Using Physical Chemistry To Differentiate Nicotinic from Cholinergic Agonists at the Nicotinic Acetylcholine Receptor

The binding of three distinct agonists - acetylcholine (ACh), nicotine, and epibatidine - to the nicotinic acetylcholine receptor has been probed using unnatural amino acid mutagenesis. ACh makes a cation−π interaction with Trp α149, while nicotine employs a hydrogen bond to a backbone carbonyl in the same region of the agonist binding site. The nicotine analogue epibatidine achieves its high potency by taking advantage of both the cation−π interaction and the backbone hydrogen bond. A simple structural model that considers only possible interactions with Trp α149 suggests that a novel aromatic C - H···O=C hydrogen bond further augments the binding of epibatidine. These studies illustrate the subtleties and complexities of the interactions between drugs and membrane receptors and establish a paradigm for obtaining detailed structural information

A fluorophore attached to nicotinic acetylcholine receptor beta M2 detects productive binding of agonist to the alpha delta site

To study conformational transitions at the muscle nicotinic acetylcholine (ACh) receptor (nAChR), a rhodamine fluorophore was tethered to a Cys side chain introduced at the beta-19' position in the M2 region of the nAChR expressed in Xenopus oocytes. This procedure led to only minor changes in receptor function. During agonist application, fluorescence increased by (Delta-F/F) approximate to 10%, and the emission peak shifted to lower wavelengths, indicating a more hydrophobic environment for the fluorophore. The dose-response relations for Delta-F agreed well with those for epibatidine-induced currents, but were shifted approximate to 100-fold to the left of those for ACh-induced currents. Because (i) epibatidine binds more tightly to the alpha-gamma-binding site than to the alpha-delta site and (ii) ACh binds with reverse-site selectivity, these data suggest that Delta-F monitors an event linked to binding specifically at the alpha-delta-subunit interface. In experiments with flash-applied agonists, the earliest detectable Delta-F occurs within milliseconds, i.e., during activation. At low [ACh] (less than or equal to 10 muM), a phase of Delta-F occurs with the same time constant as desensitization, presumably monitoring an increased population of agonist-bound receptors. However, recovery from Delta-F is complete before the slowest phase of recovery from desensitization (time constant approximate to 250 s), showing that one or more desensitized states have fluorescence like that of the resting channel. That conformational transitions at the alpha-delta-binding site are not tightly coupled to channel activation suggests that sequential rather than fully concerted transitions occur during receptor gating. Thus, time-resolved fluorescence changes provide a powerful probe of nAChR conformational changes

High-Resolution Structure of a Beta-Peptide Bundle

We recently reported that β-peptides can form discrete hetero-oligomers in aqueous solution. Here we describe the structure of such an oligomer as determined by X-ray crystallography. The structure of Zwit-1F reveals a homo-octamer of two cupped “hands” composed of both parallel and antiparallel 314-helices. The core of the assembly is composed entirely of solvent-excluded β3-homoleucine residues. The Zwit-1F assembly shares many of the physical characteristics of natural proteins

Biophysical Characterization of a Beta-Peptide Bundle: Comparison To Natural Proteins

We recently described the high-resolution X-ray structure of a helical bundle composed of eight copies of the β-peptide Zwit-1F. Like many proteins in Nature, the Zwit-1F octamer contains parallel and antiparallel helices, extensive inter-helical electrostatic interactions, and a solvent-excluded hydrophobic core. Here we explore the stability of the Zwit-1F octamer using circular dichroism (CD) spectroscopy, analytical ultracentrifugation (AU), differential scanning calorimetry (DSC), and NMR. These studies demonstrate that the thermodynamic and kinetic properties of Zwit-1F closely resemble those of α-helical bundle proteins. Together these studies should provide a model for the design of β-peptide proteins with biological functions

Biophysical and Structural Characterization of a Robust Octameric Beta-Peptide Bundle

Proteins composed of α-amino acids are essential components of the machinery required for life. Stanley Miller\u27s renowned electric discharge experiment provided evidence that an environment of methane, ammonia, water, and hydrogen was sufficient to produce α-amino acids. This reaction also generated other potential protein building blocks such as the β-amino acid β-glycine (also known as β-alanine); however, the potential of these species to form complex ordered structures that support functional roles has not been widely investigated. In this report we apply a variety of biophysical techniques, including circular dichroism, differential scanning calorimetry, analytical ultracentrifugation, NMR and X-ray crystallography, to characterize the oligomerization of two 12-mer β3-peptides, Acid-1Y and Acid-1Y*. Like the previously reported β3-peptide Zwit-1F, Acid-1Y and Acid-1Y* fold spontaneously into discrete, octameric quaternary structures that we refer to as β-peptide bundles. Surprisingly, the Acid-1Y octamer is more stable than the analogous Zwit-1F octamer, in terms of both its thermodynamics and kinetics of unfolding. The structure of Acid-1Y, reported here to 2.3 Å resolution, provides intriguing hypotheses for the increase in stability. To summarize, in this work we provide additional evidence that nonnatural β-peptide oligomers can assemble into cooperatively folded structures with potential application in enzyme design, and as medical tools and nanomaterials. Furthermore, these studies suggest that nature\u27s selection of α-amino acid precursors was not based solely on their ability to assemble into stable oligomeric structures

Caged Phosphoproteins

We present the chemical and biological synthesis of caged phosphoproteins using the in vitro nonsense codon suppression methodology. Specifically, phosphoamino acid analogues of serine, threonine, and tyrosine with a single photocleavable o-nitrophenylethyl caging group were synthesized as the amino acyl tRNA adducts for insertion into full-length proteins. For this purpose, a novel phosphitylating agent was developed. The successful incorporation of these bulky and charged amino acids into the α-subunit of the nicotinic acetyl choline receptor (nAChR) and the vasodilator-stimulated phosphoprotein (VASP) using an in vitro translation system is reported

Improving the fluorescent probe acridonylalanine through a combination of theory and experiment

Acridonylalanine (Acd) is a useful fluorophore for studying proteins by fluorescence spectroscopy, but it can potentially be improved by being made longer wavelength or brighter. Here, we report the synthesis of Acd core derivatives and their photophysical characterization. We also performed ab initio calculations of the absorption and emission spectra of Acd derivatives, which agree well with experimental measurements. The amino acid aminoacridonylalanine (Aad) was synthesized in forms appropriate for genetic incorporation and peptide synthesis. We show that Aad is a superior Förster resonance energy transfer acceptor to Acd in a peptide cleavage assay and that Aad can be activated by an aminoacyl tRNA synthetase for genetic incorporation. Together, these results show that we can use computation to design enhanced Acd derivatives, which can be used in peptides and proteins

Improving the fluorescent probe acridonylalanine through a combination of theory and experiment

Acridonylalanine (Acd) is a useful fluorophore for studying proteins by fluorescence spectroscopy, but it can potentially be improved by being made longer wavelength or brighter. Here, we report the synthesis of Acd core derivatives and their photophysical characterization. We also performed ab initio calculations of the absorption and emission spectra of Acd derivatives, which agree well with experimental measurements. The amino acid aminoacridonylalanine (Aad) was synthesized in forms appropriate for genetic incorporation and peptide synthesis. We show that Aad is a superior Förster resonance energy transfer acceptor to Acd in a peptide cleavage assay and that Aad can be activated by an aminoacyl tRNA synthetase for genetic incorporation. Together, these results show that we can use computation to design enhanced Acd derivatives, which can be used in peptides and proteins