Student Number : 9306227A - PhD thesis - School of Molecular and Cell Biology - Faculty of Science / It is well documented that N-capping motifs are stabilising local motifs for -helices. N-capping motifs have been identified within hGST A1-1 at the N-terminal ends of -helix 9 and helix 6. The conservational role of these two motifs in protein stability, folding and function was investigated. -Helix 9 is a unique structural feature to class Alpha GSTs that is important for its catalytic functioning. This amphipathic helix is highly dynamic, where upon ligand binding at the active-site, the delocalised C-terminal region becomes immobilised to form a structured helix forming a “lid” over the active-site. The specific role of the Asp N-cap motif toward the stability and dynamics of helix 9 was determined by substituting the Asp-209 for a Gly. ANS binding and urea-induced activity studies showed that by removing the N-cap motif of helix 9 in hGST A1-1, the helix 9 is destabilised rendering a less hydrophobic binding site compared to that in the wild-type. The helical content of the peptide, corresponding to helix 9 in the C-terminal region of hGST A1-1 (208 -222), decreased significantly upon the removal of the N-cap motif. The explanation for the conservation of the Asp N-cap residue can be found in its stabilising role of the C-terminal region of class Alpha GSTs. This stabilising role was however less apparent in context of the protein compared to that in the peptide. Majority of the atomic contacts owing to the stability of helix 9 appear to be governed by non-local tertiary interactions rather than local interactions, such as the N-cap motif. These tertiary interactions are likely to include short and long range contacts between residues on the surface of the protein that are already known to contribute towards the stability of the C-terminal region. In this study, the ligand displacement-studies and the molecular docking results strongly suggest that 8-aniline-1-napthalene sulfonate binds at the H-site in hGST A1-1. The N-capping motif of helix 6 identified in class Alpha GSTs is located within the core of domain 2. This motif is a common feature found amongst almost all GST-like proteins and is thought to be the folding nucleation site (Stenberg et al. J. Biol. Chem. 275 (2000), 10421-10428). The N-cap (Ser- 154) and N3 (Asp-157) residues were each substituted with an Ala in hGST A1-1 to investigate the role of this motif in the folding of hGST A1-1. Both substitutions resulted in thermal sensitive mutants compared to that of the wild-type. The N3 substitution (D157A) was however too disruptive, where the yields of this mutant were insufficient for any further studies to be carried out. For the N-cap mutant (S154A), the unfolding kinetic studies revealed a significantly destabilised core in domain 2 compared to that of the wild-type. The kinetic folding studies monitored by fluorescence spectroscopy, revealed that the N-cap motif contributes to the efficient folding and dimerisation of the subunits, and to a far lesser extent towards the final tight packing and reorganisation of tertiary interactions in hGST A1-1. Since no changes in the burst-phase of S154A was evident compared to that of the wild-type, it seems unlikely that this motif is a folding nucleation site in hGST A1-1. These results do not exclude the possibility that this motif contributes to the rapid formation secondary structure during the burst-phase of folding. Due to the highly conserved region surrounding helix 6 , the role of this motif contributing to the stability of hGST A1-1 could be a general feature for GSTs and GSTlike proteins. In this study, further insight into the mechanism of folding for hGST A1-1 was gained. The hydrophobic core packing surrounding helix 6 occurred as a late folding event, that is during the final packing and reorganisation of tertiary interactions of the protein. The N-cap motif is an important structural feature for the fast folding of domain 2. This N-cap motif is a unique structural feature important for the efficient folding of the monomers, which is exclusive to its role in stabilising helix 6 in hGST A1-1.

The host adaptive immune response to a pathogen infection comprises both cell mediated and antibody dependent components. Antibody mediated neutralization is a key component of protection against viruses and is the primary focus of this thesis. Antibodies recognize structurally defined epitopes within the context of native proteins. These may be represented by a simple linear sequence of amino acids or a discontinuous sequence of residues brought together by the conformational constraints of the protein. Many protein epitopes recognized by antibodies have been shown to be short α-helices of 3-5 turns. However corresponding synthetic peptides of this length have no structure in water because solvent competes strongly for the hydrogen bonding amides otherwise required to hydrogen bond one another to define an α-helix. This thesis is aimed primarily at (1) synthetically constraining short peptide sequences (9-13 residues) into stable α-helices of 3-4 turns; (2) structurally characterizing such constrained α-helical structures by circular dichroism and 1D and 2D NMR spectroscopy; and (3) evaluating these helix mimetics for serum stability, immunogenicity, antigenicity as well as the biological relevance of the antibodies they induce. The overall aim was to demonstrate that constrained short peptides more effectively structurally and functionally mimic known α-helical B cell epitopes from native proteins than unconstrained short peptides of the same lengths. The primary focus of Chapter 2 was to optimize in vitro ELISA conditions and immunization protocols for potentially assessing antibody responses in mice to short peptides corresponding to segments of important dengue virus proteins (NS1 and the envelope fusion protein, E). The NS1 peptide investigated had been suggested to be an α-helical epitope, but my investigations reveal that it is more likely a turn rather than a helix. While the E protein epitope chosen was not a viable epitope for testing a helix-constraining strategy, it was evaluated as a constrained turn mimic of a viral fusion epitope. Although the constrained peptides from both proteins (NS1 and E) elicited stronger antibody responses in mice than their unconstrained analogues, they still induced relatively poor antibody levels. Interestingly, mouse antibodies raised to the constrained peptide (β-turn analogue) from NS1 protein also reacted with the native protein. To evaluate a helix-constraining strategy for short peptides (less than 15 residues) that have no helix structure in water, an epitope of the HPV E7 protein was selected for mimicry. A short peptide sequence corresponding to this B cell epitope had previously been reported to have α-helical propensity but only in trifluoroethanol-water mixtures, and my initial work showed that it had no detectable helical structure at all in water. Chapter 3 presents an example of a short helical peptide as a B cell epitope, constrained into an α-helix by a side chain to side chain lactam bridge. The constraint involved cyclizing the peptide by specifically linking together side chains of lysine and aspartic acid inserted in the sequence three amino acids apart. CD and NMR structural studies highlighted significant α-helicity in the constrained short peptide, whereas the corresponding unconstrained short peptide had no structure in water. Both unconstrained and constrained short peptide epitopes were injected into mice and antibodies raised were quantified ex vivo by peptide ELISA. The helix-constrained epitope elicited higher antibody titres than the unconstrained peptide which was relatively non-immunogenic. Importantly, antibodies raised to the constrained synthetic α-helical peptide also reacted with the native E7 protein, suggesting that the helical constraint conferred on the peptide a structure analogous to that seen in the protein. In Chapter 4 a constrained α-helical peptide corresponding to a crystallographically defined α-helical sequence in the fusion, F protein of respiratory syncitial virus (RSV) was investigated for its potential to induce an antibody response. Again, while the helix-constrained peptide clearly had α-helicity by CD and NMR studies, the unconstrained short peptide had no detectable helical structure in water. To potentially boost antibody responses, relative to those generated against the dengue virus peptides examined in Chapter 3, both unconstrained and constrained peptides were coupled to the carrier protein KLH before immunizing mice. Significant levels of peptide reactive antibody were generated to both the unconstrained and constrained peptides. However, when investigated in a viral neutralization assay, the antibodies raised to the unconstrained peptide showed a higher neutralization potential than those raised to the constrained peptide. We attribute this unexpected difference to the fact that the region of the F protein corresponding to the epitope chosen, undergoes dramatic conformational changes during the viral fusion process and it is only in its post-fusion form that this helix has been observed. It is possible that the inherent flexibility of the linear, unconstrained counterpart of this epitope may more effectively mimic the conformational intermediates of the native structure on presentation to the immune system. Chapter 5 began an examination of the effects of three different adjuvants on antibody induction by short peptides. They were compared using a candidate peptide vaccine for malaria as a model system. As before, a helix-constrained peptide was compared with its unconstrained peptide sequence in immunization experiments. Higher titres of antibodies were raised to the constrained versus unconstrained peptides. In the second part of this chapter, a putative cancer vaccine peptide was similarly constrained via an ester linkage or a helix-inducing lactam bridge but both methods induced only low T-cell responses compared to their corresponding unconstrained sequences, possibly because the incorrect structure had been stabilized. The focus of this thesis was to evaluate a helix stabilization strategy for its possible application to short peptide vaccines. Using extensive circular dichroism and NMR spectroscopy measurements, we have shown in all cases that helix-constrained peptides were much more α-helical in solution than their corresponding unconstrained short peptide sequences that tended to have no or negligible α-helix structure in water. In some examples, we have compared serum stability and found that constrained peptides have higher serum stability than unconstrained peptides, a difference attributed to their greater stability towards proteolytic degradation – proteases being unable to recognize helices. We have also proven that the helix-constrained peptides induced higher mouse antibody titres than unconstrained peptides. Several attempts were made to boost antibody responses to the peptides by varying either immunization protocols, adjuvant or by attaching a carrier molecule. Further work is needed to optimize this promising new approach to short peptide vaccines.

Molecular structures of biomaterials have close relation to their functions. We are interested in how biological building blocks assemble into the structures of native biomaterials and the hierarchy of those structures. We tackled the problem mainly with X-ray diffraction experiments and developed a thorough analysis technique to assign the X-ray signals to protein secondary structures and chitin. Three different types of biomaterials were examined: vimentin fibres, squid pens, and human hair. In vimentin fibres, we found that the secondary protein structures play an important role in the strength of the fibres. In native squid pens, we found a self-similar, hierarchical structure from millimetres down to nanometres. In human hair, we compared the signals corresponding to keratin proteins, intermediate filaments, and lipids between different subjects, and found small deviations. The structures of these three biomaterials, which encompass different orders of length scales, were described both quantitatively and graphically. We hope that this work will eventually allow us to understand how and why nature builds biomaterials this way. / Thesis / Master of Science (MSc)

A refinement of a minimal model for protein folding originally proposed by Imamura is presented. The representation of the alpha-helix has been improved by adding in explicit modelling of the entire peptide unit. A four-helix bundle consisting of four alpha-helices and three loop regions is generated with the parallel tempering Monte Carlo scheme. Six native states are found for the given sequence, four U-bundle and two Z-bundle states. All six states have energies of E approx -218ε and all appear equally likely to occur in simulation. The highest probability of folding a native state is found to be at a hydrophobic strength of Ch = 0.8 which agrees with the value of Ch = 0.7 used by Imamura in his studies of alpha to beta structural conversions. Two folding stages are observed in the temperature spectrum dependent on the magnitude of the hydrophobic strength parameter. The two stages observed as temperature decreases are 1) the hydrophobic energy causes the random coil to collapse into a compact globule 2) the secondary structure starts forming below a temperature of about T = 0.52ε/kB. The temperature of the first stage, which corresponds to the characteristic collapse temperature Tθ, is highly dependent on the hydrophobic strength. The temperature of the second stage is constant with respect to hydrophobic strength. Attempts to measure the characteristic folding temperature, Tf , from the structural overlap function proved to be difficult due mostly to the presence of six minima and the complications that arose in the parallel tempering Monte Carlo scheme. However, a rough estimate of Tf is obtained at each hydrophobic strength from a native state density analysis. Tf is found to be significantly lower than Tθ.

A refinement of a minimal model for protein folding originally proposed by Imamura is presented. The representation of the alpha-helix has been improved by adding in explicit modelling of the entire peptide unit. A four-helix bundle consisting of four alpha-helices and three loop regions is generated with the parallel tempering Monte Carlo scheme. Six native states are found for the given sequence, four U-bundle and two Z-bundle states. All six states have energies of E approx -218ε and all appear equally likely to occur in simulation. The highest probability of folding a native state is found to be at a hydrophobic strength of Ch = 0.8 which agrees with the value of Ch = 0.7 used by Imamura in his studies of alpha to beta structural conversions. Two folding stages are observed in the temperature spectrum dependent on the magnitude of the hydrophobic strength parameter. The two stages observed as temperature decreases are 1) the hydrophobic energy causes the random coil to collapse into a compact globule 2) the secondary structure starts forming below a temperature of about T = 0.52ε/kB. The temperature of the first stage, which corresponds to the characteristic collapse temperature Tθ, is highly dependent on the hydrophobic strength. The temperature of the second stage is constant with respect to hydrophobic strength. Attempts to measure the characteristic folding temperature, Tf , from the structural overlap function proved to be difficult due mostly to the presence of six minima and the complications that arose in the parallel tempering Monte Carlo scheme. However, a rough estimate of Tf is obtained at each hydrophobic strength from a native state density analysis. Tf is found to be significantly lower than Tθ.

Membrane proteins roughly constitute 30% of open reading frames in a genome and form 70% of current drug targets. They are classified as integral, peripheral membrane proteins and polypeptide toxins. α-helices and β -strands are the principal secondary structures observed in integral membrane proteins. This thesis presents the results of studies on analysis and correlation of sequence and structure of helices constituting integral helical membrane proteins. The aim of this work is to understand the helix stabilization, distortion as well as packing in terms of amino acid sequences and the correlated structures they adopt. To this end, analyses of datasets of X-ray crystal structures of integral helical membrane proteins and their comparison with a dataset of representative folds of globular proteins was carried out. Initial analysis was carried out using a non-redundant dataset of 75 membrane proteins to understand sequence and structural preferences for stabilization of helix termini. The subsequent analysis of helix distortions in membrane proteins was carried out using an updated dataset of 90 membrane proteins. Chapter 1 of the thesis reviews experimental as well as theoretical studies that have provided insights into understanding the structure of helical membrane proteins. Chapter 2 details the methods used during the course of the present investigations. These include the protocol used for creation of the non-redundant database of membrane and globular proteins. Various statistical methods used to test significance of the position-wise representation of amino acids in helical regions and the differences in globular and membrane protein datasets have been listed. Based on the tests of significance, a methodology to identify differences in propensity values that are statistically significant among two datasets has been devised. Programs used for secondary structure identification of membrane proteins namely Structure Identification (STRIDE) and Assignment of Secondary Structure in Proteins (ASSP) as well as those used for characterization of helical geometry (Helanal-Plus) have also been enlisted. In Chapter 3, datasets of 865 α-helices in 75 membrane proteins and 2680 α- helices from 626 representative folds in globular proteins defined by the STRIDE program have been analyzed to study the sequence determinants at fifteen positions within and around the α-helix. The amino acid propensities have been studied for positions that are important for the process of helix initiation, propagation, stabilization and termination. Each of the 15 positions has unique sequence characteristics reflecting their role and contribution towards the stability of the α-helix. A comparison of the sequence preferences in membrane and globular proteins revealed common residue preferences in both these datasets confirming the importance of these positions and the strict residue preferences therein. However, short/medium length α-helices that initiated/terminated within the membrane showed distinct amino acid preferences at the N-terminus (Ncap, N1, N2) as well as the C-terminus ( Ccap, Ct) when compared to α-helices belonging to membrane and globular proteins. The sequence preferences in membrane proteins were governed by the helix initiating and terminating property of the amino acids as well as the external environment of the helix. Results from our analysis also conformed well with experimentally tested amino acid preferences in a position-specific amino acid preference library of the rat neurotensin receptor (Schlinkmann et al (2012) Proc Natl Acad Sci USA 109(25):1890-5) as well as crystal structures of GPCR proteins. In the light of the environment dependent amino acid preferences found at α- helix termini, a survey was carried out to find various helix capping motifs adopted at both termini of α-helices in globular and membrane proteins to stabilize these helix termini. The results from these findings have been reported in Chapter 4. A sequence dependent structural preference is found for capping motifs at helix termini embedded inside and protruding outside the membrane. The N-terminus of α-helices was capped by hydrogen bonds involving free main chain amide groups of the first helical turn as donors and amino acid side chains as acceptors, as against the C-terminus which showed position-dependent characteristic backbone conformations to cap the helix. Overall helix termini inside the membrane did not show a very high number of capping motifs; instead these termini were stabilized by helix- helix interactions contributed by the neighboring helices of the helical bundle. In Chapter 5, we examine transmembrane helical (TMH) regions to identify as well as characterize the various types of helix perturbations in membrane proteins using ASSP and Helanal-Plus. A survey of literature shows that the term ‘helix kink’ has been used rather loosely when in fact helical regions show significant amounts of variation and transitions in helical parameters. Hence a systematic analysis of TMH regions was undertaken to quantify different types of helix perturbations, based on geometric parameters such as helical twist, rise per residue and local bending angle. Results from this analysis indicated that helices are not only kinked but undergo transitions to form interspersed stretches of 310 helices and π-bulges within the bilayer. These interspersed 310 and π-helices showed unique sequence preferences within and around their helical body, and also assisted in main- taining the helical structure within the bilayer. We found that Proline not only kinked the helical regions in a characteristic manner but also caused a tightening or unwinding in a helical region to form 310 and π-helix fragments respectively. The helix distortions also resulted in backbone hydrogen bonds to be missed which were stabilized by hydrogen bonds from neighboring residues mediated by their side chain atoms. Furthermore, a packing analysis showed that helical regions with distortions were able to establish inter-helical interactions with more number of transmembrane segments in the helical bundle. The study on helix perturbations presented in the previous chapter, brought to light a previously unreported 19 amino acid π-helix fragment interspersed between α-helices in the functionally important transmembrane helix 2 (TM2) belonging to Mitochondrial cytochrome-c-oxidase (1v55). Chapter 6 describes a case study of the structurally similar but functionally different members within the Heme-Copper- Superoxidases (HCO) superfamily that were considered for a comparative analysis of TM2. An analysis of 7 family members revealed that the π-helix shortens, fragments in two shorter π-helices or was even absent in some family members. The long π-helix significantly decreased the total twist and rise of the entire helical fragment thus accommodating more hydrophobic amino acids within the bilayer to avoid hydrophobic mismatch with the bilayer. The increased radius of the TM2 helical fragment also assisted in helix packing interactions by increasing the number of residues involved in helix-helix interactions and hydrogen bonds. Chapter 7 documents the conclusions from the different analyses presented in each of the above chapters. Overall, it is found that membrane proteins optimize the biophysical and chemical constraints of the external environment to strategically place select amino acids at helix termini to ‘start’ and ‘stop’ α-helices. The stabilization of these helix termini is a consequence of sequence dependent structural preferences to form helix capping motifs. The studies on helix transitions and distortions highlight that membrane proteins are not only packed as α-helices but also accomodate 310- and π-helical fragments. These transitions and distortions help in harboring more hydrophobic amino acids and aiding inter-helical interactions important for maintaining the fold of the membrane protein. Appendix A describes a comparison of α-helix assignments in globular and membrane proteins by two algorithms, one based on Cα trace (ASSP) and the other using a combination of hydrogen bond pattern along with backbone torsion angles φ and ψ (STRIDE).

Le virus de la mosaïque jaune du navet (TYMV) possède un génome monopartite constitué d’ARN de polarité positive codant pour trois protéines, dont seule la polyprotéine 206K est indispensable à la réplication virale.Elle subit une maturation protéolytique, générant les protéines 140K et 66K, localisées au niveau de l’enveloppe des chloroplastes, siège de la réplication virale.Adressée aux chloroplastes, la protéine 140K y recrute la 66K et se comporte comme une protéine intégrale membranaire.Le domaine d’adressage aux chloroplastes (DAC) de la protéine 140K a été défini grâce à la transfection et à des protoplastes d’Arabidopsis thaliana par différentes constructions codantpour des versions délétées de la protéine fusionnées à l’EGFP, et à leur observation en microscopie confocale. Le DAC comprend deux hélices alpha amphipathiques dont la présence a été attestée par dichroïsme circulaire. Leur nécessité pour la localisation aux chloroplastes, l’association aux membranes et la réplication virale, a été étudiée. Différents patterns de distribution subcellulaire de la protéine 140K ont été observés. Ils sont corrélés au taux d’expression de la protéine. Sa dimérisation a également été démontrée.L’implication d’autres résidus du DAC dans la localisation subcellulaire, la dimérisation et la réplication virale, a également été recherchée. / Turnip yellow mosaic virus (TYMV) is a positive single-stranded RNA virus. Among the three ORFs encoded by the TYMV genome, 206K is the only protein required for viral replication. It is cleaved into 140K and 66K, which are both present at the chloroplast envelope membrane, where viral replication takes place.The 140K protein is targeted to chloroplasts, where it recruits 66K, and behaves as an integral membrane protein. The chloroplast targeting domain (DAC) of the 140K protein was defined using Arabidopsis thaliana protoplasts transfected by various constructs encoding deleted versions of 140Kfused to EGFP and subsequent confocal microscopy. The DAC comprises two amphipathic alpha helices, as confirmed by circular dichroism. Their involvement in chloroplast localisation and membrane association has been assessed, as well as their contribution to viral replication.We observed different subcellular distribution patterns of 140K protein, which correlate with the expression level of the protein. Its capability to dimerize has also been demonstrated.The involvement of other DAC residues in subcellular localisation, dimerization and viral replication has been studied.

Voltage-gated ion channels play fundamental roles in neural excitability and thus dysfunctional channels can cause disease. Understanding how the voltage-sensor of these channels activate and inactivate could potentially be useful in future drug design of compounds targeting neuronal excitability. The opening and closing of the pore in voltage-gated ion channels is caused by the arginine-rich S4 helix of the voltage sensor domain (VSD) moving in response to an external potential. Exactly how this movement is accomplished is not yet fully known and an area of hot debate. In this thesis I study how the opening and closing in voltage-gated potassium (Kv) channels occurs. Recently, both experimental and computational results have pointed to the possibility of a secondary structure transition from α- to 3(10)-helix in S4 being an important part of the gating. First, I show that the 3(10)-helix structure in the S4 helix of a Kv1.2-2.1 chimera protein is significantly more favorable compared to the α-helix in terms of a lower free energy barrier during the gating motion. Additional I suggest a new gating model for S4, moving as sliding 310-helix. Interestingly, the single most conserved residue in voltage- gated ion channels is a phenylalanine located in the hydrophobic core and directly facing S4 causing a barrier for the gating charges. In a second study, I address the problem of the energy barrier and show that mutations of the phenylalanine directly alter the free energy barrier of the open to closed transition for S4. Mutations can either facilitate the relaxation of the voltage-sensor or increase the free energy barrier, depending on the size of the mutant. These results are confirmed by new experimental data that supports that a rigid, cyclic ring at the phenylalanine position is the determining rate-limiting factor for the voltage sensor gating process. / QC 20110616

Cette thèse est consacrée à la synthèse et à l'étude structurale de peptides-beta/gamma, contenant en alternance des acides aminés-beta et -gamma, conçus pour mimer l'hélice-alpha (ou hélice-13), structure secondaire des protéines. Nous avons ainsi élaboré une stratégie de design « bottom-up » pour des peptides-beta/gamma devant se replier sous forme d'hélice-13. Ces peptides comportent un acide aminé-beta, le (1S,2S)-trans-2-aminocyclobutanecarboxylique, qui joue un rôle clé de brique constitutive en apportant des contraintes conformationnelles. Dans un premier temps, la synthèse énantiomériquement pure du trans-ACBC basée sur une étape clé de photocycloaddition [2+2] a été optimisée. Il a alors été possible de synthétiser des peptides-beta/gamma incorporant en alternance le trans-ACBC et le GABA, qui est un acide aminé-gamma dépourvu de toute contrainte. Des études expérimentales et théoriques fines de ces peptides-beta/gamma ont révélé une structuration inédite sous forme de rubban-9/8, en solution. Il a été démontré que ces nouveaux foldamères adoptent une forme plus ou moins courbe gouvernée par un code combinant configuration et conformation des acides aminés constitutifs de ces peptides. Dans un deuxième temps, des contraintes sur l'acide aminé-gamma ont été introduites par la préparation de peptides-beta/gamma alternant le trans-ACBC et des acides aminés-gamma4. Des études expérimentales et théoriques de ces peptides-beta/gamma en solution ont révélé une préférence conformationnelle sous forme d'hélice-13. La stabilité de cette structure hélicoïdale augmente avec la longueur de la chaîne peptidique. Ces hélices-13 sont en effet fortement stabilisées à partir de 5 liaisons hydrogènes inter-résidus. Enfin, des peptides-alpha/beta/gamma capables de mimer l'hélice-alpha du peptide p53(15-31) ont été conçus et synthétisés, afin de vérifier expérimentalement leur hélicité prédite par modélisation moléculaire. Une fois leur résistance à la dégradation protéolytique démontrée, ces peptides-alpha/beta/gamma ont été testés comme inhibiteur de l'interaction p53/hDM2. Un candidat a particulièrement été capable d'inhiber cette interaction en se liant au site naturel de fixation avec la protéine hDM2. Ce résultat illustre la réussite de notre stratégie de construction de mimes de l'hélice-alpha. / This thesis is devoted to the synthesis and the structural characterisation of beta/gamma-peptides, constructed from beta- and gamma-amino acids in alternation, designed to mimic the alpha-helix secondary structure which is present in many native proteins. The alpha-helix can be defined as a 13-helix and a bottom-up foldamer design strategy to target a 13-helical structure was examined, whereby beta/gamma-peptides were proposed in which (1S,2S)-trans-2-aminocyclobutanecarboxylic acid (trans-ACBC) was incorporated as a conformationally-restricted beta-amino acid component. The scalable synthesis of enantiomerically pure trans-ACBC using a [2+2] photocycloaddition strategy was successfully optimized. beta/gamma-Peptides incorporating trans-ACBC and GABA, the latter being the gamma-amino acid component devoid of any constraint, were then synthesised. Experimental and theoretical investigations of their solution-state folding behaviour revealed an unprecedented 9/8-ribbon foldamer structure that adopts curved shapes governed by a combined configuration-conformation code. Additional constraints on the gamma-amino acid component were then considered and beta/gamma-peptides incorporating trans-ACBC and gamma4-amino acids were synthesised. Experimental and theoretical investigations of these beta/gamma-peptides in solution unveiled a preference for 13-helix folding behaviour, which increased commensurately with the peptide chain length; robust 13-helices were stabilised by a minimum of five intramolecular hydrogen bonds. In the last part of this thesis, molecular modelling was used to design helical alpha/beta/gamma-peptides intended to reproduce as closely as possible the hot-spot residues of the known alpha-helical peptide sequence p53(15-31). These peptides were synthesised and their predicted helical folding was verified experimentally along with their resistance to proteolytic enzymes. The alpha/beta/gamma-peptides were tested as inhibitors of the p53/hDM2 interaction. One peptide was found to behave as potent inhibitor and to bind to the native peptide binding pocket of the hDM2 protein, providing a successful proof of concept of the alpha-helix mimetic design strategy.