Biomimetic magnetite formation: from biocombinatorial approaches to mineralization effects.

Biological materials typically display complex morphologies and hierarchical architectures, properties that are hardly matched by synthetic materials. Understanding the biological control of mineral properties will enable the development of new synthetic approaches toward biomimetic functional materials. Here, we combine biocombinatorial approaches with a proteome homology search and in vitro mineralization assays to assess the role of biological determinants in biomimetic magnetite mineralization. Our results suggest that the identified proteins and biomimetic polypeptides influence nucleation in vitro. Even though the in vivo role cannot be directly determined from our experiments, we can rationalize the following design principles: proteins, larger complexes, or membrane components that promote nucleation in vivo are likely to expose positively charged residues to a negatively charged crystal surface. In turn, components with acidic (negatively charged) functionality are nucleation inhibitors, which stabilize an amorphous structure through the coordination of iron.

ß sheets not required: combined experimental and computational studies of self-assembly and gelation of the ester-containing analogue of an Fmoc-dipeptide hydrogelator.

In our work toward developing ester-containing self-assembling peptides as soft biomaterials, we have found that a fluorenylmethoxycarbonyl (Fmoc)-conjugated alanine-lactic acid (Ala-Lac) sequence self-assembles into nanostructures that gel in water. This process occurs despite Fmoc-Ala-Lac's inability to interact with other Fmoc-Ala-Lac molecules via ß-sheet-like amide-amide hydrogen bonding, a condition previously thought to be crucial to the self-assembly of Fmoc-conjugated peptides. Experimental comparisons of Fmoc-Ala-Lac to its self-assembling peptide sequence analogue Fmoc-Ala-Ala using a variety of microscopic, spectroscopic, and bulk characterization techniques demonstrate distinct features of the two systems and show that while angstrom-scale self-assembled structures are similar, their nanometer-scale size and morphological properties diverge and give rise to different bulk mechanical properties. Molecular dynamics simulations were performed to gain more insight into the differences between the two systems. An analysis of the hydrogen-bonding and solvent-surface interface properties of the simulated fibrils revealed that Fmoc-Ala-Lac fibrils are stronger and less hydrophilic than Fmoc-Ala-Ala fibrils. We propose that this difference in fibril amphiphilicity gives rise to differences in the higher-order assembly of fibrils into nanostructures seen in TEM. Importantly, we confirm experimentally that ß-sheet-type hydrogen bonding is not crucial to the self-assembly of short, conjugated peptides, and we demonstrate computationally that the amide bond in such systems may act mainly to mediate the solvation of the self-assembled single fibrils and therefore regulate a more extensive higher-order aggregation of fibrils. This work provides a basic understanding for future research in designing highly degradable self-assembling materials with peptide-like bioactivity for biomedical applications.

Charge and sequence effects on the self-assembly and subsequent hydrogelation of Fmoc-depsipeptides.

Herein we report on the self-assembly of a family of Fmoc-depsipeptides into nanofibers and hydrogels. We show that fiber formation occurs in depsipeptide structures in which the fluorenyl group is closely associated and that side-chain charge and sequence affect the extent of self-assembly and subsequent gelation. Using fluorescence emission spectroscopy and circular dichroism, we show that self-assembly can be monitored and is observed in these slow-gelling systems prior to hydrogel formation. We also demonstrate that the ionic strength of salt-containing solutions affects the time at which self-assembly results in gelation of the bulk solution. From transmission electron microscopy, we report that morphological changes progress over time and are observed as micelles transitioning to fibers prior to the onset of gelation. Gelled depsipeptides degraded at a slower rate than non-gelled samples in the presence of salt, while hydrolysis in water of both gels and solution samples was minimal even after 14 days. Our work shows that while incorporating ester functionality within a peptide backbone reduces the number of hydrogen bonding sites available for forming and stabilizing supramolecular assemblies, the substitution does not prohibit self-assembly and subsequent gelation.

Experimental and computational studies reveal an alternative supramolecular structure for fmoc-dipeptide self-assembly.

We have investigated the self-assembly of fluorenylmethoxycarbonyl-conjugated dialanine (Fmoc-AA) molecules using combined computational and experimental approaches. Fmoc-AA gels were characterized using transmission electron microscopy (TEM), circular dichroism (CD), Fourier transform infrared (FTIR), and wide-angle X-ray scattering (WAXS). Computationally, we simulated the assembly of Fmoc-AA using molecular dynamics techniques. All simulations converged to a condensed fibril structure in which the Fmoc groups stack mostly within in the center of the fibril. However, the Fmoc groups are partially exposed to water, creating an amphiphilic surface, which may be responsible for the aggregation of fibrils into nanoscale fibers observed in TEM. From the fibril models, radial distribution calculations agree with d-spacings observed in WAXS for the fibril diameter and π-stacking interactions. Our analyses show that dialanine, despite its short length, adopts a mainly extended polyproline II conformation. In contrast to previous hypotheses, these results indicate that ß-sheet-like hydrogen bonding is not prevalent. Rather, stacking of Fmoc groups, inter-residue hydrogen bonding, and hydrogen bonding with water play the important roles in stabilizing the fibril structure of supramolecular assemblies of short conjugated peptides.

Design and Evaluation of Short Self-Assembling Depsipeptides as Bioactive and Biodegradable Hydrogels.

Described herein is the design of a cell-adherent and degradable hydrogel. Our goal was to create a self-assembling, backbone ester-containing analogue of the cell adhesion motif, arginine-glycine-aspartic acid (RGD). Two depsipeptides containing Fmoc (N-(fluorenyl)-9-methoxycarbonyl), Fmoc-FR-Glc-D, and Fmoc-F-Glc-RGD (where "Glc" is glycolic acid) were designed based on the results of integrin-binding affinity and cell interaction analyses. Two candidate molecules were synthesized, and their gelation characteristics, degradation profiles, and ability to promote cell attachment were analyzed. We found that ester substitution within the RGD sequence significantly decreases the integrin-binding affinity and subsequent cell attachment, but when the ester moiety flanks the bioactive sequence, the molecule can maintain its integrin-binding function while permitting nonenzymatic hydrolytic degradation. A self-assembled Fmoc-F-Glc-RGD hydrogel showed steady, linear degradation over 60 days, and when mixed with Fmoc-diphenylalanine (Fmoc-FF) for improved mechanical stiffness, the depsipeptide gel exhibited improved cell attachment and viability. Though the currently designed depsipeptide has several inherent limitations, our results indicate the potential of depsipeptides as the basis for biologically functional and degradable self-assembling hydrogel materials.