A Model Study to Assess Fibrillation and Product Stability to Support Peptide Drug Design.

The fibrillation of therapeutic peptides can present significant quality concerns and poses challenges for manufacturing and storage. A fundamental understanding of the mechanisms of fibrillation is critical for the rational design of fibrillation-resistant peptide drugs and can accelerate product development by guiding the selection of solution-stable candidates and formulations. The studies reported here investigated the effects of structural modifications on the fibrillation of a 29-residue peptide (PepA) and two sequence modified variants (PepB, PepC). The C-terminus of PepA was amidated, whereas both PepB and PepC retained the carboxylate, and Ser16 in PepA and PepB was substituted with a helix-stabilizing residue, α-aminoisobutyric acid (Aib), in PepC. In thermal denaturation studies by far-UV CD spectroscopy and fibrillation kinetic studies by fluorescence and turbidity measurements, PepA and PepB showed heat-induced conformational changes and were found to form fibrils, whereas PepC did not fibrillate and showed only minor changes in the CD signal. Pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) showed a high degree of protection from HD exchange in mature PepA fibrils and its proteolytic fragments, indicating that most of the sequence had been incorporated into the fibril structure and occurred nearly simultaneously throughout the sequence. The effects of the net peptide charge and formulation pH on fibrillation kinetics were investigated. In real-time stability studies of two formulations of PepA at pH's 7.4 and 8.0, analytical methods detected significant changes in the stability of the formulations at different time points during the study, which were not observed during accelerated studies. Additionally, PepA samples were withdrawn from real-time stability and subjected to additional stress (40 °C, continuous shaking) to induce fibrillation; an approach that successfully amplified oligomers or prefibrillar species previously undetected in a thioflavin T assay. Taken together, these studies present an approach to differentiate and characterize fibrillation risk in structurally related peptides under accelerated and real-time conditions, providing a model for rapid, iterative structural design to optimize the stability of therapeutic peptides.

Deciphering the Molecular Mechanism of HCV Protease Inhibitor Fluorination as a General Approach to Avoid Drug Resistance.

Third generation Hepatitis C virus (HCV) NS3/4A protease inhibitors (PIs), glecaprevir and voxilaprevir, are highly effective across genotypes and against many resistant variants. Unlike earlier PIs, these compounds have fluorine substitutions on the P2-P4 macrocycle and P1 moieties. Fluorination has long been used in medicinal chemistry as a strategy to improve physicochemical properties and potency. However, the molecular basis by which fluorination improves potency and resistance profile of HCV NS3/4A PIs is not well understood. To systematically analyze the contribution of fluorine substitutions to inhibitor potency and resistance profile, we used a multi-disciplinary approach involving inhibitor design and synthesis, enzyme inhibition assays, co-crystallography, and structural analysis. A panel of inhibitors in matched pairs were designed with and without P4 cap fluorination, tested against WT protease and the D168A resistant variant, and a total of 22 high-resolution co-crystal structures were determined. While fluorination did not significantly improve potency against the WT protease, PIs with fluorinated P4 caps retained much better potency against the D168A protease variant. Detailed analysis of the co-crystal structures revealed that PIs with fluorinated P4 caps can sample alternate binding conformations that enable adapting to structural changes induced by the D168A substitution. Our results elucidate molecular mechanisms of fluorine-specific inhibitor interactions that can be leveraged in avoiding drug resistance.

Cα-Methyl-l-valine: A Preferential Choice over α-Aminoisobutyric Acid for Designing Right-Handed α-Helical Scaffolds.

In synthetic peptides containing Gly and coded α-amino acids, one of the most common practices to enhance their helical extent is to incorporate a large number of l-Ala residues along with noncoded, strongly foldameric α-aminoisobutyric acid (Aib) units. Earlier studies have established that Aib-based peptides, with propensity for both the 310- and α-helices, have a tendency to form ordered three-dimensional structure that is much stronger than that exhibited by their l-Ala rich counterparts. However, the achiral nature of Aib induces an inherent, equal preference for the right- and left-handed helical conformations as found in Aib homopeptide stretches. This property poses challenges in the analysis of a model peptide helical conformation based on chirospectroscopic techniques like electronic circular dichroism (ECD), a very important tool for assigning secondary structures. To overcome such ambiguity, we have synthesized and investigated a thermally stable 14-mer peptide in which each of the Aib residues of our previously designed and reported analogue ABGY (where B stands for Aib) is replaced by Cα-methyl-l-valine (L-AMV). Analysis of the results described here from complementary ECD and 1H nuclear magnetic resonance spectroscopic techniques in a variety of environments firmly establishes that the L-AMV-containing peptide exhibits a significantly stronger preference compared to that of its Aib parent in terms of conferring α-helical character. Furthermore, being a chiral α-amino acid, L-AMV shows an intrinsic, extremely strong bias for a quite specific (right-handed) screw sense. These findings emphasize the relevance of L-AMV as a more appropriate unit for the design of right-handed α-helical peptide models that may be utilized as conformationally constrained scaffolds.

The first non-helical Aib-containing hexapeptide: The crystal structure of Z-Gly-Aib-Gly-Aib-Gly-Aib-OtBu.

The synthetic peptide Z-Gly-Aib-Gly-Aib-Gly-Aib-OtBu was crystallized from a mixture of ethyl acetate and n-hexane. The crystals belong to the centrosymmetric space group Pbca. There are three molecules in the asymmetric unit. The three molecules differ mainly in the Z-group conformation. The first Gly residue adopts a fully extended conformation, residues 2 and 3 lie in the left-handed helical region, residues 4 and 5 in the right-handed helical region, and residue 6 again in the left-handed helical region of the Ramachandran plot. There are only two of four possible intramolecular hydrogen bonds formed, namely, between Aib4 and Gly1 forming a ß-turn of type III' and between Aib6 and Gly3 forming a ß-turn of type I. The inverted molecules (by space group symmetry) lie in the regions with opposite handedness and form ß-turns of type III and I'. In contrast to all known long synthetic and naturally occurring Aib-containing peptides that fold as 310 - or α-helix, Z-(Gly-Aib)3 -OtBu folds in a quite flat structure from which only the protecting groups bulge out.

Removing the C-terminal protecting group enlarges the crystal size: Z-(Gly-Aib)2-OH·H2O.

The achiral tetrapeptide monohydrate N-(benzyloxycarbonyl)glycyl-α-aminoisobutyrylglycyl-α-aminoisobutyric acid monohydrate, Z-Gly-Aib-Gly-Aib-OH·H2O (Z is benzyloxycarbonyl, Aib is α-aminoisobutyric acid and Gly is glycine) or C20H28N4O7·H2O, exhibits two conformations related by the symmetry operation of an inversion centre. It adopts only one of two possible intramolecular hydrogen bonds in a type I (and I') ß-turn and forms a maximum of intermolecular hydrogen bonds partly mediated by water. The space group, the molecular structure and the crystal packing differ from two already described (Gly-Aib)2 peptides which vary only in the protecting groups. This structure confirms the high structural flexibility of Gly-Aib peptides and points to a strong relationship between intermolecular hydrogen bonding and crystal quality and size.

Automated solid-phase concatenation of Aib residues to form long, water-soluble, helical peptides.

Iterative coupling of 2-aminoisobutyric acid (Aib) has been achieved rapidly and efficiently using automated solid-phase peptide synthesis, employing diisopropylcarbodiimide (DIC) in the presence of ethyl cyanohydroxyiminoacetate (Oxyma). This method has allowed the first total synthesis of the fungal antibiotic Cephaibol D, and enabled the synthesis of water-soluble oligomers of Aib containing up to an unprecedented sequence of 17 consecutive Aib residues. Conformational analysis of the Aib oligomers in aqueous solution shows a length dependence in their CD spectra, with oligomers of more than 14 Aib residues apparently adopting structured helical conformations.

Synthesis and Solid State Conformation of Tetrapeptide Amides Containing two Aib and two (αMe)Phe Residues - Use of Enantiomerically Pure 2-Benzyl-2-methyl-2H-azirin-3-amines as (αMe)Phe-Synthons.

A series of tetrapeptide amides containing two aminoisobutyric acids (Aib) and two α-methylphenylalanine ((αMe)Phe) units were prepared through the 'azirine/oxazolone method'. New 2-benzyl-2-methyl-2H-azirin-3-amines have been used for the selective introduction of (S)- and (R)-(αMe)Phe, respectively. The solid-state conformations of five tetrapeptide amides were determined by X-ray crystallography. In all cases, two ß-turns stabilize 310 -helical conformations and it was confirmed that, in contrast to proteinogenic amino acids, the configuration of (αMe)Phe does not determine the screw sense of the helix.

Helical Peptides Design for Molecular Dipoles Functionalization of Wide Band Gap Oxides.

The use of helical hexapeptides to establish a surface dipole layer on a TiO2 substrate, with the goal of influencing the energy levels of a coadsorbed chromophore, is explored. Two helical hexapeptides, synthesized from 2-amino isobutyric acid (Aib) residues, were protected at the N-terminus with a carboxybenzyl group (Z) and at the C-terminus carried either a carboxylic acid or an isophthalic acid (Ipa) anchor group to form Z-(Aib)6-COOH or Z-(Aib)6-Ipa, respectively. Using a combination of vibrational and photoemission spectroscopies, bonding of the two peptides to TiO2 surfaces (either nanostructured or single-crystal TiO2(110)) was found to be highly dependent on the anchor group, with Ipa establishing a monolayer much more efficiently than COOH. Furthermore, a monolayer of Z-(Aib)6-Ipa on TiO2(110) was exposed for different binding times to a solution of a zinc tetraphenylporphyrin (ZnTPP) derivative terminated with an Ipa anchor group (ZnTPP-P-Ipa). Photoemission spectroscopy revealed that ZnTPP-P-Ipa partly displaced Z-(Aib)6-Ipa, forming a coadsorbed monolayer on the oxide surface. The presence of the peptide molecular dipole shifted the HOMO levels of the ZnTPP group to lower energy by ∼300 meV, in accordance with a simple parallel plate capacitor model. These results suggest that a mixed-layer approach, involving coadsorption of a strong molecular dipole compound with a chromophore, is a versatile method to shift the energy levels of such chromophores with respect to the band edges of the substrate.

Molecular and Structural Mechanism of Pan-Genotypic HCV NS3/4A Protease Inhibition by Glecaprevir.

Hepatitis C virus, causative agent of chronic viral hepatitis, infects 71 million people worldwide and is divided into seven genotypes and multiple subtypes with sequence identities between 68 to 82%. While older generation direct-acting antivirals had varying effectiveness against different genotypes, the newest NS3/4A protease inhibitors including glecaprevir (GLE) have pan-genotypic activity. The structural basis for pan-genotypic inhibition and effects of polymorphisms on inhibitor potency were not well-known due to lack of crystal structures of GLE-bound NS3/4A or genotypes other than 1. In this study, we determined the crystal structures of NS3/4A from genotypes 1a, 3a, 4a, and 5a in complex with GLE. Comparison with the highly similar grazoprevir indicated the mechanism of GLE's drastic improvement in potency. We found that, while GLE is highly potent against wild-type NS3/4A of all genotypes, specific resistance-associated substitutions (RASs) confer orders of magnitude loss in inhibition. Our crystal structures reveal molecular mechanisms behind pan-genotypic activity of GLE, including potency loss due to RASs at D168. Our structures permit for the first time analysis of changes due to polymorphisms among genotypes, providing insights into design principles that can aid future drug development and potentially can be extended to other proteins.

Ambidextrous α,γ-Hybrid Peptide Foldamers.

Molecular chirality is ubiquitous in nature. The natural biopolymers, proteins and DNA, preferred a right-handed helical bias due to the inherent stereochemistry of the monomer building blocks. Here, we are reporting a rare co-existence of left- and right-handed helical conformations and helix-terminating property at the C-terminus within a single molecule of α,γ-hybrid peptide foldamers composed of achiral Aib (α-aminoisobutyric acid) and 3,3-dimethyl-substituted γ-amino acid (Adb; 4-amino-3,3-dimethylbutanoic acid). At the molecular level, the left- and right-handed helical screw sense of α,γ-hybrid peptides are representing a macroscopic tendril perversion. The pronounced helix-terminating behaviour of C-terminal Adb residues was further explored to design helix-Schellman loop mimetics and to study their conformations in solution and single crystals. The stereochemical constraints of dialkyl substitutions on γ-amino acids showed a marked impact on the folding behaviour of α,γ-hybrid peptides.

α-Aminoisobutyric Acid-Containing Amphipathic Helical Peptide-Cyclic RGD Conjugation as a Potential Drug Delivery System for MicroRNA Replacement Therapy in Vitro.

Replacement therapy with tumor suppressive microRNA (TS-miRNA) might be the next-generation oligonucleotide therapy; however, a novel drug delivery system (DDS) is required. Recently, we developed the cell-penetrating peptide, model amphipathic peptide with α-aminoisobutyric acid (MAP(Aib)), as a carrier for oligonucleotide delivery to cells. In this study, we examined whether a modified MAP(Aib) analogue, MAP(Aib)-cRGD, could be a DDS for TS-miRNA replacement therapy. MIR145-5p, a representative TS-miRNA especially in colorectal cancer, was selected. The MAP(Aib)-cRGD dose was adjusted for MIR145-5p delivery to cells using peripheral blood mononuclear cells and degradation analysis. AlexaFluor488-labeled MIR145-5p incorporation into cells and negative regulation of MIR145-5p-targeting genes demonstrated MAP(Aib)-cRGD's functionality as a miRNA DDS. Treating MIR145-5p with MAP(Aib)-cRGD also revealed various anticancer effects, such as cell viability, invasion inhibition, and apoptosis induction in WiDr cells. Altogether, these findings suggest that MAP(Aib)-cRGD could be a DDS for TS-miRNA replacement therapy, but in vivo investigations are required.

Designed Helical Peptides as Functional Probes for γ-Secretase.

γ-Secretase is a membrane-embedded aspartyl protease complex with presenilin as the catalytic component that cleaves within the transmembrane domain (TMD) of >90 known substrates, including the amyloid precursor protein (APP) of Alzheimer's disease. Processing by γ-secretase of the APP TMD produces the amyloid ß-peptide (Aß), including the 42-residue variant (Aß42) that pathologically deposits in the Alzheimer brain. Complex proteolysis of APP substrate by γ-secretase involves initial endoproteolysis and subsequent carboxypeptidase trimming, resulting in two pathways of Aß production: Aß49 → Aß46 → Aß43 → Aß40 and Aß48 → Aß45 → Aß42 → Aß38. Dominant mutations in APP and presenilin cause early onset familial Alzheimer's disease (FAD). Understanding how γ-secretase processing of APP is altered in FAD is essential for elucidating pathogenic mechanisms in FAD and developing effective therapeutics. To improve our understanding, we designed synthetic APP-based TMD substrates as convenient functional probes for γ-secretase. Installation of the helix-inducing residue α-aminoisobutyric acid provided full TMD helical substrates while also facilitating their synthesis and increasing the solubility of these highly hydrophobic peptides. Through mass spectrometric analysis of proteolytic products, synthetic substrates were identified that were processed in a manner that reproduced physiological processing of APP substrates. Validation of these substrates was accomplished through mutational variants, including the installation of two natural APP FAD mutations. These FAD mutations also resulted in increased levels of formation of Aß-like peptides corresponding to Aß45 and longer, raising the question of whether the levels of such long Aß peptides are indeed increased and might contribute to FAD pathogenesis.

Synthesis, in vitro biological activity and docking of new analogs of BIM-23052 containing unnatural amino acids.

Somatostatin (SST) is an endogenous cyclic tetradecapeptide hormone that exerts multiple biological activities via a family of five receptors. BIM-23052 (DC-23-99) D-Phe-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-NH2 is a linear SST analog with established in vitro GH-inhibitory activity and high affinity to sstr5, sstr3 and sstr2. The different SSTR subtypes are expressed in different tissues and in some tumor cells. Based on this finding, a series of new analogs of BIM-23052 with expected antitumor activity have been synthesized. The Thr at position 6 in BIM-23052 was replaced by the conformationally hindered Tle, Aib, Ac5c and Ac6c of the new analogs. The peptides were synthesized by standard solid-phase peptide chemistry methods, Fmoc strategy. The cytotoxic effects of the compounds were tested in vitro against a panel of tumor cell lines: HT-29, MDA-MB-23, Hep-G2, HeLa and the normal human diploid cell line Lep-3. All five somatostatin receptor subtypes were modeled and docking was performed to determine the binding affinity of the analogs. The new peptides exhibited different concentration-dependent antiproliferative effect on the tumor cell lines after 24 h of treatment. The compound 3B (Aib6) demonstrated the most pronounced antiproliferative effects on HepG-2 cells with the IC50 = 0.01349 nM. Docking confirmed that all compounds bind well to SST receptors with preference to sstr3 and sstr5, which is most probably the reason for the observed biological effects.

Development of 2-aminoisobutyric acid (Aib)-rich cell-penetrating foldamers for efficient siRNA delivery.

We have designed and synthesized a set of cell-penetrating foldamers (CPFs), Blocks 1-8, composed of the common amino acids Leu, Arg, and Gly, as well as the helicogenic amino acid 2-aminoisobutyric acid. The findings showed that Block 3 could deliver siRNA into cells without significant cytotoxicity. We also demonstrated that Block 3 could be applied to selectively kill the oncogene-driven cancer cells.

Single-Conformation Spectroscopy of Capped Aminoisobutyric Acid Dipeptides: The Effect of C-Terminal Cap Chromophores on Conformation.

Aminoisobutyric acid (Aib) oligomers are known to form racemic mixtures of enantiomeric left- and right-handed structures. The introduction of a chiral cap converts the enantiomeric structures into diastereomers that, in principle, afford spectroscopic differentiation. Here, we screen different C-terminal caps based on a model Aib dipeptide using double resonance laser spectroscopy in the gas phase to record IR and UV spectra of individual conformations present in the supersonic expansion: NH-benzyl (NHBn) as a reference structure because of its common use as a fluorophore in similar studies, NH- p-fluorobenzyl (NHBn-F), and α-methylbenzylamine (AMBA). For both the NHBn and NHBn-F caps, a single conformer is observed, with infrared spectra assignable to an enantiomeric pair of type II/II' ß-turns in these molecules lacking a chiral center. The higher oscillator strength of the NHBn-F cap enabled UV-UV hole burning, not readily accomplished with the NHBn cap. The AMBA-capped structure, with its chiral center, produced two unique conformers, one of which was a nearly identical left-handed type II ß-turn, while the minor conformer is assigned to a C7-C7 sequential double ring, which is an emergent form of a 27-ribbon. Although not observed, the type II' ß-turn diastereomer, with opposite handedness, is calculated to be 11 kJ/mol higher in energy, a surprisingly large difference. This destabilization is attributed primarily to steric interference between the C-terminal acyl oxygen of the peptide and the chirality-inducing methyl of the AMBA group. Last, computational evidence indicates that the use of an N-terminal aromatic cap hinders the formation of a 310-helix in Ac-Aib2 dipeptides.

L amino acid transporter structure and molecular bases for the asymmetry of substrate interaction.

L-amino acid transporters (LATs) play key roles in human physiology and are implicated in several human pathologies. LATs are asymmetric amino acid exchangers where the low apparent affinity cytoplasmic side controls the exchange of substrates with high apparent affinity on the extracellular side. Here, we report the crystal structures of an LAT, the bacterial alanine-serine-cysteine exchanger (BasC), in a non-occluded inward-facing conformation in both apo and substrate-bound states. We crystallized BasC in complex with a nanobody, which blocks the transporter from the intracellular side, thus unveiling the sidedness of the substrate interaction of BasC. Two conserved residues in human LATs, Tyr 236 and Lys 154, are located in equivalent positions to the Na1 and Na2 sites of sodium-dependent APC superfamily transporters. Functional studies and molecular dynamics (MD) calculations reveal that these residues are key for the asymmetric substrate interaction of BasC and in the homologous human transporter Asc-1.

Evaluation of Aib and PEG-polymer insect kinin analogs on mosquito and tick GPCRs identifies potent new pest management tools with potentially enhanced biostability and bioavailability.

Insect kinins modulate aspects of diuresis, digestion, development, and sugar taste perception in tarsi and labellar sensilla in mosquitoes. They are, however, subject to rapid biological degradation by endogenous invertebrate peptidases. A series of α-aminoisobutyric (Aib) acid-containing insect kinin analogs incorporating sequences native to the Aedes aegypti mosquito aedeskinins were evaluated on two recombinant kinin invertebrate receptors stably expressed in cell lines, discovering a number of highly potent and biostable insect kinin mimics. On the Ae. aegypti mosquito kinin receptor, three highly potent, biostable Aib analogs matched the activity of the Aib-containing biostable insect kinin analog 1728, which previously showed disruptive and/or aversive activity in aphid, mosquito and kissing bug. These three analogs are IK-Aib-19 ([Aib]FY[Aib]WGa, EC50 = 18 nM), IK-Aib-12 (pQKFY[Aib]WGa, EC50 = 23 nM) and IK-Aib-20 ([Aib]FH[Aib]WGa, EC50 = 28 nM). On the Rhipicephalus (Boophilus) microplus tick receptor, IK-Aib-20 ([Aib]FH[Aib]WGa, EC50 = 2 nM) is more potent than 1728 by a factor of 3. Seven other potentially biostable analogs exhibited an EC50 range of 5-10 nM, all of which match the potency of 1728. Among the multi-Aib hexapeptide kinin analogs tested the tick receptor has a preference for the positively-charged, aromatic H over the aromatic residues Y and F in the X1 variable position ([Aib]FX1[Aib]WGa), whereas the mosquito receptor does not distinguish between them. In contrast, in a mono-Aib pentapeptide analog framework (FX1[Aib]WGa), both receptors exhibit a preference for Y over H in the variable position. Among analogs incorporating polyethylene glycol (PEG) polymer attachments at the N-terminus that can confer enhanced bioavailability and biostability, three matched or surpassed the potency of a positive control peptide. On the tick receptor IK-PEG-9 (P8-R[Aib]FF[Aib]WGa) was the most potent. Two others, IK-PEG-8 (P8-RFFPWGa) and IK-PEG-6 (P4-RFFPWGa), were most potent on the mosquito receptor, with the first surpassing the activity of the positive control peptide. These analogs and others in the IK-Aib series expand the toolbox of potent analogs accessible to invertebrate endocrinologists studying the structural requirements for bioactivity and the as yet unknown role of the insect kinins in ticks. They may contribute to the development of selective, environmentally friendly pest arthropod control agents.

The Shape of the Simplest Non-proteinogenic Amino Acid α-Aminoisobutyric Acid (Aib).

The simplest non-proteinogenic amino acid α-aminoisobutyric acid (Aib), an analogue of glycine and alanine, has been vaporized by laser ablation and probed by high-resolution Fourier transform microwave spectroscopic techniques. Comparison of the experimental rotational and 14 N nuclear quadrupole constants with that predicted ab initio has allowed the identification of three conformers of Aib exhibiting three types of hydrogen-bond interactions I (NH⋅⋅⋅O=C, cis-COOH), II (OH⋅⋅⋅N, trans-COOH), and III (N-H⋅⋅⋅O-H, cis-COOH) within the amino acid backbone. The observation of conformer III, not detected previously for related proteinogenic amino acids with a nonpolar side chain in a supersonic expansion, indicates that the presence of the methyl groups should restrict the conformational relaxation from conformer Aib-III to Aib-I. For conformer Aib-II, the rotational spectra of the 13 C isotopomers reveal a tunneling motion arising from the two equivalent methyl groups in the molecule. The observation of a single spectrum at the midpoint between those predicted for the two 13 C of the methyl groups has been explained by considering a double-minimum potential function with a low-energy interconversion barrier for a large amplitude internal motion. This singular fact has been corroborated by the anomalous centrifugal distortion effects determined in conformer Aib-II.

Effect of oscillation dynamics on long-range electron transfer in a helical peptide monolayer.

Electron transfer (ET) reactions via helical peptides composed of -(Aib-Pro)n- were studied in self-assembled monolayers and compared with -(Ala-Aib)n- peptides. Short Aib-Pro peptides showed slightly higher ET rates due to the better electronic coupling of the Pro residue. But, the 24mer Aib-Pro peptide showed a smaller ET rate than the corresponding Ala-Aib peptide. On the basis of DFT calculations, the deceleration of the ET rate of the longer Aib-Pro peptide is considered to be due to the smaller number of active modes of accordion-like oscillations than the Ala-Aib peptide, which has a strong influence on a long-range ET reaction.

Tuning the Morphology of Nanostructured Peptide Films by the Introduction of a Secondary Structure Conformational Constraint: A Case Study of Hierarchical Self-Assembly.

Peptide self-assembly is ubiquitous in nature. It governs the organization of proteins, controlling their folding kinetics and preserving their structural stability and bioactivity. In this connection, model oligopeptides may give important insights into the molecular mechanisms and elementary forces driving the formation of supramolecular structures. In this contribution, we show that a single residue substitution, that is, Aib (α-aminoisobutyric acid) in place of Ala at position 4 of an -(l-Ala)5-homo-oligomer, strongly alters the aggregation process. In particular, this process is initiated by the formation of small peptide clusters that promote aggregation on the nanometer scale and, through a hierarchical self-assembly, lead to mesoscopic structures of micrometric dimensions. Furthermore, we show that the use of the well-established Langmuir-Blodgett technique represents an effective strategy for coating extended areas of inorganic substrates by densely packed peptide layers, thus paving the way for application of peptide films as templates for biomineralization, biocompatible coating of surfaces, and scaffolds for tissue engineering.