Phenylalanine (Phe)-derived molecules have been exploited as low molecular weight hydrogelators. Perturbing the hydrophobic and p-p interactions that promote self-assembly and hydrogelation of these derivatives will facilitate improved understanding of hydrogelation phenomena and the design of small molecule hydrogelators with novel properties. The efficient self-assembly and hydrogelation of Fmoc-protected pentafluorophenylalanine (Fmoc-F 5 -Phe) are reported herein. Suspensions of Fmoc-F 5 -Phe in water undergo rapid self-assembly to entangled fibrillar structures within minutes, giving rise to rigid supramolecular gels. Self-assembly occurs at concentrations as low as 2 mM (0.1 wt%). Variation of the fluorinated aromatic side chain or N-terminal functionalization perturbs hydrogelation, implicating fluorous and p-p interactions as the primary determinants for molecular recognition and self-assembly. The hydrophobic and electronic properties of F 5 -Phe provide remarkable potential for functional self-assembly in a minimal amino acid scaffold.

Aromatic amino acids have been shown to promote self-assembly of amyloid peptides, although the basis for this amyloid-inducing behavior is not understood. We adopted the amyloid-β 16-22 peptide (Aβ(16-22), Ac-KLVFFAE-NH(2)) as a model to study the role of aromatic amino acids in peptide self-assembly. Aβ(16-22) contains two consecutive Phe residues (19 and 20) in which Phe 19 side chains form interstrand contacts in fibrils while Phe 20 side chains interact with the side chain of Va l18. The kinetic and thermodynamic effect of varying the hydrophobicity and aromaticity at positions 19 and 20 by mutation with Ala, Tyr, cyclohexylalanine (Cha), and pentafluorophenylalanine (F(5)-Phe) (order of hydrophobicity is Ala < Tyr < Phe < F(5)-Phe < Cha) was characterized. Ala and Tyr position 19 variants failed to undergo fibril formation at the peptide concentrations studied, but Cha and F(5)-Phe variants self-assembled at dramatically enhanced rates relative to wild-type. Cha mutation was thermodynamically stabilizing at position 20 (ΔΔG = -0.2 kcal mol(-1) relative to wild-type) and destabilizing at position 19 (ΔΔG = +0.2 kcal mol(-1)). Conversely, F(5)-Phe mutations were strongly stabilizing at both positions (ΔΔG = -1.3 kcal mol(-1) at 19, ΔΔG = -0.9 kcal mol(-1) at 20). The double Cha and F(5)-Phe mutants showed that the thermodynamic effects were additive (ΔΔG = 0 kcal mol(-1) for Cha 19,20 and -2.1 kcal mol(-1) for F(5)-Phe 19,20). These results indicate that sequence hydrophobicity alone does not dictate amyloid potential, but that aromatic, hydrophobic, and steric considerations collectively influence fibril formation.

The self-assembly of amyloid peptides is influenced by hydrophobicity, charge, secondary structure propensity, and sterics. Previous experiments have shown that increasing hydrophobicity at the aromatic positions of the amyloid-β 16-22 fragment (Aβ(16-22)) without introducing steric restraints greatly increases the rate of self-assembly and thermodynamically stabilizes the resulting fibrils [Senguen et al., Mol. BioSyst., 2011, DOI: 10.1039/c0mb00080a]. Conversely, when increasing side chain hydrophobicity coincides with an increase in side chain volume, the increase in the rate of self-assembly is offset by a thermodynamic destabilization of the resulting amyloid fibrils when direct cross-strand side chain interactions occur. These findings indicate that steric effects also influence the self-assembly of amyloidogenic peptides. Herein, the aromatic Phe residues at positions 19, 20, and 19,20 of Aβ(16-22) have been systematically replaced by Val, Leu, Ile, or hexafluoroleucine (Hfl) and amyloid formation has been characterized. The Val variants, despite the high β-sheet propensity of Val, were thermodynamically destabilized (ΔΔG = +0.1-0.4 kcal mol(-1)) relative to the wild-type with the double mutant failing to self-assemble at the concentrations studied. Conversely, the Leu and Ile variants formed fibrils at enhanced rates relative to wild-type and exhibited similar, or in some cases enhanced thermodynamic stabilities relative to the wild-type (ΔΔG = 0-0.6 kcal mol(-1)). The more hydrophobic Hfl variants were greatly stabilized (ΔΔG = -0.3-2.1 kcal mol(-1)) relative to the wild-type. These data indicate that hydrophobicity and steric effects both influence peptide self-assembly processes, including nucleation and fibrillization rates and the thermodynamic stability of the resulting fibrils.

Calmodulin (CaM), a member of the EF-hand superfamily, regulates many aspects of the cell function by responding specifically to micromolar concentrations of Ca2+ in the presence of ~1000× higher concentration of cellular Mg2+. To explain the structural basis of metal ion binding specificity we have solved the X-ray structures of the N-terminal domain of calmodulin (N-CaM) in complexes with Mg2+, Mn2+ and Zn2+. In contrast to Ca2+, which induces domain opening in CaM, octahedrally coordinated Mg2+ and Mn2+ stabilize the closed-domain, apo-like conformation, while tetrahedrally coordinated Zn2+ ions bind at the protein surface and do not compete with Ca2+. The relative positions of bound Mg2+ and Mn2+ within the EF-hand loops are similar to those of Ca2+, however the Glu sidechain in the 12th position of the loop, whose bidentate interaction with Ca2+ is critical for domain opening, does not bind directly to either Mn2+ or Mg2+ and the vacant ligand position is occupied by a water molecule. We conclude that this critical interaction is prevented by specific stereochemical constraints imposed on the ligands by the EF-hand-β-scaffold. The structures suggest that Mg2+ contributes to the switching off of calmodulin activity and possibly other EF-hand proteins at the resting levels of Ca2+. The Mg2+-bound N-CaM structure also provides a unique view of a transiently bound hydrated metal ion and suggests a role for the hydration water in the metal induced conformational change.

While protein film voltammetry (PFV) frequently makes use of pyrolytic graphite edge (PGE) electrodes as a suitable surface for the study of biological macromolecules, 1 few studies have reported the comparison of PFV as a function of electrode material, with the notable exception of blue copper protein azurin. 2 Here we report that upon PGE electrodes, bacterial cytochromes c 551 (cyts c) and mitochondrial cyt c can undergo a spontaneous chemical reaction at neutral pH values, which we describe in terms of the loss of the methionine ligand of the heme iron. Additionally, the native conformation of the bacterial cyts c can be studied in the same experiment. These data contrast with reports of cyts c electrochemistry that utilize alkanethiol modified gold electrodes, 3 which do not show the spontaneous lower-potential product. Further, by comparison with horse heart cyt c, we show that there is a systematic reciprocal correlation of the relative stability of the Met-Fe interaction, and the propensity for generation of a stable, low-potential form of cyts c at the PGE electrode.Monoheme, Class I ferricytochromes c were studied by direct adsorption upon a freshly polished, chilled PGE electrode, and subjected to PFV. Figure 1 illustrates that for a series of analogous cyts c, the PFV experiment revealed not one electrochemical response, but two: a pair of reversible voltammetric features at approximately +250 mV, the typical cyt c Fe(II/III) couple, and a lower potential feature at approximately -100 mV (vs SHE). Baseline-subtracted data for these features are shown in the insets of Figure 1.As shown in Table 1, the higher potential redox couples (Figure 1, blue traces) measured here with PGE electrodes agree with previous measurements conducted using gold self-assembled monolayer-modified gold electrodes (Au-SAMs) for cyt c 551 from Pseudomonas aeruginosa (PA) and cyt c 552 from Hydrogenobacter thermophilus (HT). For example, HT wild-type cyt c shows a modest shift (25 mV) downward in potential when comparing PGE to Au-SAM measurements, and the PA cyt c measurements are identical within experimental uncertainty. 3 Thus, part of the PFV response indicates the anticipated Fe(II/III) couple, though it is clear that the horse heart (HH) cyt c results differ ( Figure 1C), showing a lower potential signal at the PGE electrode only.All of the His/Met ligated c-type cytochromes studied here at graphitic electrodes show the existence of the lower-potential form, and HH cyt c yields essentially 100% of the lowerpotential signal. Similar PFV studies using PGE to interrogate cyts c 6 (from A. thaliana and P. laminosum) have revealed an unknown signal that mirrors the lower-potential couples reported here. 4 Our observation of similar, lower-potential couples in different His/Met cyts c suggests that a similar state of cyt c can be attained at PGE electrodes, regardless of the source elliott@bu.edu. of the cyt c. We hypothesize that the extent to which the lower-potential form is present at the electrode correlates with the pr...