Evaluation of the membrane-binding properties of the proximal region of the angiotensin II receptor (AT1A) carboxyl terminus by surface plasmon resonance.

The proximal region of the angiotensin II receptor (AT1A) carboxyl-terminus (known as helix VIII) is important for receptor function. In this study, we used surface plasmon resonance (SPR) to examine the interaction of helix VIII-derived peptides with three model lipid membranes. The membrane-binding properties of these synthetic peptides, as well as a series of peptide analogues with modified amino acid sequences, could be explained by both amino acid sequence and kinetic binding data by SPR. The helix VIII peptides showed a higher affinity for lipid membranes that contained negatively charged phospholipid, rather than zwitterionic phospholipid. The findings of an SPR study may be useful for estimating the cooperative binding of intracellular receptor domains with G proteins and the components of the lipid bilayer.     

Structure-activity relationships of alpha-human atrial natriuretic peptide (alpha-hANP) analogs: role of charged groups in alpha-hANP.

To determine whether the addition of a methylene unit in the side chain of the Asp or Arg residue in alpha-human atrial natriuretic peptide (alpha-hANP) influences its biological activity, analogs of alpha-hANP, [Glu13]-alpha-hANP (7-28) (1), [Aad13]-alpha-hANP (7-28) (2), and [Harn]-alpha-hANP(7-28) (where n is any possible combination of 11, 14 and 27) (3-9), where the original Asp or Arg residue was replaced by a homo-amino acid, were synthesized by the solid-phase synthesis method. All the analogs were evaluated for their receptor binding, cyclic guanosine monophosphate (cGMP) accumulation activity in rat vascular smooth muscle cells (VSMC), and for vasorelaxant activity employing rat aorta. 1 and 2 were 0.9 and 0.03 times as potent as alpha-hANP (7-28), respectively, in binding. Har-containing analogs (3-9) were as potent as alpha-hANP (7-28) in binding. Among the Har-containing analogs, [Har11,14]-alpha-hANP (7-28) (6) and [Har11,27]-alpha-hANP (7-28) (7) were remarkably vasorelaxant active, being 4.2 and 5.3 times potent than alpha-hANP (7-28), respectively, in spite of relatively lower cGMP accumulation activity in the case of 7. The roles of the chargeable amino acid residues in biological activity are discussed.     

Micro-enzyme immunoassay of vasoactive intestinal polypeptide (VIP)-like immunoreactive substance in bovine milk

A sensitive and specific enzyme immunoassay for vasoactive intestinal polypeptide (VIP)-like immunoreactivity was developed with the use of synthetic carboxy-terminal (C-terminal) fragment (residue 11-28) of porcine VIP conjugated with beta-D-galactosidase and a second antibody-coated immunoplate. Using 4-methylumbelliferyl beta-D-galactopyranoside as a fluorogenic substrate, the minimum amount of VIP-like immunoreactive substance (VIP-IS) detectable by this method was 0.1 fmol/well (2.5 pmol/l). The level of VIP-IS in bovine foremilk was above 100 pmol/l, which was more than eightfold higher than that in normal milk.     

Characterisation of adducts of the lipid peroxidation product 4-hydroxy-2-nonenal and amyloid beta-peptides by liquid chromatography/electrospray ionisation mass spectrometry

Alzheimer's disease is characterised by brain neuritic plaques composed of a 39-44 amino acid peptide (Abeta). Lipid peroxidation is an early event induced by these amyloid beta-peptides, leading to the formation of 4-hydroxy-2-nonenal (HNE), which is one of the major end products of this process. HNE has been reported to form adducts via a stable covalent binding to proteins through a Michael addition to amino acid residues with a nucleophilic side chain. The present study reports an investigation of the conditions for formation of Abeta-HNE (Abeta 1-28 and Abeta 1-42) adducts, and their characterisation by liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS). The results suggest that one or more HNE moieties are localised in the 6-16 region of these adducts, while Asp-1, Lys-16 and Lys-28 are not modified under the described reaction conditions.     

Synthesis of covalent probes for the radiolabeling of the cannabinoid receptor

The main psychoactive constituent of marijuana, (-)-Delta(9)-tetrahydrocannabinol, produces most of its physiological effects by interacting with the CB1 cannabinoid receptor, a membrane protein belonging to the large superfamily of G-protein coupled receptors. The 3-D structure of the receptor binding site is of value in the design of novel medications for a variety of therapeutic indications. To obtain information on the amino acid residues associated with this binding site, we have designed and synthesized a cannabinergic CB1 ligand prototype carrying an electrophilic isothiocyanato group capable of reacting covalently with amino acid residues bearing thiol or unprotonated amino groups. The ligand also incorporates an iodide atom, which can serve as a high-activity radiolabel. The key step in our synthesis involves a rapid intramolecular Diels-Alder reaction of a transiently formed o-quinone methide, which proceeds stereospecifically with the formation of the tricyclic cannabinoid template. Introduction of the iodo group is the last step in the sequence and is compatible with the use of (125)I-radiolabel.     

Synthesis and pharmacological characterization at glutamate receptors of erythro- and threo-tricholomic acid and homologues thereof

The erythro- and threo-amino-(3′-hydroxy-4′,5′-dihydro-isoxazol-5′-yl)-acetic acids, stereoisomers of tricholomic acid, were synthesized along with the corresponding higher homologues erythro- and threo-amino-(3′-carboxy-4′,5′-dihydro-isoxazol-5′-yl)-acetic acids. The target compounds were prepared via the 1,3-dipolar cycloaddition of a suitable nitrile oxide to (±)-2-tert-butoxycarbonylamino-3-buten-1-ol. Such a strategy allowed the synthesis of the two stereoisomeric amino acids in comparable amounts. The pharmacological activity of these compounds was investigated at ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs) by means of receptor binding assays to rat cortical membranes, electrophysiological tests and second messenger assays at cloned receptors expressed in CHO cells. Their pharmacological profiles were compared to those of l-glutamate and of the previously described selective NMDA receptor antagonists 5-(2-amino-2-carboxyethyl)-4,5-dihydroisoxazole-3-carboxylic acids in order to highlight the effect of increasing/reducing the distance between the amino acid moiety and the distal acid group, which represent the two pharmacophoric entities.     

Tunable inhibition and denaturation of alpha-chymotrypsin with amino acid-functionalized gold nanoparticles

Water-soluble gold nanoparticles bearing diverse l-amino acid terminals have been fabricated to probe the effect of receptor surface on protein surface binding. The interaction of these nanoparticles with alpha-chymotrypsin (ChT) was investigated by activity assay, gel electrophoresis, zeta-potential, circular dichroism, and fluorescence spectroscopy. The results show that both electrostatic and hydrophobic interactions between the hydrophobic patches of receptors and the protein contribute to the stability of the complex. The microscopic binding constants for these receptor-protein systems are 10(6)-10(7) M(-1), with the capacity of the nanoparticle receptors to bind proteins determined by both their surface area and their surface charge density. Furthermore, it is found that the hydrophilic side chains destabilize the ChT structure through either competitive hydrogen bonding or breakage of salt bridges, whereas denaturation was much slower with hydrophobic amino acid side chains. Significantly, correlation between the hydrophobicity index of amino acid side chains and the binding affinity and denaturation rates was observed.     

High-affinity mu opioid receptor ligands discovered by the screening of an exhaustively stereodiversified library of 1,5-enediols

In this communication, we report the synthesis of an exhaustively stereodiversified library of 16 1,5-enediols (2) and the screening of these compounds for mu opioid receptor (MOR) binding. The stereochemical configuration of 2 strongly impacted the binding affinity, and (S,S,S,R)-2 exhibited a Ki of 8.8 nM for MOR, comparable to that of endomorphin-2 (Ki = 1.2 nM). Moreover, compounds 2 exhibited 5-86-fold selectivity for MOR over delta opioid receptor (DOR) and 16-150-fold selectivity for MOR over kappa opioid receptor (KOR). Additionally, analogues of 2 were synthesized which showed the importance of the trans olefin for receptor binding but that modifications of the C-terminal amino acid were well tolerated. Ligand 11 is noteworthy because it retains only one of the amide bonds present in 1, but binds MOR with an affinity of 10 nM and 110- and 600-fold selectivity for MOR over DOR and KOR. These results demonstrate the utility of stereochemical diversity in the discovery of bioactive small molecules.     

Computational analysis of binding affinity and neural response at the l-alanine receptor

Summary A model of analogue-receptor binding is developed for the l-alanine receptor in the channel catfish using the AM1-SM2 and ab initio SCRF computational methods. Besides interactions involving the zwitterionic moiety of the amino acid analogue and complementary subsites on the receptor, the model suggests the presence of a hydrophobic pocket with dispersion interactions between the receptor and the residue on the amino acid analogue. Conformational analysis suggests not only a small compact active site on the receptor, but also that the analogues with the highest affinity occupy nearly identical regions of space. Although the binding interaction is dominated by the ionic terms, AM1-SM2 calculations indicate that free energy terms associated with cavity formation, solvent reorganization, and dispersion interactions can be correlated to activation and neural response. From a consideration of this model, molecular features of the analogues that are important for binding and neural response were deduced and other analogues or ligands were developed and tested.     

CHEMICAL DERIVATION OF HUMAN INSULIN SUPERAGONISM

The role of three highly conserved insulin residues Tyr^B26 was studied to better understand the relationship between insulin and receptor from rat adipose tissue plasma membranes, lnsulin analogues with a single amino acid substitution or single N-methylation of the peptide bond in the position B26 were all shortened in the C-terminus of the B-chain by four amino acids. The effect of modifications was followed by the binding to the insulin receptor. From our results, we can deduce several conclusions： （1） the replacement of tyrosine in the position B26 by histidine, [N-MeHis^B26]-des-tetrapeptide-（B27-B30）-insulin-B26-amide and [N-MeGlu^B26]-des-tetrapeptide- （B2-B30）-insulin-B26-amide, have no significant effect on the binding affinity and they show binding affinity 105%, 190% and 208%, respectively, of that of human insulin; （2） [Aad^B26] -des-tetrapeptide-（B27-B30）-insulin-B26-amide and [Phe（4-carboxy^B26）]-des-tetrapeptide- （B27~B30）-insulin-B26-amide affect the potency highly positively in vitro studies; they show binding affinity 529 and 289 %, respectively, of that of human insulin.     

Selection of synthetic receptors capable of sensing the difference in binding of d-Ala-d-Ala or d-Ala-d-Lac ligands by screening of a combinatorial CTV-based library

Screening of a combinatorial CTV-based artificial, synthetic receptor library 1 {1-13, 1-13, 1-13} for binding of a variety d-Ala-d-Ala and d-Ala-d-Lac containing ligands (6-11) was carried out in phosphate buffer (0.1 N, pH=7.0). After screening and Edman sequencing, synthetic receptors were found containing amino acid sequences, which are either characteristic for binding dye labeled d-Ala-d-Ala or d-Ala-d-Lac containing ligands. For example, receptors capable of binding d-Ala-d-Ala containing ligands 6, 7, 9 and 11 contained—almost in all cases—at least one basic amino acid residue—predominantly Lys—in their arms. This was really a striking difference with the arms of the receptors capable of binding d-Ala-d-Lac containing ligands 8 and 10, which usually contained a significant number of polar amino acids (Gln and Ser), especially in ligand 8, but hardly any basic amino acids. Use of different (fluorescent) dye labels showed that the label has a profound, albeit not decisive, influence on the binding by the receptor. A hit from the screening of the CTV-library with FITC-peptidoglycan (6) was selected for resynthesis and validation.     

Carboxylate binding by 2-(guanidiniocarbonyl)pyrrole receptors in aqueous solvents: improving the binding properties of guanidinium cations through additional hydrogen bonds

A series of guanidiniocarbonyl pyrrole receptors has been synthesized which bind carboxylates by ion pairing in combination with multiple hydrogen bonds. Their binding properties with various carboxylates have been investigated using NMR titration studies in 40% water/DMSO (v/v). The best receptor has association constants which are in the order of K approximately/= 10(3) mol(-1) and hence some 30 times larger than with the simple acetyl guanidinium cation. Through a systematic variation of the receptor structure, semiquantitative estimates for the energetic contributions of the individual binding interactions could be derived. These data show that the various hydrogen bonds are not equally important for the binding but differ significantly in their energetic contribution to the overall complexation process. Furthermore, the receptor can be made chiral and shows selectivity upon binding of enantiomeric amino acid carboxylates. Molecular modeling was used to obtain structural information for the various receptor carboxylate complexes and served as a basis to explain the observed differences in binding constants.     

Effects of hydrogen-bonding and stacking interactions with amino acids on the acidity of uracil

The effects of hydrogen-bonding interactions with amino acids on the (N1) acidity of uracil are evaluated using (B3LYP) density functional theory. Many different binding arrangements of each amino acid to three uracil binding sites are considered. The effects on the uracil acidity are found to significantly depend upon the nature of the amino acid and the binding orientation, but weakly depend on the binding site. Our results reveal that in some instances small models for the amino acids can be used, while for other amino acids larger models are required to properly describe the binding to uracil. The gas-phase acidity of uracil is found to increase by up to approximately 60 kJ mol(-1) due to discrete hydrogen-bonding interactions. Although (MP2) stacking interactions with aromatic amino acids decrease the acidity of uracil, unexpected increases in the acidity are found when any of the aromatic amino acids, or the backbone, hydrogen bond to uracil. Consideration of enzymatic and aqueous environments leads to decreases in the effects of the amino acids on the acidity of uracil. However, we find that the magnitude of the decrease varies with the nature of the molecule bound, as well as the (gas-phase) binding orientations and strengths, and therefore solvation effects should be considered on a case-by-case basis in future work. Nevertheless, the effects of amino acid interactions within enzymatic environments are as much as approximately 35 kJ mol(-1). The present study has general implications for understanding the nature of active site amino acids in enzymes, such as DNA repair enzymes, that catalyze reactions involving anionic nucleobase intermediates.     

Dynamics of Zn(II) binding as a key feature in the formation of amyloid fibrils by Aβ11-28

Supramolecular assembly of peptides and proteins into amyloid fibrils is of multifold interest, going from materials science to physiopathology. The binding of metal ions to amyloidogenic peptides is associated with several amyloid diseases, and amyloids with incorporated metal ions are of interest in nanotechnology. Understanding the mechanisms of amyloid formation and the role of metal ions can improve strategies toward the prevention of this process and enable potential applications in nanotechnology. Here, studies on Zn(II) binding to the amyloidogenic peptide Aβ11-28 are reported. Zn(II) modulates the Aβ11-28 aggregation, in terms of kinetics and fibril structures. Structural studies suggest that Aβ11-28 binds Zn(II) by amino acid residues Glu11 and His14 and that Zn(II) is rapidly exchanged between peptides. Structural and aggregation data indicate that Zn(II) binding induces the formation of the dimeric Zn(II)(1)(Aβ11-28)(2) species, which is the building block of fibrillar aggregates and explains why Zn(II) binding accelerates Aβ11-28 aggregation. Moreover, transient Zn(II) binding, even briefly, was enough to promote fibril formation, but the final structure resembled that of apo-Aβ11-28 amyloids. Also, seeding experiments, i.e., the addition of fibrillar Zn(II)(1)(Aβ11-28)(2) to the apo-Aβ11-28 peptide, induced aggregation but not propagation of the Zn(II)(1)(Aβ11-28)(2)-type fibrils. This can be explained by the dynamic Zn(II) binding between soluble and aggregated Aβ11-28. As a consequence, dynamic Zn(II) binding has a strong impact on the aggregation behavior of the Aβ11-28 peptide and might be a relevant and so far little regarded parameter in other systems of metal ions and amyloidogenic peptides.     

Heteroditopic receptor based on crown ether and cyclen units for the recognition of zwitterionic amino acids

The molecular recognition of five targeted amino acids differing in the nature of the side (R)- group and in the size of the aliphatic chain, glycine (Hgly), phenylalanine (Hphe), glutamic acid (Hglu^?), 4-aminobutyric acid (Hgaba), and 6-aminohexanoic acid (Heahx), has been studied with a new heteroditopic receptor based in two distinct macrocycles, a cyclen and a crown ether moiety. The bismacrocycle L was synthesized via the bis-aminal route allowing to obtain the designed compound in gram scale and in good yield. Protonation constants of L and its binding constants with amino acids were determined by potentiometry in H₂O/MeOH (1:1 v/v) solutions at 298.2 K and I=0.10 mol dm^?3 in NMe₄NO₃. Stronger binding ability of the H_nLⁿ⁺ receptor for α-amino acids, Hgly and Hphen, than for the other studied substrates were found. Structural data derived from NMR studies showed that the binding of α-amino acids result from the cooperative participation of hydrogen bonds between the carboxylate group of amino acids and the polyammonium sites of cyclen, and the ion-dipole interactions between the ammonium group of the amino acids and the oxygen atoms of the crown ether.     

TERS Detection of αVβ3 Integrins in Intact Cell Membranes

Integrins are important membrane receptors that form focal adhesions with the extracellular matrix and are transmembrane signaling proteins. We demonstrate that nanoparticles functionalized with c‐RGDfC ligands bind to intact cell membranes and selectively enhance the amino acid signals of the integrin receptor when coupled with tip‐enhanced Raman scattering (TERS) detection. Controlling the plasmonic interaction between the functionalized nanoparticle and the TERS tip provides a clear Raman signal from α_Vβ₃ integrins in the cell membrane that matches the signal of the purified integrin receptor. Random aggregation of nanoparticles on the cell does not provide the same spectral information. Chemical characterization of membrane receptors in intact cellular membranes is important for understanding membrane signaling and drug targeting. These results provide a new method to investigate the chemical interactions associated with ligand binding to membrane receptors in cells.     

Synthetic receptors for the differentiation of phosphorylated peptides with nanomolar affinities

Artificial ditopic receptors for the differentiation of phosphorylated peptides varying in i+3 amino acid side chains were synthesized, and their binding affinities and selectivities were determined. The synthetic receptors show the highest binding affinities to phosphorylated peptides under physiological conditions (HEPES, pH 7.5, 154 mM NaCl) reported thus far for artificial systems. The tight and selective binding was achieved by high cooperativity of the two binding moieties in the receptor molecules. All receptors interact with phosphorylated serine by bis(ZnII-cyclen) complex coordination and a second binding site recognizing a carboxylate or imidazole amino acid side chain functionality.     

Chiral Recognition of Amino Acid Esters in Organic Solvents Using a Glucose-Based Receptor

Due to the chemical and biological relevance of amino acids, efficient methods for the recognition and separation of their enantiomers are highly sought after. Chiral receptors based on extended molecular scaffolds are typically employed for this purpose. These receptors are often effective only in specific environments and towards a narrow scope of amino acid guests. Recently we reported a simple, glucose-based macrocycle capable of enantioselective binding of a broad range of amino acid methyl esters in water. Herein we demonstrate that the same receptor can be used for chiral recognition of amino acid esters in organic solvents. We show that the binding affinity and selectivity of the receptor are highly dependent on the coordinating strength of the solvent. An in-depth analysis of the receptor’s conformation and its interactions with amino acid methyl esters allowed us to propose a binding mode of amino acids to the receptor in CDCl₃. The binding modes in CDCl₃ and D₂O were then compared, highlighting the main interactions responsible for binding affinity and selectivity in each solvent. We envision that the insight provided by this study will facilitate the development of further amino acid receptors based on monosaccharides with improved binding affinities and both enantio- as well as chemoselectivities.     

胆甾类分子钳对氨基酸衍生物的对映选择性识别

用差紫外光谱滴定法考察了以脱氧胆酸作spacer的手性分子钳1～3对一系列α-氨基酸甲酯的对映选择性识别性能。结果表明,分子钳1和2与客体氨基酸甲酯形成1:1型超分子配合物,并显示较好的手性识别能力。分钳3对所考察的氨基酸甲酯均没有明显的识别作用。讨论了主-客体间尺寸/形状匹配、几何互补等因素对形成超分子配合物的影响,并利用计算机模拟作辅助手段对实验结果和现象进行了解释。