Investigation Of Protein-ligand Interactions Research Articles

Preparation of miniaturized hydrophilic affinity monoliths: Towards a reduction of non-specific interactions and an increased target protein density

In affinity chromatography, non-specific interactions between the ligands and the affinity column may affect the results, leading to misinterpretations during the investigation of protein-ligand interactions (detection of false positives in ligand screening, lack of specificity in purification). Such non-specific interactions may arise both from the underlying support or from the target protein itself. If the second ones are protein-dependent (and cannot be studied in a general framework), the first ones occur in the same way regardless of the immobilized target. We propose a methodology to identify the origin of such non-specific interactions with the underlying material of the affinity column. This methodology relies on the systematic investigation of the retention behavior of a set of 41 low-molecular weight compounds covering a wide chemical space (net charge, log D, functionality). We first demonstrate that the main source of non-specific interactions on the most commonly used GMA-co-EDMA monolith comes from hydrophobic effects. To reduce such non-specific interactions, we developed a new hydrophilic glycidyl methacrylate-based monolith by replacing the EDMA crosslinker by the more hydrophilic NN’ Methylenebisacrylamide (MBA). Optimization of the synthesis parameters (monomer content, initiation type, temperature) has focused on the reduction of non-specific interaction with the monolithic support while maximizing the amount of protein that can be grafted onto the monolith at the issue of its synthesis. The retention data of the 41 test solutes on the new poly(GMA-co-MBA) monolith shows a drastic reduction of non-specific interactions except for cationic compounds. The particular behavior of cationic compounds is due to their electrostatic interactions with carboxylic groups resulting from the partial acidic hydrolysis of amide groups of MBA during the epoxide ring opening step. So, the ring opening step in acidic media was replaced by a hot water treatment to avoid side reaction on MBA. The new monolith poly(GMA-co-MBA) not only has improved hydrophilic surface properties but also a higher protein density (16 ± 0.8 pmol cm−1 instead of 8 ± 0.3 pmol cm−1). To highlight the benefits of this new hydrophilic monolith for affinity chromatographic studies, frontal affinity chromatography experiments were conducted on these monoliths grafted with con A.

Expedited Nuclear Magnetic Resonance Assignment of Small- to Medium-Sized Molecules with Improved HSQC-CLIP-COSY Experiments.

Resonance assignment is a pivotal step for any nuclear magnetic resonance (NMR) analysis, such as structure elucidation or the investigation of protein-ligand interactions. Both 1H-13C heteronuclear single quantum correlation (HSQC) and 1H-1H correlation spectroscopy (COSY) two-dimensional (2D) experiments are invaluable for 1H NMR assignment, by extending the high signal dispersion of 13C chemical shifts onto 1H resonances and by providing a high amount of through-bond 1H-1H connectivity information, respectively. The recently introduced HSQC-CLIP(Clean In-Phase)-COSY method combines these two experiments, providing COSY correlations along the high-resolution 13C dimension with clean in-phase multiplets. However, two experiments need to be recorded to unambiguously identify COSY cross-peaks. Here, we propose novel variants of the HSQC-CLIP-COSY pulse sequence that edit cross-peak signs so that direct HSQC responses can be distinguished from COSY relay peaks, and/or the multiplicities of the 13C nuclei are reflected, allowing the assignment of all the peaks in a single experiment. The advanced HSQC-CLIP-COSY variants have the potential to accelerate and simplify the NMR structure-elucidation process of both synthetic and natural products and to become valuable tools for high-throughput computer-assisted structure determination.

Pharmacophore Mapping via Cross-Relaxation during Adiabatic Fast Passage

A novel NMR method is demonstrated for the investigation of protein ligand interactions. In this approach an adiabatic fast passage pulse, i.e. a long, weak pulse with a linear frequency sweep, is used to probe (1)H-(1)H NOEs. During the adiabatic fast passage the effective rotating-frame NOE is a weighted average of transverse and longitudinal cross-relaxation contributions that can be tuned by pulse power and frequency sweep rate. It is demonstrated that the occurrence of spin diffusion processes leads to sizable deviations from the theoretical relationship between effective relaxation rate and effective tilt angle in the spin lock frame and can be used to probe protein-ligand binding. This methodology comprises high sensitivity and ease of implementation. The feasibility of this technique is demonstrated with two protein complexes, vanillic acid bound to the quail lipocalin Q83 and NAD(+) and AMP binding to alcohol dehydrogenase (ADH).