Charged amino acid propensities and solubility: lysine is elevated at the termini of helices in E. coli proteins

Prebiotic cationic amino acids support formation of an ancient protein fold

Crystallographic analysis revealed that both this peptide and its Dab-substituted derivative adopted the double-Z β-barrel (DZBB) fold, a likely evolutionary intermediate between extant β-barrel folds, which might have supported the foldability of primitive proteins before the incorporation of lysine and arginine into the genetic code.

Sequence-dependent Stability and the Apparent Two-state Thermal Transition of Extended Collagen Triple Helices

The thermal stability of collagen triple helices is strongly influenced by the amino acid sequence of the repeating Gly–X–Y tripeptides, yet how these residue-specific interactions are integrated within an extended triple helix to determine thermal behavior remains poorly understood. Here, we addressed this question using recombinant collagen mimetic peptides (rCMPs) containing extended native sequences from the α1(I) and α2(I) chains of human type I collagen. Triple-helix formation was nucleated by a C-terminal foldon domain and further stabilized by interchain disulfide crosslinking, allowing the apparent melting temperature (Tₘ) to reflect interactions within the triple-helical domain independent of nucleation. The stabilizing effects of Pro and Y-position Arg identified in host-guest peptides were largely preserved in extended triple helices, whereas the proposed Lys–Gly–Glu (KGE) interchain salt bridge produced little measurable stabilization, demonstrating the influence of sequence context. Remarkably, identical triple-helical sequences exhibited markedly different thermal behavior when unfolding was initiated under different conditions. Nevertheless, extended triple helices differing substantially in sequence and length retained an apparently two-state thermal transition. These findings support a mechanism in which unfolding is preferentially initiated within regions of lower intrinsic stability, while the continuity of the triple helix couples neighboring regions into a cooperative unfolding process throughout the helix. This mechanism provides a plausible explanation for the longstanding paradox that extended collagen triple helices exhibit persistent sequence-dependent thermodynamic heterogeneity despite a two-state thermal transition, and a framework for investigating how sequence-dependent stability contributes to the structure and function of collagen molecules. TOC

Is the Bilayer Insertion of the Peptide KALP23 Under Kinetic or Thermodynamic Control?

Factors Influencing the Selection of α-Amino Acids over β-Amino Acids in Nature: Studies Involving Protein-l-isoaspartyl Methyltransferase Substrates and Related Peptides.

To understand the differences resulting from the presence of an α- or β-amino acid in a peptide, we used two sets of amino acids corresponding to Asp/isoAsp and Ala/isoAla in two types of host peptides: one containing Gly (with no side chains) and the other containing Ala (representing amino acids with side chains), and measured their tendency to form fibrils. The peptides with a β-amino acid formed fibrils, while those with an α-amino acid did not exhibit this proclivity, as inferred from the thioflavin T (ThT) fluorescence intensity measurements. The fibrillation of the hexapeptide with isoAsp (the β-amino acid corresponding to Asp) was inhibited by protein-l-isoaspartyl methyltransferase (PIMT), a repair enzyme that converts the abnormal isoAsp residue to normal Asp. Isothermal Titration Calorimetry revealed the exothermic mode of binding of A6-isoAsp (Ala-based host hexapeptide) with PIMT. Far-UV CD spectroscopy revealed a β-sheet to α-helix transformation of isoAsp-containing peptides in the presence of PIMT. The hydrophobicity of the peptides was measured by noting their distribution in a mixture of water and octanol, where the β-amino acid exhibits a more positive hydrophobicity value than the α-amino acid. This may suggest that the hydrophobic forces bring chains containing β-amino acids together to enable nucleation for fibril formation. Molecular dynamics (MD) simulations indicated that isoAsp-based host peptides, formed into fibrils, have stable structures over the course of simulations, whereas the equivalent peptide with Asp disintegrated. Models of a single β-strand of these peptides bound to the active site of the enzyme were stable.

Systematic In Silico Analysis of Interhelical Electrostatic Interactions in Leucine Zippers

Leucine zippers are parallel coiled-coil dimers composed of two α-helices with a repeating heptad pattern, designated as a–g. Hydrophobic residues at the a and d positions form the dimerization core, whereas charged residues at the e and g positions generate interhelical electrostatic interactions that influence stability and specificity. Oppositely charged e–g pairs are generally considered stabilizing, whereas like-charged pairs are expected to be destabilizing. However, their collective effects across an extended leucine zipper interface have not been systematically evaluated. Here, we designed a 40-residue homodimeric leucine zipper containing 10 e–g interaction positions. Ten e–g positions were independently assigned to one of three states—neutral, salt-bridge-forming, or repulsive—yielding 59,049 models. Each model was then analyzed with FoldX to calculate the interaction energy and component energy terms. Increasing the number of salt bridges progressively improved the interaction energy, with an average stabilization of 1.68 kcal/mol relative to neutral pairs. Unexpectedly, repulsive pairs were also modestly favorable relative to neutral pairs, improving interaction energy by 0.48 kcal/mol. Energy decomposition showed that favorable solvation and van der Waals contributions outweighed the electrostatic penalty. These effects were position-dependent, indicating that leucine zipper stability reflects a balance of electrostatics, solvation, packing, and positional context.

Thermal Stabilization of Ubiquitin in Hydrophobic Amino Acid Solutions via Coupled Modulation of Solvation Structure and Dynamics.

It is demonstrated that hydrophobic amino acids enhance protein thermal stability through a coupled modulation of solvation structure, solvent dynamics, and hydrogen-bonding interactions, with distinct mechanisms depending on side-chain size and aggregation propensity.