characterized by its specific physicochemical properties and behavior that change after the formation of the complex with the corresponding interacting partner. Based on these alterations, many different approaches to studying the interactions have been used-equilibrium dialysis [2], nuclear magnetic resonance [3], circular dichroism [4], fluorescence-based techniques [5], isothermal titration…”