CHARMM force field parameterization protocol for self-assembling peptide amphiphiles : the Fmoc moiety

Aromatic peptide amphiphiles are known to self-assemble into nanostructures but the molecular level structure and the mechanism of formation of these nanostructures is not yet understood in detail. Molecular dynamic simulations using the CHARMM force field have been applied to a wide variety of peptide-based systems to obtain molecular level details of processes that are inaccessible with experimental techniques. However, this force field does not include parameters for the aromatic moieties which dictate the self-assembly of these systems. The standard CHARMM force field parameterization protocol uses hydrophilic interactions for the non-bonding parameters evaluation. However, to effectively reproduce the self-assembling behaviour of these molecules, the balance between the hydrophilic and hydrophobic nature of the molecule is essential. In this work, a modified parameterization protocol for the CHARMM force field for these aromatic moieties is presented. This protocol is applied for the specific case of the Fmoc moiety. The resulting set of parameters satisfies the conformational and interactions analysis and is able to reproduce experimental results such as the Fmoc-S-OMe water/octanol partition free energy and the self-assembly of Fmoc-S-OH and Fmoc-Y-OH into spherical micelles and fibres, respectively, while also providing detailed information on the mechanism of these processes. The effectiveness of the parameters for the Fmoc moiety validates the protocol as a robust approach to paramterise this class of compounds

Supramolecular fibers in gels can be at thermodynamic equilibrium : a simple packing model reveals preferential fibril formation versus crystallization

Low molecular weight gelators are able to form nanostructures, typically fibers, which entangle to form gel-phase materials. These materials have wide-ranging applications in biomedicine and nanotechnology. While it is known that supramolecular gels often represent metastable structures due to the restricted molecular dynamics in the gel state, the thermodynamic nature of the nanofibrous structure is not well understood. Clearly, 3D extended structures will be able to form more interactions than 1D structures. However, self-assembling molecules are typically amphiphilic, thus giving rise to a combination of solvophobic and solvophilic moieties where a level of solvent exposure at the nanostructure surface is favorable. In this study, we introduce a simple packing model, based on prisms with faces of different nature (solvophobic and solvophilic) and variable interaction parameters, to represent amphiphile self-assembly. This model demonstrates that by tuning shape and "self" or "solvent" interaction parameters either the 1D fiber or 3D crystal may represent the thermodynamic minimum. The model depends on parameters that relate to features of experimentally known systems: The number of faces exposed to the solvent or buried in the fiber; the overall shape of the prism; and the free energy penalties associated with the interactions can be adjusted to match their chemical nature. The model is applied to describe the pH-dependent gelation/precipitation of well-known gelator Fmoc-FF. We conclude that, despite the fact that most experimentally produced gels probably represent metastable states, one-dimensional fibers can represent thermodynamic equilibrium. This conclusion has critical implications for the theoretical treatment of gels

Enzymatically activated emulsions stabilised by interfacial nanofibre networks

We report on-demand formation of emulsions stabilised by interfacial nanoscale networks. These are formed through biocatalytic dephosphorylation and self-assembly of Fmoc(9-fluorenylmethoxycarbonyl)-dipeptide amphiphiles in aqueous/organic mixtures. This is achieved by using alkaline phosphatase which transforms surfactant-like phosphorylated precursors into self-assembling aromatic peptide amphiphiles (Fmoc-tyrosine-leucine, Fmoc-YL) that form nanofibrous networks. In biphasic organic/aqueous systems, these networks form preferentially at the interface thus providing a means of emulsion stabilisation. We demonstrate on-demand emulsification by enzyme addition, even after storage of the biphasic mixture for several weeks. Experimental (Fluorescence, FTIR spectroscopy, fluorescence microscopy, electron microscopy, atomic force microscopy) and computational techniques (atomistic molecular dynamics) are used to characterise the interfacial self-assembly process

Elucidation of the bonding of a near infrared dye to hollow gold nanospheres : a chalcogen tripod

Infrared surface enhanced Raman scattering (SERS) is an attractive technique for the in situ detection of nanoprobes in biological samples due to the greater depth of penetration and reduced interference compared to SERS in the visible region. A key challenge is to understand the surface layer formed in suspension when a specific label is added to the SERS substrate in aqueous suspension. SERS taken at different wavelengths, theoretical calculations, and surface-selective sum frequency generation vibrational spectroscopy (SFG-VS) were used to define the surface orientation and manner of attachment of a new class of infrared SERS label with a thiopyrylium core and four pendant 2-selenophenyl rings. Hollow gold nanospheres (HGNs) were used as the enhancing substrate and two distinct types of SERS spectra were obtained. With excitation close to resonance with both the near infrared electronic transition in the label (max 826 nm) and the plasmon resonance maximum (690 nm), surface enhanced resonance Raman scattering (SERRS) was obtained. SERRS indicates that the major axis of the core is near to perpendicular to the surface plane and SFG-VS obtained from a dried gold film gave a similar orientation with the major axis at an angle 64°-85° from the surface plane. Longer excitation wavelengths give SERS with little or no molecular resonance contribution and new vibrations appeared with significant displacements between the thiopyrylium core and the pendant selenophene rings. Analysis using calculated spectra with one or two rings rotated indicates that two rings on one end are rotated towards the metal surface to give an arrangement of two selenium and one sulphur atoms directly facing the gold structure. The spectra, together with a space filled model, indicate that the molecule is strongly adsorbed to the surface through the selenium and sulphur atoms in an arrangement which will facilitate layer formation

Modelling Fmoc-dipeptide nanostructures, the synergistic effect of combining computational and experimental methods

Nanomaterials based on aromatic peptide amphiphiles are interesting new materials with potential applications in the areas of biomedicine and nanotechnology. These natural based materials take advantage of the properties of the peptides, as it is the ability to form the final structures spontaneously without any external stimulus by self-assembly, or the high number of functionality available due to the 20 natural building blocks, amino acids, and their possible combinations. Although it is known that the functionality of these nanostructures is highly dependent on both, the chemical groups and the topology of the nanostructure, and both vary with the amino acids side chains, the relationship between nanostructure shape and peptide chain composition is still unknown. Understanding this is necessary to be able to design Fmoc-peptide nanostructures on demand. In this thesis a combination between these experimental techniques and computational methods, molecular dynamic (MD) simulations, is used to elucidate the self-assembly motifs for a set of model systems composed of Fmoc-dipeptides. The interpretation of the experimental spectroscopic characterization is improved by using enzymatic self-assembly under thermodynamic control Fmoc-dipeptide and side-by-side comparison of nanostructures using dynamic peptide libraries (DPLs). This approach allowed to resolve which features increase the self-assembly tendency of these molecules. Both MD and DPL approaches depend on the premise that gels can be at thermodynamic equilibrium, which is not clear in the literature. It has been argued that they represent metastable states, where crystals are suggested to represent the actual thermodynamically favoured structures. Hence, the study starts with a model proposed to demonstrate that nanofibrous gels can represent the thermodynamically favoured structure. This is achieved by using a packing model where self-assembling molecules are represented by prisms with faces of different nature, solvophilic andsolvophobic to mimic the amphiphilicity of these molecules as a key feature. This approach gives rise to a combination of solvophobic and solvophilic interactions where a level of solvent exposure is favourable. The model depends on parameters which can be related with features of the system and demonstrates that the amphiphilicity is key to allow 1D objects, fibres, to be more stable than 3D objects,crystals; and hence, that MD simulations and DPLs can be applied for their study.For MD simulations, the CHARMM force field is used because it has beenapplied and validated to a wide variety of peptide-based systems. However, this forcefield does not include parameters for the Fmoc moiety. Therefore, the second steps for this study was to develop an Fmoc parameterization for the CHARMM force field, in order to be able to run all atoms self-assembling Fmoc-peptides simulations, to improve the understanding of these nanostructures and their formation. The parameterization is based in the CHARMM protocol adapted due to the amphiphilic nature of the Fmoc moiety. Experimentally, in order to get more valuable information from the experimental characterization, the study of different Fmoc-dipeptides nanostructures with specific changes in their peptide chain are compared in order to understand how these specific changes affect the self-assembled structure: phenylalanine/leucinesubstitution to understand how the aromatic side chain affects; and amide/methyl esterC-terminus substitution, to understand the role of the possible extra hydrogen bondsof the amide group. Furthermore, DPLs are also applied to rationalize the influence of these changes in the self-assembling tendency of Fmoc-dipeptides.;Then, the experimental information is used to develop a model for Fmoc-TF-NH ₂ fibre and simulate it. The analysis of the model in addition with correlation of these data with the experimental insights, allows the refinement of the model. The resulting new model is validated by comparing the simulation analysis with the previous model and,again, correlating the computational results with experimental. Finally, the new model is applied to gain understanding of the experimental observed phenomena of fibres evolving to twisted ribbons. The simulations using the developed fibre model demonstrate that those twisted ribbons are formed by lateral aggregation of the fibres. The useful information obtained using the model, supports its validity. In conclusion, in this thesis the thermodynamic nature of gels is demonstrated to be able to use MD simulations and DPLs for the molecular level study of Fmoc-dipeptide nanostructures. A parameterization of the Fmoc is also developed to allow the implementation of the MD simulations for these systems. Then, standard characterization of Fmoc-dipeptide nanostructures is combined with DPLs to gain intermolecular interaction information of these systems to then use this information for an iterative model development of a fibre model, which is correlated and validated with experimental observations. This demonstrates the synergistic effect of combining computational with experimental methods to gain understanding of supramolecular nanostructures at a level which is not accessible with any other technique.Nanomaterials based on aromatic peptide amphiphiles are interesting new materials with potential applications in the areas of biomedicine and nanotechnology. These natural based materials take advantage of the properties of the peptides, as it is the ability to form the final structures spontaneously without any external stimulus by self-assembly, or the high number of functionality available due to the 20 natural building blocks, amino acids, and their possible combinations. Although it is known that the functionality of these nanostructures is highly dependent on both, the chemical groups and the topology of the nanostructure, and both vary with the amino acids side chains, the relationship between nanostructure shape and peptide chain composition is still unknown. Understanding this is necessary to be able to design Fmoc-peptide nanostructures on demand. In this thesis a combination between these experimental techniques and computational methods, molecular dynamic (MD) simulations, is used to elucidate the self-assembly motifs for a set of model systems composed of Fmoc-dipeptides. The interpretation of the experimental spectroscopic characterization is improved by using enzymatic self-assembly under thermodynamic control Fmoc-dipeptide and side-by-side comparison of nanostructures using dynamic peptide libraries (DPLs). This approach allowed to resolve which features increase the self-assembly tendency of these molecules. Both MD and DPL approaches depend on the premise that gels can be at thermodynamic equilibrium, which is not clear in the literature. It has been argued that they represent metastable states, where crystals are suggested to represent the actual thermodynamically favoured structures. Hence, the study starts with a model proposed to demonstrate that nanofibrous gels can represent the thermodynamically favoured structure. This is achieved by using a packing model where self-assembling molecules are represented by prisms with faces of different nature, solvophilic andsolvophobic to mimic the amphiphilicity of these molecules as a key feature. This approach gives rise to a combination of solvophobic and solvophilic interactions where a level of solvent exposure is favourable. The model depends on parameters which can be related with features of the system and demonstrates that the amphiphilicity is key to allow 1D objects, fibres, to be more stable than 3D objects,crystals; and hence, that MD simulations and DPLs can be applied for their study.For MD simulations, the CHARMM force field is used because it has beenapplied and validated to a wide variety of peptide-based systems. However, this forcefield does not include parameters for the Fmoc moiety. Therefore, the second steps for this study was to develop an Fmoc parameterization for the CHARMM force field, in order to be able to run all atoms self-assembling Fmoc-peptides simulations, to improve the understanding of these nanostructures and their formation. The parameterization is based in the CHARMM protocol adapted due to the amphiphilic nature of the Fmoc moiety. Experimentally, in order to get more valuable information from the experimental characterization, the study of different Fmoc-dipeptides nanostructures with specific changes in their peptide chain are compared in order to understand how these specific changes affect the self-assembled structure: phenylalanine/leucinesubstitution to understand how the aromatic side chain affects; and amide/methyl esterC-terminus substitution, to understand the role of the possible extra hydrogen bondsof the amide group. Furthermore, DPLs are also applied to rationalize the influence of these changes in the self-assembling tendency of Fmoc-dipeptides.;Then, the experimental information is used to develop a model for Fmoc-TF-NH ₂ fibre and simulate it. The analysis of the model in addition with correlation of these data with the experimental insights, allows the refinement of the model. The resulting new model is validated by comparing the simulation analysis with the previous model and,again, correlating the computational results with experimental. Finally, the new model is applied to gain understanding of the experimental observed phenomena of fibres evolving to twisted ribbons. The simulations using the developed fibre model demonstrate that those twisted ribbons are formed by lateral aggregation of the fibres. The useful information obtained using the model, supports its validity. In conclusion, in this thesis the thermodynamic nature of gels is demonstrated to be able to use MD simulations and DPLs for the molecular level study of Fmoc-dipeptide nanostructures. A parameterization of the Fmoc is also developed to allow the implementation of the MD simulations for these systems. Then, standard characterization of Fmoc-dipeptide nanostructures is combined with DPLs to gain intermolecular interaction information of these systems to then use this information for an iterative model development of a fibre model, which is correlated and validated with experimental observations. This demonstrates the synergistic effect of combining computational with experimental methods to gain understanding of supramolecular nanostructures at a level which is not accessible with any other technique

Assessing the utility of infrared spectroscopy as a structural diagnostic tool for β-sheets in self-assembling aromatic peptide amphiphiles

beta-Sheets are a commonly found structural motif in self-assembling aromatic peptide amphiphiles, and their characteristic "amide I" infrared (IR) absorption bands are routinely used to support the formation of supramolecular structure. In this paper, we assess the utility of IR spectroscopy as a structural diagnostic tool for this class of self-assembling systems. Using 9-fluorene-methyloxycarbonyl dialanine (Fmoc-AA) and the analogous 9-fluorene-methylcarbonyl dialanine (Fmc-AA) as examples, we show that the origin of the band around 1680-1695 cm(-1) in Fourier transform infrared (FTIR) spectra, which was previously assigned to an antiparallel beta-sheet conformation, is in fact absorption of the stacked carbamate group in Fmoc-peptides. IR spectra from C-13-labeled samples support our conclusions. In addition, DFT frequency calculations on small stacks of aromatic peptides help to rationalize these results in terms of the individual vibrational modes

Assessing the Utility of Infrared Spectroscopy as a Structural Diagnostic Tool for β‑Sheets in Self-Assembling Aromatic Peptide Amphiphiles

β-Sheets are a commonly found structural motif in self-assembling aromatic peptide amphiphiles, and their characteristic “amide I” infrared (IR) absorption bands are routinely used to support the formation of supramolecular structure. In this paper, we assess the utility of IR spectroscopy as a structural diagnostic tool for this class of self-assembling systems. Using 9-fluorene-methyloxycarbonyl dialanine (Fmoc-AA) and the analogous 9-fluorene-methylcarbonyl dialanine (Fmc-AA) as examples, we show that the origin of the band around 1680–1695 cm^–1 in Fourier transform infrared (FTIR) spectra, which was previously assigned to an antiparallel β-sheet conformation, is in fact absorption of the stacked carbamate group in Fmoc-peptides. IR spectra from ¹³C-labeled samples support our conclusions. In addition, DFT frequency calculations on small stacks of aromatic peptides help to rationalize these results in terms of the individual vibrational modes