分子印迹技术用于蛋白质的识别

分子印迹技术是一种新型的高效分离技术。合成含识别蛋白质的分子印迹聚合物具有极大的应用价值,又极具挑战性,已成为许多分子识别领域工作者的研究热点。本文总结了近10年来该领域的研究进展,讨论了已有不同方法的发展状况以及相对优缺点,阐明了其可能发展的方向和前景。     

Macromolecular Recognition: Discrimination between Human and Bovine Serum Albumins by Cyclodextrins

The interactions of cyclodextrins (CDs) with human serum albumin (HSA) and bovine serum albumin (BSA) were investigated by two‐dimensional nuclear Overhauser effect spectroscopy (2D NOESY), steady state fluorescence, isothermal titration calorimetry, and circular dichroism spectroscopy. 2D NOESY indicates that α‐CD and β‐CD, but not γ‐CD, interact with the aromatic amino acid residues on HSA. On the other hand, the 2D NOESY signals between CDs and BSA are not significantly correlated. Hence, CDs can discriminate the subtle protein structural differences of HSA and BSA.

     

A Disulfide Bridge Allows for Site‐Selective Binding in Liver Bile Acid Binding Protein Thereby Stabilising the Orientation of Key Amino Acid Side Chains

The presence of a disulfide bridge in liver bile acid binding protein (L‐BABP/S‐S) allows for site‐selective binding of two bile acids, glycochenodeoxycholic (GCDA) and glycocholic acid (GCA), differing only in the presence of a hydroxyl group. The protein form devoid of the disulfide bridge (L‐BABP) binds both bile salts without discriminating ability. We investigate the determinants of the molecular recognition process in the formation of the heterotypic L‐BABP/S‐S complex with GCA and GCDA located in the superficial and inner protein sites, respectively. The comparison of the NMR spectroscopy structure of heterotypic holo L‐BABP/S‐S, the first reported for this protein family, with that of the homotypic L‐BABP complex demonstrates that the introduction of a S–S link between adjacent strands changes the conformation of three key residues, which function as hot‐spot mediators of molecular discrimination. The favoured χ₁ rotameric states (t, g⁺ and g^? for E99, Q100 and E109 residues, respectively) allow the onset of an extended intramolecular hydrogen‐bond network and the consequent stabilisation of the side‐chain orientation of a buried histidine, which is capable of anchoring a specific ligand.     

Site‐Specific Investigation of the Steady‐State Kinetics and Dynamics of the Multistep Binding of Bile Acid Molecules to a Lipid Carrier Protein

The investigation of multi‐site ligand–protein binding and multi‐step mechanisms is highly demanding. In this work, advanced NMR methodologies such as 2D ¹H–¹⁵N line‐shape analysis, which allows a reliable investigation of ligand binding occurring on micro‐ to millisecond timescales, have been extended to model a two‐step binding mechanism. The molecular recognition and complex uptake mechanism of two bile salt molecules by lipid carriers is an interesting example that shows that protein dynamics has the potential to modulate the macromolecule–ligand encounter. Kinetic analysis supports a conformational selection model as the initial recognition process in which the dynamics observed in the apo form is essential for ligand uptake, leading to conformations with improved access to the binding cavity. Subsequent multi‐step events could be modelled, for several residues, with a two‐step binding mechanism. The protein in the ligand‐bound state still exhibits a conformational rearrangement that occurs on a very slow timescale, as observed for other proteins of the family. A global mechanism suggesting how bile acids access the macromolecular cavity is thus proposed.     

Molecular Recognition of Rosmarinic Acid from Salvia sclareoides Extracts by Acetylcholinesterase: A New Binding Site Detected by NMR Spectroscopy

Acetylcholinesterase (AChE) inhibition is one of the most currently available therapies for the management of Alzheimer’s disease (AD) symptoms. In this context, NMR spectroscopy binding studies were accomplished to explain the inhibition of AChE activity by Salvia sclareoides extracts. HPLC‐MS analyses of the acetone, butanol and water extracts eluted with methanol and acidified water showed that rosmarinic acid is present in all the studied samples and is a major constituent of butanol and water extracts. Moreover, luteolin 4′‐O‐glucoside, luteolin 3′,7‐di‐O‐glucoside and luteolin 7‐O‐(6′′‐O‐acetylglucoside) were identified by MS² and MS³ data acquired during the LC‐MSⁿ runs. Quantification of rosmarinic acid by HPLC with diode‐array detection (DAD) showed that the butanol extract is the richest one in this component (134 μg mg^?1 extract). Saturation transfer difference (STD) NMR spectroscopy binding experiments of S. sclareoides crude extracts in the presence of AChE in buffer solution determined rosmarinic acid as the only explicit binder for AChE. Furthermore, the binding epitope and the AChE‐bound conformation of rosmarinic acid were further elucidated by STD and transferred NOE effect (trNOESY) experiments. As a control, NMR spectroscopy binding experiments were also carried out with pure rosmarinic acid, thus confirming the specific interaction and inhibition of this compound against AChE. The binding site of AChE for rosmarinic acid was also investigated by STD‐based competition binding experiments using Donepezil, a drug currently used to treat AD, as a reference. These competition experiments demonstrated that rosmarinic acid does not compete with Donepezil for the same binding site. A 3D model of the molecular complex has been proposed. Therefore, the combination of the NMR spectroscopy based data with molecular modelling has permitted us to detect a new binding site in AChE, which could be used for future drug development.     

Unravelling the Time Scale of Conformational Plasticity and Allostery in Glycan Recognition by Human Galectin-1

The interaction of human galectin-1 with a variety of oligosaccharides, from di-(N-acetyllactosamine) to tetra-saccharides (blood B type-II antigen) has been scrutinized by using a combined approach of different NMR experiments, molecular dynamics (MD) simulations, and isothermal titration calorimetry. Ligand- and receptor-based NMR experiments assisted by computational methods allowed proposing three-dimensional structures for the different complexes, which explained the lack of enthalpy gain when increasing the chemical complexity of the glycan. Interestingly, and independently of the glycan ligand, the entropy term does not oppose the binding event, a rather unusual feature for protein-sugar interactions. CLEANEX-PM and relaxation dispersion experiments revealed that sugar binding affected residues far from the binding site and described significant changes in the dynamics of the protein. In particular, motions in the microsecond-millisecond timescale in residues at the protein dimer interface were identified in the presence of high affinity ligands. The dynamic process was further explored by extensive MD simulations, which provided additional support for the existence of allostery in glycan recognition by human galectin-1.     

Studies Related to Norway Spruce Galactoglucomannans: Chemical Synthesis,Conformation Analysis,NMR Spectroscopic Characterization,and Molecular Recognition of Model Compounds

Galactoglucomannan (GGM) is a polysaccharide mainly consisting of mannose, glucose, and galactose. GGM is the most abundant hemicellulose in the Norway spruce (Picea abies), but is also found in the cell wall of flax seeds, tobacco plants, and kiwifruit. Although several applications for GGM polysaccharides have been developed in pulp and paper manufacturing and the food and medical industries, attempts to synthesize and study distinct fragments of this polysaccharide have not been reported previously. Herein, the synthesis of one of the core trisaccharide units of GGM together with a less‐abundant tetrasaccharide fragment is described. In addition, detailed NMR spectroscopic characterization of the model compounds, comparison of the spectral data with natural GGM, investigation of the acetyl‐group migration phenomena that takes place in the polysaccharide by using small model compounds, and a binding study between the tetrasaccharide model fragment and a galactose‐binding protein (the toxin viscumin) are reported.     

The Plasticity of the Carbohydrate Recognition Domain Dictates the Exquisite Mechanism of Binding of Human Macrophage Galactose-Type Lectin

The human macrophage galactose-type lectin (MGL), expressed on macrophages and dendritic cells (DCs), modulates distinct immune cell responses by recognizing N-acetylgalactosamine (GalNAc) containing structures present on pathogens, self-glycoproteins, and tumor cells. Herein, NMR spectroscopy and molecular dynamics (MD) simulations were used to investigate the structural preferences of MGL against different GalNAc-containing structures derived from the blood group A antigen, the Forssman antigen, and the GM2 glycolipid. NMR spectroscopic analysis of the MGL carbohydrate recognition domain (MGL-CRD, C181-H316) in the absence and presence of methyl α-GalNAc (α-MeGalNAc), a simple monosaccharide, shows that the MGL-CRD is highly dynamic and its structure is strongly altered upon ligand binding. This plasticity of the MGL-CRD structure explains the ability of MGL to accommodate different GalNAc-containing molecules. However, key differences are observed in the recognition process depending on whether the GalNAc is part of the blood group A antigen, the Forssman antigen, or GM2-derived structures. These results are in accordance with molecular dynamics simulations that suggest the existence of a distinct MGL binding mechanism depending on the context of GalNAc moiety presentation. These results afford new perspectives for the rational design of GalNAc modifications that fine tune MGL immune responses in distinct biological contexts, especially in malignancy.     

Site‐Resolved Backbone and Side‐Chain Intermediate Dynamics in a Carbohydrate‐Binding Module Protein Studied by Magic‐Angle Spinning NMR Spectroscopy

Magic‐angle spinning solid‐state NMR spectroscopy has been applied to study the dynamics of CBM3b–Cbh9A from Clostridium thermocellum (ctCBM3b), a cellulose binding module protein. This 146‐residue protein has a nine‐stranded β‐sandwich fold, in which 35 % of the residues are in the β‐sheet and the remainder are composed of loops and turns. Dynamically averaged ¹H‐¹³C dipolar coupling order parameters were extracted in a site‐specific manner by using a pseudo‐three‐dimensional constant‐time recoupled separated‐local‐field experiment (dipolar‐chemical shift correlation experiment; DIPSHIFT). The backbone‐Cα and Cβ order parameters indicate that the majority of the protein, including turns, is rigid despite having a high content of loops; this suggests that restricted motions of the turns stabilize the loops and create a rigid structure. Water molecules, located in the crystalline interface between protein units, induce an increased dynamics of the interface residues thereby lubricating crystal water‐mediated contacts, whereas other crystal contacts remain rigid.     

Monitoring Glycan–Protein Interactions by NMR Spectroscopic Analysis: A Simple Chemical Tag That Mimics Natural CH–π Interactions

Detection of molecular recognition processes requires robust, specific, and easily implementable sensing methods, especially for screening applications. Here, we propose the difluoroacetamide moiety (an acetamide bioisoster) as a novel tag for detecting by NMR analysis those glycan–protein interactions that involve N‐acetylated sugars. Although difluoroacetamide has been used previously as a substituent in medicinal chemistry, here we employ it as a specific sensor to monitor interactions between GlcNAc‐containing glycans and a model lectin (wheat germ agglutinin). In contrast to the widely employed trifluoroacetamide group, the difluoroacetamide tag contains geminal ¹H and ¹⁹F atoms that allow both ¹H and ¹⁹F NMR methods for easy and robust detection of molecular recognition processes involving GlcNAc‐ (or GalNAc‐) moieties over a range of binding affinities. The CHF₂CONH‐ moiety behaves in a manner that is very similar to that of the natural acetamide fragment in the involved aromatic‐sugar interactions, providing analogous binding energy and conformations, whereas the perfluorinated CF₃CONH‐ analogue differs more significantly.     

Molecular Recognition by a Short Partial Peptide of the Adrenergic Receptor: A Bottom‐Up Approach

Receptor–neurotransmitter molecular recognition is key for neurotransmission. Although crystal structures of the receptors are known, the mechanism for recognition is not clear. Reported here is the ultraviolet (UV) and infrared (IR) spectra of complexes between a partial peptide (SIVSF), mimicking the binding motif of a catechol ring in the adrenergic receptor, and various ligands. The UV spectra show that two isomers coexist in the complex of SIVSF with properly recognized ligands, such as protonated adrenaline (adrenalineH⁺). From IR spectra, they are assigned to catechol‐ and amino‐bound structures. The catechol‐bound structure is not observed when the ligand is replaced by nonproper molecules, such as noradrenalineH⁺. The results suggest that SIVSF not only recognizes the catechol ring but can distinguish differences in the amine side chain. The method provides a new possibility for screening molecules as potential therapeutics for activating the receptor.     

Molecular Recognition of Nucleotides in Water by Scorpiand‐Type Receptors Based on Nucleobase Discrimination

The detection of nucleotides is of crucial importance because they are the basic building blocks of nucleic acids. Scorpiand‐based polyamine receptors functionalized with pyridine or anthracene units are able to form stable complexes with nucleotides in water, based on coulombic, π–π stacking, and hydrogen‐bonding interactions. This behavior has been rationalized by means of an exploration with NMR spectroscopy and DFT calculations. Binding constants were determined by potentiometry. Fluorescence spectroscopy studies have revealed the potential of these receptors as sensors to effectively and selectively distinguish guanosine‐5′‐triphosphate (GTP) from adenosine‐5′‐triphosphate (ATP).