Foldamers controlled by functional triamino acids: structural investigation of α/γ-hybrid oligopeptides.

Peptide-like foldamers controlled by normal amide backbone hydrogen bonding have been extensively studied, and their folding patterns largely rely on configurational and conformational constraints induced by the steric properties of backbone substituents at appropriate positions. In contrast, opportunities to influence peptide secondary structure by functional groups forming individual hydrogen bond networks have not received much attention. Here, peptide-like foldamers consisting of alternating α,ß,γ-triamino acids 3-amino-4-(aminomethyl)-2-methylpyrrolidine-3-carboxylate (AAMP) and natural amino acids glycine and alanine are reported, which were obtained by solution phase peptide synthesis. They form ordered secondary structures, which are dominated by a three-dimensional bridged triazaspiranoid-like hydrogen bond network involving the non-backbone amino groups, the backbone amide hydrogen bonds, and the relative configuration of the α,ß,γ-triamino and α-amino acid building blocks. This additional stabilization leads to folding in both nonpolar organic as well as in aqueous environments. The three-dimensional arrangement of the individual foldamers is supported by X-ray crystallography, NMR spectroscopy, chiroptical methods, and molecular dynamics simulations.

Efficiently Computing NMR 1H and 13C Chemical Shifts of Saccharides in Aqueous Environment.

Determining the structure of saccharides in their native environment is crucial to understanding their function and more accurately targeting their utilization. Nuclear magnetic resonance observables such as the nuclear Overhauser effect or spin-spin coupling constants are routinely utilized to study saccharides in their native water environment. However, while highly sensitive to the local environment, chemical shifts are mostly overlooked, despite being commonly measured for compounds identification. Although chemical shifts carry considerable structural information, their direct association with structure is notoriously difficult. This is mostly due to the similarity in the chemical nature of most saccharides causing similar physicochemical environments close to sugar C and H atoms, resulting in comparable chemical shifts. The rise of computational power allows one to compute reliable chemical shifts and use them to determine atomistic details of these sugars in solution. However, any prediction is severely limited by the computational protocol used and its accuracy. In this work, we studied a set of 31 saccharides on which we evaluated various computational protocols to calculate the total number of 375 1H and 327 13C chemical shifts of sugars in an aqueous environment. Our study proposes two cost-effective protocols for simulating 1H and 13C chemical shifts that we recommend for further use. These protocols can help with the interpretation of experimental spectra, but we also show that they are also capable of structure prediction independently. This is possible because of the low mean absolute deviations of calculated shifts from the experiment (0.06 ppm for 1H and 1.09 ppm for 13C). We explore different solvation methods, basis sets, and optimization schemes to reach such accuracy. A correct sampling of the conformation phase space of flexible sugar molecules is also key to obtaining accurately converged theoretical chemical shifts. The linear regression method was applied to convert the calculated isotropic nuclear magnetic shielding constants to simulated chemical shifts comparable with the experiment. The achieved level of accuracy can help in utilizing chemical shifts for elucidating the 3D atomistic structure of saccharides in aqueous solutions. All linear regression parameters obtained on our extensive set of sugars for all the tested protocols can be reutilized in future works.

Use of Raman and Raman optical activity to extract atomistic details of saccharides in aqueous solution.

Sugars are crucial components in biosystems and industrial applications. In aqueous environments, the natural state of short saccharides or charged glycosaminoglycans is floating and wiggling in solution. Therefore, tools to characterize their structure in a native aqueous environment are crucial but not always available. Here, we show that a combination of Raman/ROA and, on occasions, NMR experiments with Molecular Dynamics (MD) and Quantum Mechanics (QM) is a viable method to gain insights into structural features of sugars in solutions. Combining these methods provides information about accessible ring puckering conformers and their proportions. It also provides information about the conformation of the linkage between the sugar monomers, i.e., glycosidic bonds, allowing for identifying significantly accessible conformers and their relative abundance. For mixtures of sugar moieties, this method enables the deconvolution of the Raman/ROA spectra to find the actual amounts of its molecular constituents, serving as an effective analytical technique. For example, it allows calculating anomeric ratios for reducing sugars and analyzing more complex sugar mixtures to elucidate their real content. Altogether, we show that combining Raman/ROA spectroscopies with simulations is a versatile method applicable to saccharides. It allows for accessing many features with precision comparable to other methods routinely used for this task, making it a viable alternative. Furthermore, we prove that the proposed technique can scale up by studying the complicated raffinose trisaccharide, and therefore, we expect its wide adoption to characterize sugar structural features in solution.

Binding of divalent cations to acetate: molecular simulations guided by Raman spectroscopy.

In spite of the biological importance of the binding of Zn2+, Ca2+, and Mg2+ to the carboxylate group, cation-acetate binding affinities and binding modes remain actively debated. Here, we report the first use of Raman multivariate curve resolution (Raman-MCR) vibrational spectroscopy to obtain self-consistent free and bound metal acetate spectra and one-to-one binding constants, without the need to invoke any a priori assumptions regarding the shapes of the corresponding vibrational bands. The experimental results, combined with classical molecular dynamics simulations with a force field effectively accounting for electronic polarization via charge scaling and ab initio simulations, indicate that the measured binding constants pertain to direct (as opposed to water separated) ion pairing. The resulting binding constants do not scale with cation size, as the binding constant to Zn2+ is significantly larger than that to either Mg2+ or Ca2+, although Zn2+ and Mg2+ have similar radii that are about 25% smaller than Ca2+. Remaining uncertainties in the metal acetate binding free energies are linked to fundamental ambiguities associated with identifying the range of structures pertaining to non-covalently bound species.

Raman Optical Activity of Glucose and Sorbose in Extended Wavenumber Range.

Raman optical activity (ROA) is pursued as a promising method for structural analyses of sugars in aqueous solutions. In the present study, experimental Raman and ROA spectra of glucose and sorbose obtained in an extended range (50-4000 cm-1 ) are interpreted using molecular dynamics and density functional theory, with the emphasis on CH stretching modes. A reasonable theoretical basis for spectral interpretation was obtained already at the harmonic level. Anharmonic corrections led to minor shifts of band positions (up to 25 cm-1 ) below 2000 cm-1 , while the CH stretching bands shifted more, by ∼180 cm-1 , and better reproduced the experiment. However, the anharmonicities could be included on a relatively low approximation level only, and they did not always improve the harmonic band shapes. The dependence on the structure and conformation shows that the CH stretching ROA spectral pattern is a sensitive marker useful in saccharide structure studies.

Simulation of Raman and Raman optical activity of saccharides in solution.

Structural studies of sugars in solution are challenging for most of the traditional analytical techniques. Raman and Raman optical activity (ROA) spectroscopies were found to be extremely convenient for this purpose. However, Raman and ROA spectra of saccharides are challenging to interpret and model due to saccharides' flexibility and polarity. In this study, we present an optimized computational protocol that enables the simulation of the spectra efficiently. Our protocol, which results in good agreement with experiments, combines molecular dynamics and density functional theory calculations. It further uses a smart optimization procedure and a novel adaptable scaling function. The numerical stability and accuracy of individual computational steps are evaluated by comparing simulated and experimental spectra of d-glucose, d-glucuronic acid, N-acetyl-d-glucosamine, methyl ß-d-glucopyranoside, methyl ß-d-glucuronide, and methyl ß-N-acetyl-d-glucosaminide. Overall, our Raman and ROA simulation protocol allows one to routinely and reliably calculate the spectra of small saccharides and opens the door to advanced applications, such as complete 3-dimensional structural determination by direct interpretation of the experimental spectra.

Can Arginine Inhibit Insulin Aggregation? A Combined Protein Crystallography, Capillary Electrophoresis, and Molecular Simulation Study.

The oligomeric state of the storage form of human insulin in the pancreas, which may be affected by several endogenous components of ß-cell storage granules such as arginine, is not known. Here, the effect of arginine on insulin oligomerization is investigated independently by protein crystallography, molecular dynamics simulations, and capillary electrophoresis. The combined results point to a strong effect of ionic strength on insulin assembly. Molecular simulations and electrophoretic measurements at low/mM salt concentrations show no significant effect of arginine on insulin aggregation. In contrast, crystallographic data at high/molar ionic strength indicate inhibition of insulin hexamerization by arginine due to its binding at the site relevant for intermolecular contacts, which was also observed in MD simulations. Our results thus bracket the in vivo situation in pancreatic ß-cell storage granules, where the ionic strength is estimated to be in the hundreds of millimolar to submolar range. The present findings add to a molecular understanding of in vivo insulin oligomerization and storage, with additional implications for insulin stability in arginine-rich injections.

Computational and structural evidence for neurotransmitter-mediated modulation of the oligomeric states of human insulin in storage granules.

Human insulin is a pivotal protein hormone controlling metabolism, growth, and aging and whose malfunctioning underlies diabetes, some cancers, and neurodegeneration. Despite its central position in human physiology, the in vivo oligomeric state and conformation of insulin in its storage granules in the pancreas are not known. In contrast, many in vitro structures of hexamers of this hormone are available and fall into three conformational states: T6, T3Rf3, and R6 As there is strong evidence for accumulation of neurotransmitters, such as serotonin and dopamine, in insulin storage granules in pancreatic ß-cells, we probed by molecular dynamics (MD) and protein crystallography (PC) if these endogenous ligands affect and stabilize insulin oligomers. Parallel studies independently converged on the observation that serotonin binds well within the insulin hexamer (site I), stabilizing it in the T3R3 conformation. Both methods indicated serotonin binding on the hexamer surface (site III) as well. MD, but not PC, indicated that dopamine was also a good site III ligand. Some of the PC studies also included arginine, which may be abundant in insulin granules upon processing of pro-insulin, and stable T3R3 hexamers loaded with both serotonin and arginine were obtained. The MD and PC results were supported further by in solution spectroscopic studies with R-state-specific chromophore. Our results indicate that the T3R3 oligomer is a plausible insulin pancreatic storage form, resulting from its complex interplay with neurotransmitters, and pro-insulin processing products. These findings may have implications for clinical insulin formulations.

DNA lesion can facilitate base ionization: vertical ionization energies of aqueous 8-oxoguanine and its nucleoside and nucleotide.

8-Oxoguanine is one of the key products of indirect radiation damage to DNA by reactive oxygen species. Here, we describe ionization of this damaged nucleobase and the corresponding nucleoside and nucleotide in aqueous phase, modeled by the nonequilibrium polarizable continuum model, establishing their lowest vertical ionization energies of 6.8-7.0 eV. We thus confirm that 8-oxoguanine has even lower ionization energy than the parental guanine, which is the canonical nucleobase with the lowest ionization energy. Therefore, it can act as a trap for the cationic hole formed by ionizing radiation and thus protect DNA from further radiation damage. We also model using time-dependent density functional theory and measure by liquid jet photoelectron spectroscopy the valence photoelectron spectrum of 8-oxoguanine in water. We show that the calculated higher lying ionization states match well the experiment which, however, is not sensitive enough to capture the electron signal corresponding to the lowest ionization process due to the low solubility of 8-oxoguanine in water.