Rational Design of α-Helix-Stabilized Exendin-4 Analogues

Exendin-4 (Ex4) is a potent glucagon-like peptide-1 receptor agonist, a drug regulating the plasma glucose level of patients suffering from type 2 diabetes. The molecule’s poor solubility and its readiness to form aggregates increase the likelihood of unwanted side effects. Therefore, we designed Ex4 analogues with improved structural characteristics and better water solubility. Rational design was started from the parent 20-amino acid, well-folded Trp cage (TC) miniprotein and involved the step-by-step N-terminal elongation of the TC head, resulting in the 39-amino acid Ex4 analogue, E19. Helical propensity coupled to tertiary structure compactness was monitored and quantitatively analyzed by electronic circular dichroism and nuclear magnetic resonance (NMR) spectroscopy for the 14 peptides of different lengths. Both 15N relaxation- and diffusion-ordered NMR measurements were established to investigate the inherent mobility and self-association propensity of Ex4 and E19. Our designed E19 molecule has the same tertiary structure as Ex4 but is more helical than Ex4 under all studied conditions; it is less prone to oligomerization and has preserved biological activity. These conditions make E19 a perfect lead compound for further drug discovery. We believe that this structural study improves our understanding of the relationship between local molecular features and global physicochemical properties such as water solubility and could help in the development of more potent Ex4 analogues with improved pharmacokinetic properties

Assignment of vibrational circular dichroism cross-referenced electronic circular dichroism spectra of flexible foldamer building blocks: towards assigning foldamers’ pure chiroptical properties

The assignment of the most established ECD spectra of polypeptides and foldamers is either “evidence based” or rely on the 3D-structures of longer oligomers of limited internal dynamics, derived from NMR (or X-ray) data. Critics warn that using NMR and ECD side by side have severe limitations for flexible molecules as the explicit knowledge of the conformational ensembles is a challenge. Herein we present the old-new method of comparing ab initio computed and measured VCD data to validate both structures and, conf(i), and their relative weights, c(i), making up the conformational ensemble. Based on the array of {conf(i), c(i)} the pure ECD spectra, g(i)conf(i), can be ab initio calculated. The reconstructed spectrum Σc(i)*g(i)conf(i) can thus help to assign any experimental ECD counterpart. Here we present such a protocol successfully applied for flexible foldamer building blocks of sugar β-amino acid diamides. The epimeric pair of our model system was selected because these molecules are conformationally tunable by simple chemical modification (N-methylation) and thus, the robustness of our current approach could be probed. The initial H-bond (NH..O) eliminated by N-methylation reorients the amide plain influencing the chiroptical properties of the foldamer building block, a structural change successfully monitored by the VCD- and ECD-transition changes now assigned to pure conformers. The current method seems general and effective without requiring extensive CPU and spectroscopic resources

Predictable Conformational Diversity in Foldamers of Sugar Amino Acids

Systematic conformational search was carried out for monomers and homohexamers of furanoid β-amino acids: cis-(S,R) and trans-(S,S) stereoisomers of aminocyclopentane carboxylic acid (ACPC), two different aminofuranuronic-acids (AFU(alpha) and AFUβ), their isopropylidene derivatives (AFU(ip)) as well as the key intermediate β-aminotetrahydrofurancarboxylic acid (ATFC). Stereochemistry of the building blocks was chosen to match with that of natural sugar amino acid (xylose and ribose) precursors (XylAFU and RibAFU). Results show that hexamers of cis furanoid beta-amino acids show great variability: while hydrophobic cyclopentane (cis(ACPC)6), and hydrophilic (XylAFU(alpha)/(beta))6 foldamers favor two different zigzagged conformation as hexamers, the backbone fold turns into a helix in case of (cisATFC)6 (10-helix) and (XylAFU(ip))6 (14-helix). Trans stereochemistry resulted in hexamers exclusively of right-handed helix conformation, (H12P)6, regardless of their polarity. We found that the preferred oligomeric structure of XylAFU(alpha)/(beta) is conformationally compatible with beta-pleated sheets, while that of the trans/(S,S) units match with alpha-helices of proteins

Can NO2+ exist in bent or cyclic forms?

Calculations of NO2+ at HF, CBS-4, CASSCF, MBPT(2), MBPT(3), and MBPT(4) theory levels, using 3-21G and 6-31G(d) basis sets, found two C-2V structures along with the linear geometry. Computations using MBPT(2) and CCSD(T) approaches and the aug-cc-pvtz basis set confirmed these results. Harmonic vibrational frequency calculations, performed with MBPT(2) and CCSD(T) theories, indicated that the linear structure was the global minimum while one of the bent structures (angle ONO = 80 degrees) was a higher energy local minimum. The second C-2V structure (angle ONO = 45 degrees) exhibited a large imaginary vibrational frequency along the asymmetric stretching (B-2) mode, indicating its saddle point nature. (C) 2001 Elsevier Science B.V. All rights reserved

The route from the folded to the amyloid state: exploring the potential energy surface of a drug-like miniprotein

The amyloid formation of the folded segment of a variant of Exenatide (a marketed drug for Type-2 Diabetes Mellitus ) was studied by ECD and NMR. We found that the optimum temperature for E5 protein amyloidosis coincides with body temperature and requires well below physiological salt concentration. Decomposition of the ECD spectra and its barycentric representation on the folded-unfolded-amyloid potential energy surface allowed us to monitor the full range of molecular transformation of amyloidogenesis. We identified points of no return ( e.g. T =37°C, pH =4.1, c E5 =250µM, c NaCl =50mM, t >4-6 h) which will inevitably gravitate into the amyloid-state. The strong B-type FUV-ECD spectra and an unexpectedly strong NUV-ECD signal (Θ ~275-285nm ) indicate that the amyloid phase of E5 is built from monomers of quasi -elongated backbone structure ( φ ~-145°, ψ ~+145°) with strong interstrand Tyr↔Trp interaction. Misfolded intermediers and the buildup of "toxic" early-stage oligomers leading to self-association were identified and monitored as function of time. Results indicate that the amyloid transition is triggered by subtle misfolding of the α-helix exposing aromatic and hydrophobic side chains that may provide the first centers for an intermolecular reorganization. These initial clusters provide the spatial closeness and sufficient time for a transition to the β-structured amyloid nucleus thus the process follows a nucleated growth mechanism

Proteins’ fold compactness alters disulfide-bond reducibility by 3 orders of magnitude: a comprehensive kinetic case study on the reduction of different size Trp-cage model proteins

We derived a novel approach to monitor disulfide bond reduction in the vicinity of aromatic cluster(s) by using the near-UV range (266–293 nm) of ECD spectra. By using combined NMR- and ECD-spectroscopy we have determined the 3D-fold characteristics and the associated reduction rate constants (k) of E19_SS – a highly thermostable; disulfide bond reinforced 39-amino acid long Exenatide mimetic – and its N-terminally truncated derivatives at different experimental conditions. Single SS-bond reduction of the E19_SS model (using 18-fold excess of TCEP, pH = 7, 37 °C) takes hours, 20-30 times longer than expected, thus would not reach completion applying the commonly used reduction protocols. We found that structural, steric and electrostatic factors influence the reduction rate, resulting in magnitude differences in reduction halftimes (900 > t½ > 1 min) even for structurally similar, well-folded derivatives of a small model-protein

A mathematical and computational review of Hartree-Fock SCF methods in Quantum Chemistry

We present here a review of the fundamental topics of Hartree-Fock theory in Quantum Chemistry. From the molecular Hamiltonian, using and discussing the Born-Oppenheimer approximation, we arrive to the Hartree and Hartree-Fock equations for the electronic problem. Special emphasis is placed in the most relevant mathematical aspects of the theoretical derivation of the final equations, as well as in the results regarding the existence and uniqueness of their solutions. All Hartree-Fock versions with different spin restrictions are systematically extracted from the general case, thus providing a unifying framework. Then, the discretization of the one-electron orbitals space is reviewed and the Roothaan-Hall formalism introduced. This leads to a exposition of the basic underlying concepts related to the construction and selection of Gaussian basis sets, focusing in algorithmic efficiency issues. Finally, we close the review with a section in which the most relevant modern developments (specially those related to the design of linear-scaling methods) are commented and linked to the issues discussed. The whole work is intentionally introductory and rather self-contained, so that it may be useful for non experts that aim to use quantum chemical methods in interdisciplinary applications. Moreover, much material that is found scattered in the literature has been put together here to facilitate comprehension and to serve as a handy reference.Comment: 64 pages, 3 figures, tMPH2e.cls style file, doublesp, mathbbol and subeqn package

Pseudouridylation defect due to DKC1 and NOP10 mutations causes nephrotic syndrome with cataracts, hearing impairment, and enterocolitis

Research UK Innovation and Project; Cancer Research U

Cooperativity among Short Amyloid Stretches in Long Amyloidogenic Sequences

Amyloid fibrillar aggregates of polypeptides are associated with many neurodegenerative diseases. Short peptide segments in protein sequences may trigger aggregation. Identifying these stretches and examining their behavior in longer protein segments is critical for understanding these diseases and obtaining potential therapies. In this study, we combined machine learning and structure-based energy evaluation to examine and predict amyloidogenic segments. Our feature selection method discovered that windows consisting of long amino acid segments of ∼30 residues, instead of the commonly used short hexapeptides, provided the highest accuracy. Weighted contributions of an amino acid at each position in a 27 residue window revealed three cooperative regions of short stretch, resemble the β-strand-turn-β-strand motif in A-βpeptide amyloid and β-solenoid structure of HET-s(218–289) prion (C). Using an in-house energy evaluation algorithm, the interaction energy between two short stretches in long segment is computed and incorporated as an additional feature. The algorithm successfully predicted and classified amyloid segments with an overall accuracy of 75%. Our study revealed that genome-wide amyloid segments are not only dependent on short high propensity stretches, but also on nearby residues