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Abstract: Hereditary mutations in the transforming growth factor beta induced (TGFBI) gene cause phenotypically distinct corneal dystrophies characterized by protein deposition in cornea. We show here that the Arg555Trp mutant of the fourth fasciclin 1 (FAS1-4) domain of the protein (TGFBIp/keratoepithelin/βig-h3), associated with granular corneal dystrophy type 1, is significantly less susceptible to proteolysis by thermolysin and trypsin than the WT domain. High-resolution liquid-state NMR of the WT and Arg555Trp mutant FAS1-4 domains revealed very similar structures except for the region around position 555. The Arg555Trp substitution causes Trp555 to be buried in an otherwise empty hydrophobic cavity of the FAS1-4 domain. The first thermolysin cleavage in the core of the FAS1-4 domain occurs on the N-terminal side of Leu558 adjacent to the Arg555 mutation. MD simulations indicated that the C-terminal end of helix α3′ containing this cleavage site is less flexible in the mutant domain, explaining the observed proteolytic resistance. This structural change also alters the electrostatic properties, which may explain increased propensity of the mutant to aggregate in vitro with 2,2,2-trifluoroethanol. Based on our results we propose that the Arg555Trp mutation disrupts the normal degradation/turnover of corneal TGFBIp, leading to accumulation and increased propensity to aggregate through electrostatic interactions. [Copyright &y& Elsevier]

Abstract: The well-ordered cross β-strand structure found in amyloid aggregates is stabilized by many different side chain interactions, including hydrophobic interactions, electrostatic charge and the intrinsic propensity to form β-sheet structures. In addition to the side chains, backbone interactions are important because of the regular hydrogen-bonding pattern. β-Sheet breaking peptide analogs, such as those formed by N-methylation, interfere with the repetitive hydrogen bonding pattern of peptide strands. Here we test backbone contributions to fibril stability using analogs of the 6–10 residue fibril core of human islet amyloid polypeptide, a 37 amino acid peptide involved in the pathogenesis of type II diabetes. The Phe–Gly peptide bond has been replaced by a hydroxyethylene or a ketomethylene group and the nitrogen-atom has been methylated. In addition, we have prepared peptoids where the side chain is transferred to the nitrogen atom. The backbone turns out to be extremely sensitive to substitution, since only the minimally perturbed ketomethylene analog (where only one of the −NH− groups has been replaced by −CH2−) can elongate wildtype fibrils but cannot fibrillate on its own. The resulting fibrils displayed differences in both secondary structure and overall morphology. No analog could inhibit the fibrillation of the parent peptide, suggesting an inability to bind to existing fibril surfaces. In contrast, side chain mutations that left the backbone intact but increased backbone flexibility or removed stabilizing side-chain interactions had very small effect on fibrillation kinetics. We conclude that fibrillation is very sensitive to even small modifications of the peptide backbone. [Copyright &y& Elsevier]

Abstract: Many small cationic peptides, which are unstructured in aqueous solution, have antimicrobial properties. These properties are assumed to be linked to their ability to permeabilize bacterial membranes, accompanied by the transition to an α-helical folding state. Here we show that there is no direct link between folding of the antimicrobial peptide Novicidin (Nc) and its membrane permeabilization. N-terminal acylation with C8–C16 alkyl chains and the inclusion of anionic lipids both increase Nc''s ability to form α-helical structure in the presence of vesicles. Nevertheless, both acylation and anionic lipids reduce the extent of permeabilization of these vesicles and lead to slower permeabilization kinetics. Furthermore, acylation significantly decreases antimicrobial activity. Although acyl chains of increasing length also increase the tendency of the peptides to aggregate in solution, this cannot rationalize our results since permeabilization and antimicrobial activities are observed well below concentrations where aggregation occurs. This suggests that significant induction of α-helical structure is not a prerequisite for membrane perturbation in this class of antimicrobial peptides. Our data suggests that for Nc, induction of α-helical structure may inhibit rather than facilitate membrane disruption, and that a more peripheral interaction may be the most efficient permeabilization mechanism. Furthermore, acylation leads to a deeper embedding in the membrane, which could lead to an anti-permeabilizing “plugging” effect. [Copyright &y& Elsevier]