Biomimetic materials that recapitulate the complex mechanical and biochemical cues in load-bearing tissues are of significant interest in regenerative medicine and tissue engineering applications. Several investigators have endeavored to not only emulate the mechanical properties of the vasculature, but to also mimic the biologic responsiveness of the blood vessel in creating vascular substitutes. Previous studies in our lab generated the elastin-like protein polymer LysB10, which was designed with the capability of physical and chemical crosslinks, and was shown to display a range of elastomeric properties that more closely matched those of the native artery. While extensive validation of the mechanical properties of elastin-mimetic polymers has demonstrated their functionality in a number of tissue engineering applications, limited cell growth on the surfaces of the polymers has motivated further optimization for biological interaction. Recent biologically-inspired surface strategies have focused on functionalizing material surfaces with extracellular matrix molecules and bioactive motifs in order to encourage integrin-mediated cellular responses that trigger precise intracellular signaling processes, while limiting nonspecific biomaterial interactions. Consequently, this dissertation addresses three approaches to modulating cellular behavior on elastin-mimetic analogs with the goal of promoting vascular wall healing and tissue regeneration: genetic engineering of elastin-like protein polymers (ELPs) with cell-binding domains, biofunctionalization of elastin-like protein polymers via chemoselective ligation of bioactive ligands, and incorporation of matrix protein fibronectin for engineering of cell-seeded multilamellar collagen-reinforced elastin-like constructs. The synthesis of recombinant elastin-like protein polymers that integrate biologic functions of the extracellular matrix provides a novel design strategy for generating clinically durable vascular substitutes. Ultimately, the synthesis of model protein networks provides new insights into the relationship between molecular architecture, biomimetic ligand presentation, and associated cellular responses at the cell-material interface. Understanding how each of these design parameters affects cell response will contribute significantly to the rational engineering of bioactive materials. Potential applications for polymer blends with enhanced mechanical and biological properties include surface coatings on vascular grafts and stents, as well as composite materials for tissue engineered scaffolds and vascular substitutes.

Measuring the amount of information and of shared information in biological strings, as well as relating information to structure, function and evolution, are fundamental computational problems in the post-genomic era. Classical analyses of the information content of biosequences are grounded in Shannon's statistical telecommunication theory, while the recent focus is on suitable specializations of the notions introduced by Kolmogorov, Chaitin and Solomonoff, based on data compression and compositional redundancy. Symmetrically, classical estimates of mutual information based on string editing are currently being supplanted by compositional methods hinged on the distribution of controlled substructures. Current compositional analyses and comparisons of biological strings are almost exclusively limited to short sequences of contiguous solid characters. Comparatively little is known about longer and sparser components, both from the point of view of their effectiveness in measuring information and in separating biological strings from random strings, and from the point of view of their ability to classify and to reconstruct phylogenies. Yet, sparse structures are suspected to grasp long-range correlations and, at short range, they are known to encode signatures and motifs that characterize molecular families. In this thesis, we introduce and study compositional measures based on the repertoire of distinct subsequences of any length, but constrained to occur with a predefined maximum gap between consecutive symbols. Such measures highlight previously unknown laws that relate subsequence abundance to string length and to the allowed gap, across a range of structurally and functionally diverse polypeptides. Measures on subsequences are capable of separating only few amino acid strings from their random permutations, but they reveal that random permutations themselves amass along previously undetected, linear loci. This is perhaps the first time in which the vocabulary of all distinct subsequences of a set of structurally and functionally diverse polypeptides is systematically counted and analyzed. Another objective of this thesis is measuring the quality of phylogenies based on the composition of sparse structures. Specifically, we use a set of repetitive gapped patterns, called motifs, whose length and sparsity have never been considered before. We find that extremely sparse motifs in mitochondrial proteomes support phylogenies of comparable quality to state-of-the-art string-based algorithms. Moving from maximal motifs -- motifs that cannot be made more specific without losing support -- to a set of generators with decreasing size and redundancy, generally degrades classification, suggesting that redundancy itself is a key factor for the efficient reconstruction of phylogenies. This is perhaps the first time in which the composition of all motifs of a proteome is systematically used in phylogeny reconstruction on a large scale. Extracting all maximal motifs, or even their compact generators, is infeasible for entire genomes. In the last part of this thesis, we study the robustness of measures of similarity built around the dictionary of LZW -- the variant of the LZ78 compression algorithm proposed by Welch -- and of some of its recently introduced gapped variants. These algorithms use a very small vocabulary, they perform linearly in the input strings, and they can be made even faster than LZ77 in practice. We find that dissimilarity measures based on maximal strings in the dictionary of LZW support phylogenies that are comparable to state-of-the-art methods on test proteomes. Introducing a controlled proportion of gaps does not degrade classification, and allows to discard up to 20% of each input proteome during comparison.

Peptides have assumed considerable importance in pharmaceutical industry and vaccine research. Understanding the structural features of these peptide molecules can be effective not only in mimicking natural proteins but also in the design of new biomaterials. Polypeptide sequences consisting of twenty genetically coded amino acids possess structural flexibility, which makes the predictions difficult. However, the introduction of non-protein amino acids into the peptide chain restricts the available range of backbone conformations and acts as stereochemical directors of polypeptide chain folding. Such conformationally rigid residues allow the formation of well defined structures like helices, strands etc, which further assemble into super secondary structural motifs by flexible linkages. Crystal structure determination of the oligopeptides by X-ray diffraction gives insight into the specific conformational states, modes of aggregation, hydrogen bond interactions, solvation of peptides and various weakly polar interactions involving the side chains of aromatic residues (Phe, Trp and Tyr). In β-, γ- and higher ω-amino acids, due to the insertion of one or more methylene groups between the N- and Cα-atoms into the peptide backbone the accessible conformational space is greater than the α-amino acids. The β-, γ-, δ-…. amino acid residues belong to the family of ω-amino acids. Extensive research in the field of β-peptides, which have been experimentally verified or theoretically postulated, has assigned several helices, turns and sheets. The use of ω-amino acid residues in conjunction with α-residues permits systematic exploration of the effects of introducing additional backbone atoms into well-characterized α-peptide structures. The observation of new families of hydrogen bonded motifs in the hybrid peptides containing α- and ω-amino acids are the recent interest in this regard. This thesis reports results of X-ray crystallographic studies of eighteen designed peptides and four protected ω-amino acids listed below. Within brackets are given the abbreviations used for the sequences (Symbol U represents Aib). The ω-amino acids reported in this thesis are: (S)-β3-HAla (β3-homoalanine), (R)-β3-HVal, (S)-β3-HVal (β3-homovaline), (S)-β3-HPhe (β3-homophenylalanine), (S)-β3-HPro (β3-homoproline), βGly (β-homoglycine), γAbu (gamma aminobutyric acid) and δAva (delta aminovaleric acid). 1. Boc-Leu-Trp-Val-OMe (LWV), C28H42N4O6 2. Ac-Leu-Trp-Val-OMe (Space group P21) (LWV1), C25H36N4O5 3. Ac-Leu-Trp-Val-OMe (Space group P212121) (LWV2), C25H36N4O5 4. Boc-Leu-Phe-Val-OMe (LFV), C26H41N3O6 5. Ac-Leu-Phe-Val-OMe (LFV1), C23H35N3O5 6. Boc-Ala-Aib-Leu-Trp-Val-OMe (AULWV), C35H54N6O8 7. Boc-Trp-Trp-OMe (WW), C28H32N4O5 8. Boc-Trp-Aib-Gly-Trp-OMe. (WUGW), C34H42N6O7 9. Boc-Leu-Trp-Val-Ala-Aib-Leu-Trp-Val-OMe (H8AU), C57H84N10O11 10. Boc-(S)-β3-HAla-NHMe (BANH), C10H20N2O3 11. Boc-(R)-β3-HVal-NHMe (BVNH), C12H24N2O3 12. Boc-(S)-β3-HPhe-NHMe (BFNH), C16H24N2O3 13. Boc-(R)-β3-HVal-(R)-β3-HVal-OMe (BVBV), C18H34N2O5 14. Boc-(R)-β3-HVal-(S)-β3-HVal-OMe (LVDV), C18H34N2O5 15. Boc-(S)-β3-HPro-OH (BPOH), C11H19N1O4 16. Boc-Leu-Phe-Val-Aib-(S)-β3-HPhe-Leu-Phe-Val-OMe (UBF8), C60H88N8O11 17. Piv-Pro-Gly-NHMe (PA1), C13H23N3O3 18. Piv-Pro-βGly-NHMe (PB1), C14H25N3O3 19. Piv-Pro-βGly-OMe (PBO), C14H24N2O4 20. Piv-Pro-δAva-OMe (PDAVA), C16H28N2O4 21. Boc-Pro-γAbu-OH (BGABU), C14H24N2O5 22. Boc-Aib-γAbu-OH (UG), C13H24N2O5 23. Boc-Aib-γAbu-Aib-OMe (UGU), C18H33N3O6 The thesis is divided into seven chapters. Chapter 1 gives a general introduction to the stereochemistry of polypeptide chains and the secondary structure classification: helices, β-sheets and β-turns followed by an overview of different types of weakly polar interactions involving the side chains of aromatic amino acid residues. This section also provides a brief overview of the conformational analysis of β-, γ- and higher ω-amino acid residues in oligomeric β-peptides and in α,ω-hybrid peptides. A brief discussion on X-ray diffraction and solution to the phase problem is also presented. Chapter 2 describes the crystal structures of the peptides, Boc-Leu-Trp-Val-OMe (LWV), the two polymorphs of Ac-Leu-Trp-Val-OMe (LWV1 and LWV2), Boc-Leu-Phe-Val-OMe (LFV), Ac-Leu-Phe-Val-OMe (LFV1) and Boc-Ala-Aib-Leu-Trp-Val-OMe (AULWV), in order to explore the nature of interactions between aromatic rings, specifically the indole side chain of Trp residues [1]. Peptide LWV adopts a type I β-turn conformation, stabilized by an intramolecular 4→1 hydrogen bond. Molecules of LWV pack into helical columns stabilized by two intermolecular hydrogen bonds, Leu(1)NH…O=CTrp(2) and Indole NH…O=CLeu(1). The superhelical columns further pack into the tetragonal space group P43 by means of a continuous network of indole - indole interactions. The peptide Ac-Leu-Trp-Val-OMe crystallized in two polymorphic forms: P21 (LWV1) and P212121 (LWV2). In both forms, the peptide backbone is extended and the crystal packing shows anti-parallel β-sheet arrangement. Similarly, extended strand conformation and anti-parallel β-sheet formation are also observed in the Phe containing analogs, LFV and LFV1. The pentapeptide AULWV adopts a short stretch of 310-helix. Analysis of aromatic - aromatic and aromatic - amide interactions in the structures of peptides LWV, LWV1 and LWV2 are reported along with the examples of 12 Trp containing peptides from the Cambridge Structural Database. The results suggest that there is no dramatic preference for the orientation of two proximal indole rings. In Trp containing peptides specific orientations of the indole ring, with respect to the preceding and succeeding peptide units, appear to be preferred in β-turns and extended structures. Crystal parameters LWV: C28H42N4O6; P43; a = 14.698(1) Å, b = 14.698(1) Å, c = 13.975(2) Å; Z = 4; R = 0.0737, wR2 = 0.1641. LWV1: C25H36N4O5; P21; a =10.966(3) Å, b = 9.509(2) Å; c = 14.130(3) Å, β = 104.94(1)°; Z = 2; R = 0.0650, wR2 = 0.1821. LWV2: C25H36N4O5; P212121; a = 9.533(6) Å, b = 14.148(9) Å, c = 19.53(1) Å, Z = 4; R = 0.0480, wR2 = 0.1365. LFV: C26H41N3O6; C2; a = 31.318(8) Å, b = 10.022(3) Å, c = 9.657(3) Å, β = 107.41(1)°; Z = 4; R = 0.0536, wR2 = 0.1328. LFV1: C23H35N3O5; P212121; a = 9.514(8) Å, b = 13.56(1) Å, c = 20.04(2) Å, Z = 4; R = 0.0897, wR2 = 0.1960. AULWV: C35H54N6O8.2H2O; P21; a = 9.743(3) Å, b = 22.807(7) Å, c = 10.106(3) Å, β = 105.73(2)°; Z = 2; R = 0.0850; wR2 = 0.2061. Chapter 3 describes the crystal structures of three peptides containing Trp residues at both N- and C-termini of the peptide backbone: Boc-Trp-Trp-OMe (WW), Boc-Trp-Aib-Gly-Trp-OMe (WUGW) and Boc-Leu-Trp-Val-Ala-Aib-Leu-Trp-Val-OMe (H8AU). Peptide WW adopts an extended conformation and the molecules pack into an arrangement of parallel β-sheet in crystals, stabilized by three intermolecular N-H…O hydrogen bonds. The potential hydrogen bonding group NE1H of Trp(1), which does not take part in hydrogen bonding interaction with an oxygen acceptor participate in an intermolecular N-H…π interaction. Peptide WUGW adopts a folded structure, stabilized by a consecutive type II-I’ β-turn conformation. The crystal of WUGW contains a stoichiometric amount of chloroform in two distinct sites each with an occupancy factor of 0.5 and the structure provides examples of N-H…π, C-H…π, π…π, N-H…Cl, C-H…Cl and C-H…O interactions [2]. The molecular conformation of H8AU reveals a 310-helix. The crystal structure of H8AU reveals an interesting packing motif in which helical columns are stabilized by side chain - backbone hydrogen bond involving the indole NH of Trp(2) as donor and C=O group of Leu(6) as acceptor of a neighboring molecule, which closely resembles the hydrogen bonding pattern obtained in the tripeptide LWV [1]. Helical columns also associate laterally and strong interactions are observed between the Trp(2) and Trp(7) residues on neighboring molecules [3]. The edge-to-face aromatic interactions between the indoles suggest a potential C-H…π interaction involving the CE3H of Trp (2) Crystal parameters WW: C28H32N4O5; P212121; a = 5.146(1) Å, b = 14.039(2) Å, c = 35.960(5) Å; Z = 4; R = 0.0503, wR2 = 0.1243. WUGW: C34H42N6O7.CHCl3; P21; a = 12.951(5) Å, b = 11.368(4) Å, c = 14.800(5) Å, β = 101.41(2)°; Z = 2; R = 0.1095, wR2 = 0.2706. H8AU: C57H84N10O11; P1; a = 10.494(7) Å, b = 11.989(7) Å, c = 13.834(9) Å, α = 70.10(1)°, β = 82.74(1)°, γ = 78.96(1)°; Z = 1; R = 0.0855, wR2 = 0.1965. Chapter 4 describes the crystal structures of four protected β-amino acid residues, Boc-(S)-β3-HAla-NHMe (BANH); Boc-(R)-β3-HVal-NHMe (BVNH); Boc-(S)-β3-HPhe-NHMe (BFNH); Boc-(S)-β3-HPro-OH (BPOH) and two β-dipeptides, Boc-(R)-β3-HVal-(R)-β3-HVal-OMe (BVBV); Boc-(R)-β3-HVal-(S)-β3-HVal-OMe (LVDV). Gauche conformations about the Cβ-Cα bonds (θ ~ ± 60°) are observed for the β3-HPhe residue in BFNH and all four β3-HVal residues in the dipeptides BVBV and LVDV. Trans conformations (θ ~ 180°) are observed for β3-HAla residues in both independent molecules in BANH and for the β3-HVal and β3-HPro residues in BVNH and BPOH, respectively. In all these cases except for BPOH, molecules associate in the crystals via intermolecular backbone hydrogen bonds leading to the formation of sheets. The polar strands formed by β3-residues aggregate in both parallel (BANH, BFNH, LVDV) and anti-parallel (BVNH, BVBV) fashion. Sheet formation accommodates both the trans and gauche conformations about the Cβ - Cα bonds [4]. Crystal parameters BANH: C10H20N2O3; P1; a = 5.104(2) Å, b = 9.469(3) Å, c = 13.780(4) Å, α = 80.14(1)°, β = 86.04(1)°, γ = 89.93(1)°; Z =2; R = 0.0489, wR2 = 0.1347. BVNH: C12H24N2O3; P212121; a = 8.730(2) Å, b = 9.741(3) Å, c = 17.509(5) Å; Z = 4; R = 0.0479, wR2 = 0.1301. BFNH: C16H24N2O3; C2; a = 20.54(1) Å, b = 5.165(3) Å, c = 16.87(1) Å, β = 109.82(1)°; Z = 4; R = 0.0909, wR2 = 0.1912. BVBV: C18H34N2O5; P212121; a = 9.385(2) Å, b = 11.899(2) Å, c = 19.199(4) Å; Z = 4; R = 0.0583, wR2 = 0.1589. LVDV: C18H34N2O5; P212121; a = 5.170(4) Å, b = 10.860(8) Å, c = 37.30(3) Å; Z = 4; R = 0.0787, wR2 = 0.1588. BPOH: C11H19N1O4; P1; a = 5.989(2) Å, b = 6.651(2) Å, c = 8.661(3) Å, α = 70.75(1)°, β = 77.42(1)°, γ = 86.98(1)°; Z = 1; R = 0.0562, wR2 = 0.1605. Chapter 5 describes a new class of polypeptide helices in hybrid sequences containing α-, β- and γ-residues. The molecular conformation in crystals determined for the octapeptide Boc-Leu-Phe-Val-Aib-(S)-β3-HPhe-Leu-Phe-Val-OMe (UBF8) reveals an expanded helical turn in the hybrid sequence (ααβ)n. A repetitive helical structure composed of C14 hydrogen bonded units is observed. Using experimentally determined backbone torsion angles for the hydrogen bonded units formed by hybrid sequences, the energetically favorable hybrid helices have been generated. Conformational parameters are provided for C11, C12, C13, C14 and C15 helices in hybrid sequences [5]. Crystal parameters UBF8: C60H88N8O11; P212121; a = 12.365(1) Å, b = 18.940(2) Å, c = 27.123(3) Å; Z = 4; R = 0.0625, wR2 = 0.1274. Chapter 6 describes the crystal structures of five model peptides Piv-Pro-Gly-NHMe (PA1), Piv-Pro-βGly-NHMe (PB1), Piv-Pro-βGly-OMe (PBO), Piv-Pro-δAva-OMe (PDAVA) and Boc-Pro-γAbu-OH (BGABU). A comparison of the structures of peptides PA1 and PB1 illustrates the dramatic consequences upon backbone homologation in short sequences. The molecule PA1 adopts a type II β-turn conformation in the crystal state, while in PB1, the molecule adopts an open conformation with the β-residue being fully extended. The peptide PBO, which differs from PB1 by replacement of the C-terminal NH group by an O-atom, adopts an almost identical molecular conformation and packing arrangement in the crystal state. In peptide PDAVA, the observed conformation resembles that determined for PB1 and PBO, with the δAva residue being fully extended. In peptide BGABU, the molecule undergoes a chain reversal, revealing a β-turn mimetic structure stabilized by a C-H…O hydrogen bond [6]. Crystal parameters PA1: C13H23N3O3; P1; a = 5.843(1) Å, b = 7.966(2) Å, c = 9.173(2) Å, α = 114.83(1)°, β = 97.04(1)°, γ = 99.45(1)°; Z = 1; R = 0.0365, wR2 = 0.0979. PB1: C14H25N3O3.H2O; P212121; a = 6.297(3) Å, b = 11.589(5) Å, c = 22.503(9) Å; Z = 4; R = 0.0439, wR2 = 0.1211. PBO: C14H24N2O4.H2O; P212121; a = 6.157(2) Å, b = 11.547(4) Å, c = 23.408(8) Å; Z = 4; R = 0.050, wR2 = 0.1379. PDAVA: C16H28N2O4.H2O; P21212; a = 11.33(1) Å, b = 25.56(2) Å, c = 6.243(6) Å; Z = 4; R = 0.0919, wR2 = 0.2344. BGABU: C14H24N2O5; P61; a = 9.759(2) Å, b = 9.759(2) Å, c = 29.16(1) Å; Z = 6; R = 0.0773, wR2 = 0.1243. Chapter 7 describes the crystal structures of a dipeptide, Boc-Aib-γAbu-OH (UG) and a tripeptide, Boc-Aib-γAbu-Aib-OMe (UGU) containing a single γAbu residue in each sequence. The structure of UG forms a reverse turn stabilized by a 10-membered intramolecular C-H…O hydrogen bonded ring. The peptide UGU crystallized in the triclinic space group P⎯1 with two molecules in the asymmetric unit resulting in a parallel assembly of sheets in crystals. Notably, the insertion of a single Aib residue at the C-terminus drastically changes the overall conformation of the structures. Crystal parameters UG: C13H24N2O5; P21/c; a = 16.749(3) Å, b = 5.825(1) Å, c = 16.975(3) Å; β = 111.82(1); Z = 4; R = 0.0507; wR2 = 0.1294. UGU: C18H33N3O6; P⎯1; a = 9.576(6) Å, b = 13.98(1) Å, c = 17.83(1); α = 85.31 (1); β = 77.46 (1); γ = 71.39 (1); Z = 4; R = 0.0648; wR2 = 0.1837.

<p>The cellular response to a biomaterial, such as a dental implant, is mainly governed by the surface properties, and can thus be altered by the introduction of a surface coating. In this thesis the buildup of a biomacromolecule-based coating formed by layerby-layer (LbL) deposition of the charged polypeptides poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA) has been studied. In an attempt to make these coatings bioactive and useful for bone-anchored implants, an amelogenin protein mixture (EMD), has been immobilized in these thin polyelectrolyte multilayer (PEM) films. Multilayers were also built by LbL deposition of the natural biomacromolecules collagen (Col) and hyaluronic acid (HA). Multilayer films of these two extra-cellular biomacromolecules should be of interest for use as a scaffold for tissue engineering.</p><p>The buildup of the multilayer films has been followed in situ, using ellipsometry, quartz crystal microbalance with dissipation (QCM-D), and dual polarization interferometry (DPI). The studied PLL/PGA multilayers were found to be highly hydrated, and to exhibit a two-regime buildup behavior, with an initial “slow-growing” regime, and a second “fast-growing” regime with a linear growth in film thickness and more than linear growth in mass. A net diffusion of polypeptides into the film during the buildup led to an increase in density of the films for each layer adsorbed. A change in density was also observed in the Col/HA film, where HA penetrated and diffused into the porous fibrous Col network.</p><p>The formed PLL/PGA films were further found to be rather stable during drying, and post-buildup changes in temperature and pH, not losing any mass as long as the temperature was not raised too rapidly. The film thickness responded to changes in the ambient media and collapsed reversibly when dried. A swelling/de-swelling behavior of the film was also observed for changes in the temperature and pH.</p><p>The EMD protein adsorbed to silica surfaces as nanospheres, and could by itself form multilayers. The adsorption of EMD onto PLL/PGA multilayer films increased at lower pH (5.0), and EMD could be immobilized in several layers by alternate deposition of EMD and PGA.</p>

1. Viruses are obligate parasites that hijack the host cell machinery to synthesize their own gene products and for their propagation. In order to establish a successful viral infection, viruses have evolved different strategies to evade host check points. Further more, their success also relies in employing varied strategies to express maximum number of functional proteins from their small constrained genome. Polyprotein processing is a widely used strategy of expression by many plant viruses. With limited information available on this aspect for sobemoviruses, the present study was undertaken. 2. The present thesis deals with the mechanism of Sesbania mosaic virus (SeMV) polyprotein processing and functional characterization of the polyprotein domains. SeMV infects Sesbania grandiflora that belongs to the Fabaceae family. It is a positive sense ssRNA virus with a genome length of 4149 nucleotides. The genome encodes four potential overlapping open reading frames (ORFs). ORF1 codes for an 18 kDa protein that is proposed to be involved in the movement of the virus. ORF 3 codes for the coat protein (CP) that encapsidates the viral genomic RNA to form the viral particles. The central ORF codes for polyprotein that has a serine protease domain at its Nterminus that cleaves the polyprotein at specific E-T/S sites to release the functional domains. So far only in SeMV, the E. coli expressed polyprotein, Protease-VPg-RdRp was shown to undergo processing at E325-T326, E402-T403 and E498-S499 releasing protease, VPg, P10 and RdRp domains respectively. 3. Based on the arrangement of the central ORF, the genome organization of SeMV was earlier shown to be like that of SCPMV type. However, recent sequencing data from the laboratory showed that the organization of SeMV gRNA was like that of CfMV type. This would imply that in SeMV the central two ORFs will be translated to give two polyproteins, 2a (Protease-VPg-C-terminal domain) and 2ab (Protease-VPg-RdRp) the C-terminus of 2a and N-terminus of RdRp being different from what was reported previously. Therefore, in the light of the new genome organization for SeMV, the mechanism of processing of polyprotein 2a and 2ab needs to be revisited. 4. SeMV protease domain was shown to require natively unfolded VPg at its Cterminus for its activity. Aromatic stacking interactions between protease and VPg (via W43 residue) were shown to confer the activity to the protease. However, the residues in the protease domain involved in these interactions have not been identified. 5. The objectives of the present studies are • To elucidate the mechanism of processing of polyproteins 2a and 2ab in E. coli and in planta. • To identify residues in the protease domain involved in mediating aromatic stacking interactions with VPg. • To functionally characterize the C-terminal domain of polyprotein 2a. 6. Polyprotein 2a when expressed in E. coli, from the new cDNA clone, got cleaved at the earlier identified sites E325-T326, E402-T403 and E498-S499 to release protease, VPg, P10 and P8 respectively. The specificities of the cleavage sites were established by mutational analysis. 7. Additionally, a novel cleavage was identified within the protease domain at position E132-S133. The polyprotein 2a that was mutated for this site (ΔN70 2a-E132A) showed no release of P8 protein though the polyprotein was intact for E498-S499 site. Unlike other cleavage site mutants, ΔN70 2a-E132A mutant also revealed large accumulation of intact polyprotein, again implying that the mutation not only abolished the proteolytic cleavage at that site but hampered the processing at other sites. The results confirmed that the cleavage at N-terminus of the protease/polyprotein is crucial for an efficient processing in particular for the cleavage between P10-P8. 8. Interestingly, though the sites in polyprotein 2ab are exactly the same as identified in polyprotein 2a, the former got cleaved between Protease-VPg but not between VPg-RdRp. This cleavage site appeared to be rather masked in polyprotein 2ab. Also, the cleavage at E132-S133 site appeared to be rather slow. These results indicate to a differential cleavage pattern, governed probably by the conformation of 2ab. In other words, the local context of the cleavage site and just not the sequence per se could be playing a key role in 2ab polyprotein processing. 9. Products, corresponding to all cleavages identified in E. coli (E132-S133, E325-T326, E402-T403 and E498-S499) were also detected in infected Sesbania leaves. Products corresponding to the sizes of ΔN132 Protease and ΔN132 Protease-VPg were detected suggesting that the removal of the membrane anchoring domain from the protease does occur in planta. Also, detection of band corresponding P8, confirmed that the cleavage between P10-P8 indeed occured in planta too. 10. The trans cleavage experiments suggested that not all of the four cleavages in polyprotein 2a occur in trans (intermolecular). Cleavages at E132-S133 and E498-S499 do not occur in trans impling that cleavages at these sites could only occur in cis (intramolecular) by auto-proteolysis of the polyprotein. 11. The Thr at P1’ did not make a site trans cleavable. Interestingly, SeMV protease was found to cleave even an E-S site in trans but only when present at positions 324-325 and 402-403, suggesting that trans cleavage in SeMV is governed by the context rather than the Thr at P1’position of the cleavage site. The E498-S499 site was found to be highly stringent not only for the mode of its cleavage (cis cleavage) but also for its sequence (E-S only). A Thr substitution for Ser at this site, made it non cleavable in cis. 12. The results reveal that the polyprotein processing in SeMV is regulated by a number of strategies, viz. a) availability of the cleavage site depending on the conformation of the flanking domains (E132-S133 and E402-T403 cleavages in 2ab). b) Mode of recognition (cis or trans). c) Context/position of the cleavage site. 13. Based on the sequences of all four cleavage sites identified, a consensus has been drawn for SeMV serine protease cleavage site, i.e., N/Q-E-T/S-X (where X is an aliphatic residue) at P2-P1-P1’-P2’ position respectively. 14. With a view to understand the structural reasons for such high specificity, the residues in the S1 and S2 binding pocket, that recognize the substrate P1 and P2 residues respectively, were identified based on the structural comparison of SeMV protease with other Glu/Gln specific proteases. Mutational analysis of these residues clearly demonstrated that H298, T279 and N308 of the S1-binding pocket that would bind the substrate glutamate are crucial for the protease activity. R309 that forms the S2 binding pocket is also crucial for protease activity. 15. Also, the P2 (Asn/Gln) residue recognized by R309 plays an important role in determining the substrate specificity. A positively charged residue Lys was not tolerated at this position. SeMV protease was also shown to efficiently cleave the peptide bond C-terminus to an uncharged Gln in vivo suggesting that it is a Glu/Gln specific protease. 16. An interesting feature of the SeMV protease domain is the presence of a disulphide bond that holds the S1-binding pocket. However, unlike for the cellular counterparts like trypsin, the disulphide was found to be not essential for either the SeMV protease activity or structural stability. 17. Protease and VPg domains were proposed to be involved in aromatic interactions that conferred activity to the protease. The structure of protease revealed a stack of aromatic residues (W271, F269. Y315 and Y319) exposed to the solvent. Mutational analysis was performed to identify their role in mediating the interactions and hence the activity of protease. H275, though not a part of exposed aromatic stack in the protease, was chosen for mutational analysis as it lies close to the W271 in sequence and is conserved in the protease domain across all the known sobemoviruses. The in vivo and trans cleavage assays suggested that residues W271 and H275 but not Y315 or Y319 are crucial for protease activity. 18. The Far-UV CD spectrum of protease-VPg is characterized by a positive peak at 230 nm, signifying the aromatic interactions. Far-UV CD spectral analysis of the aromatic mutants showed that W271 and H275, but not F269 and Y319 are the major contributors of the 230 nm positive peak, confirming the direct involvement of these residues in the stacking interactions with W43 of VPg. Thermal stability studies, fluorescence spectroscopy and 1D-NMR spectroscopy studies also confirmed the histidine aromatic interactions between W271, H275 of protease with W43 of VPg. 19. The loss in aromatic interactions in the mutants caused Protease-VPg to aggregate, suggesting that the aromatic interactions between protease and VPg not only conferred activity to the protease but also the active oligomeric status. 20. In silico analysis of the C-terminal domain showed that it has no significant similarities with any known functional proteins. The region corresponding to P8 was amplified and cloned in pRSET C vector, over-expressed and purified. 21. The purified His-tagged P8 showed mass abnormality on the SDS-PAGE. However, the mass spectrometric analysis of the purified protein showed that it had a molecular mass of 9.766 kDa as is expected for a His-tagged P8. P8 is highly basic, which could possibly explain its anomalous behaviour on the SDS-PAGE. The purified recombinant P8 protein was found to be natively unfolded. In vitro binding studies revealed that P8 had nucleic acid binding property. The protein was also found to be phosphorylated both in vitro and in vivo conditions. 22. Interestingly, P18, (a precursor of P8) but not P8, was found to possess an inherent ATP hydrolyzing property. Optimum conditions for the ATPase assay were found to be Tris HCl pH 8.0, 37 ºC, 5 mM MgCl2. The activity was linear upto 20 mins. P18 could utilize all NTPs and dNTPs. Studies revealed that ATPase activity resided in the P10 domain of P18, though P8 region could enhance the activity. Conclusively, the results demonstrate that the C-terminal domains of polyprotein 2a have ATPase and nucleic acid binding activity and could therefore have possible roles in movement and replication.

This work reports a novel biocompatible and biodegradable arborescent amphiphilic polypept(o)ides-based polymer poly(γ-benzyl L-glutamate)-co-poly(γ-tert-butyl L-glutamate)-g-polysarcosine (P(BG-co-Glu(OtBu))-g-PSar) as a smart dual-responsive targeting drug vehicle. The synthesis pathway in this work highlighted the grafting reaction improvement of the polypeptides core and using polysarcosine (PSar) corona as a coating agent. The responsiveness of the polymer is caused by the pH sensitivity of the polypeptides and the reducible linker introduced between the core and corona. While adding the tripeptides arginine, glycine, and aspartate (RGD) as a ligand on the unimolecular micelles’ surface increases the targeting ability of the polymer. During the building of the arborescent, the coupling sites were controlled by using γ-tert-butyl L-glutamate (Glu(OtBu)-NCA) as a second monomer besides γ-benzyl L-glutamate (BG-NCA) since the deprotection conditions are different for Bz and tBu groups. Knowing the coupling sites provides accuracy in calculating the molecular weight (MW) of graft polymers since it facilitates the determination of the grafting yield (Gy). The arborescent unimolecular micelles were formulated by coating the hydrophobic core with PSar hydrophilic corona. The distribution of the coupling sites on the substrates in the last generation yielded end-grafted and randomly-grafted unimolecular micelles. A comparison between those micelles by DLS, TEM, and AFM revealed that the end-grafted micelles showed more uniformity in terms of morphology and size distribution. Also, the surface modification achieved via RGD addition increased the shape uniformity and contributed to avoiding the particles’ aggregation. The sizes and shapes of end-grafted unimolecular micelles match the drug delivery systems (DDSs) requirements. Doxorubicin (DOX) was encapsulated physically into the unimolecular micelles to study the drug loading capacity (DLC) and drug loading efficiency (DLE). The maximum DLC and DLE were 14% and 28% w/w, respectively. The drug release profiles were investigated in healthy- and cancer-mimicking media. The results showed that in cancer-mimicking microenvironment (low pH and high glutathione (GSH) content), the drug diffused out the micelles faster. In addition, a slower drug release was noticed for RGD decorated unimolecular micelles. Finally, the biocompatibility, cytotoxicity, and cellular uptake of the unimolecular micelles were studied. The obtained results were promising as the arborescent unimolecular micelles showed excellent biocompatibility; meanwhile, the DOX-loaded unimolecular micelles have good cytotoxicity compared to free DOX. RGD targeting ligand contributes to increasing the cellular uptake and supports the sustained release.

Le repliement des protéines est principalement gouverné par les interactions spécifiques des structures secondaires. 1, 2 Toutefois, il existe expérimentalement peu d’informations sur les propriétés mécaniques fondamentales des hélices α et des feuillets β isolées. Les recherches antérieures sur l'étude du déploiement des hélices sont peu concluantes 3-5 et à notre connaissance l'étude des propriétés mécaniques d'un feuillet β isolé, intramoléculaire est sans précédent. Les copolymères PEG114-b-poly(L-lysine)134-(2-pyridyl disulfure),PEG114-b-poly(L-lysine)-b-PEG114 et poly(L-acide glutamique)85-b-(2-pyridyldisulfure) été synthétisés et utilisés comme systèmes modèles pour tester les propriétés mécaniques des motifs secondaires de type hélice α et feuillet β. Les résultats obtenus se sont révélés être en bon accord avec les résultats théoriques obtenus en utilisant un modèle statistique basé sur AGAGIR 6. La différence de force de déroulement comparant les hélices de poly(L-Lysine) ≈ 30 pN et de poly(L-acide glutamique) ≈ 20 pN des copolymères diblocs a été attribuée à l'hydrophobicité différente des chaînes latérales. La plus grande hydrophobie dumotif lysine conduit à de plus grandes interactions entre les chaînes latérales qui empêchent les fluctuations aléatoires au sein de l’hélice, et conduisent à une stabilité supérieure de l'hélice α. Lorsque les expériences ont été conduites dans des conditions favorisant la solubilité des chaînes latérales de lysine, les interactions ont diminué à une force de ≈ 20 pN, similaire à la force des interactions observées pour le poly(L-acide glutamique). Nous supposons qu'un minimum de ≈ 20 pN est nécessaire pour rompre la liaison hydrogène en maintenant l'hélice α, car cette force a été obtenue dans des conditions où les interactions de la chaîne latérale étaient minimisées. La présence de plateaux de force constants et d'inflexions correspondantes démontre une force de dépliement indépendante de la longueur, qui supporte un mécanisme de déroulement tour-par-tour pour l'hélice. De plus, la plus grande hydrophobie des chaînes latérales a été suggérée non seulement pour stabiliser la structure en hélice, mais également pour inhiber la formation d'une structure de type β-turn métastable intermédiaire lorsque les forces entropiques dominent. Des études préliminaires ont été effectuées sur le système de PEG114-bpoly(L-Lysine)134-(2-pyridyl disulfure) après induction d’une transition - β par un traitement thermique dans des conditions basiques. Une inflexion à une force≈ 70 pN a été obtenue, ce qui suggère la formation d'une interaction de type feuillet β. Une stratégie bottom-up a ainsi été proposée avec succès, démontrant le potentiel d'utilisation de tels systèmes artificiels pour simplifier et modéliser des systèmes biologiques réels. La compréhension de ces modèles isolés plus simples aidera sans doute la compréhension de systèmes plus complexes. / Proteins fold by the initial, preferential folding of secondarystructures 1, 2, however surprisingly little is known about the basic mechanicalproperties of isolated α-helices and β-sheets from an experimental standpoint.Previous investigations into studying the generic unfolding behaviour of α-heliceshave proved inconclusive 3-5, and to our knowledge the study of an isolated,intramolecular β-sheet is unprecedented.Bioinspired PEG114-b-poly(L-glutamic acid)85-(2-pyridyl disulphide),PEG114-b-poly(L-lysine)134-(2-pyridyl disulphide) and PEG114-b-poly(Llysine)134–b-PEG114 were designed, synthesized and utilized as model systems toprobe the mechanical properties of α-helix and β-sheet secondary motifs. Theobtained results were shown to be in good agreement with theoretical resultsobtained by utilizing a AGAGIR-based statistical mechanical model 6. Thedifference in unravelling force comparing the helices of poly(L-Lysine) ≈30 pNand poly(L-glutamic acid) ≈20 pN diblock copolymers was attributed to thediffering hydrophobicity of the side chains. The greater hydrophobicity of thelysine allowed greater interactions between the side chains and sterically hinderedrandom helix-coil fluctuations, which lead to a superior α-helix stability. Whenexperiments were conducted in conditions promoting the solubility of the lysineside chains, the interactions decreased to a force of ≈20 pN, similar to the force ofinteractions observed for the poly(L-glutamic acid). We infer that a minimum of≈20 pN is needed to rupture the hydrogen bonding maintaining the α-helix as thisforce was obtained in conditions where the side chain interactions wereminimized.The presence of constant force plateaus and corresponding inflectionsdemonstrates a length independent unfolding force, which supports a turn-by-turnunfolding mechanism for the α-helix.In addition, the greater hydrophobicity of the side chains was suggestedto not only stabilize the α-helix structure, but also to inhibit the formation of anintermediate metastable β-hairpin-like structure when entropic forces dominate.Preliminary studies were also conducted on the PEG114-b-poly(LLysine)134-(2-pyridyl disulphide) system after a α-β transition had been inducedby heat in basic conditions, where an inflection at a much higher force of ≈ 70 pNwas obtained suggesting the formation of a β-sheet interaction.A bottom-up, investigative strategy has thus been successfully proposeddemonstrating the potential of utilizing such artificial systems to simplify andexemplify real biological systems. The comprehension of these simpler isolatedmodels will no doubt aid the understanding of more complex systems.

<p>This thesis examines the isolation and characterisation (biological and chemical) of polypeptides from plants. A fractionation protocol was developed and applied on 100 plant materials with the aim of isolating highly purified polypeptide fractions from small amounts of plant materials. The polypeptide fractions were analysed and evaluated for peptide content and biological activities. A multitarget functional bioassay was optimised as a method for detecting substances interacting with the inflammatory process of activated neutrophil granulocytes. In this assay, the neutrophil was challenged with an inflammatory mediator, <i>N</i>-formyl methionyl-leucyl-phenylalanine (fMLP), or with platelet activating factor (PAF), to induce exocytotic release of the enzyme elastase, which then was quantified by photometric determination of the product p-nitroanilide (pNA) formed from a chromogenic substrate for elastase. Of the tested extracts, 41% inhibited pNA formation more than 60%, and 3% stimulated formation.</p><p>Phoratoxin B and four new peptides, phoratoxins C-F, were isolated from <i>Phoradendron tomentosum</i>. In addition, the cardiac glycoside digitoxin was isolated from <i>Digitalis purpurea</i>. All these substances expressed cytotoxicity and a neutrophil challenging activity.</p><p>Phoratoxins C-F were similar to earlier described phoratoxins A and B, which belong to the group of thionins. All the peptides were evaluated for cytotoxicity in a human cell line panel. Phoratoxin C was the most potent towards the cell lines (mean IC₅₀: 160 nM), and was therefore investigated further on tumour cells from patients. Correlation analysis of the log IC₅₀ values indicated a mechanism of action different from clinically used archetypal cytotoxic drugs. Phoratoxin C also showed selective toxicity to the solid tumours when compared to the haematological cancer types. The phoratoxin C was 18 times more potent towards the solid tumour samples from breast cancer cells (87 nM) compared to the tested haematological malignancies.</p><p>The structure-activity relationship concerning cytotoxicity was evaluated for digitoxin and related cardiac glycosides. Digitoxin was shown to be potent, with the average IC₅₀ 37 nM being within the therapeutic concentration used for cardiac congestion (13-45 nM). Digitoxin expressed selective toxicity towards solid tumours from patients compared to haematological malignancies.</p>

This thesis examines the isolation and characterisation (biological and chemical) of polypeptides from plants. A fractionation protocol was developed and applied on 100 plant materials with the aim of isolating highly purified polypeptide fractions from small amounts of plant materials. The polypeptide fractions were analysed and evaluated for peptide content and biological activities. A multitarget functional bioassay was optimised as a method for detecting substances interacting with the inflammatory process of activated neutrophil granulocytes. In this assay, the neutrophil was challenged with an inflammatory mediator, N-formyl methionyl-leucyl-phenylalanine (fMLP), or with platelet activating factor (PAF), to induce exocytotic release of the enzyme elastase, which then was quantified by photometric determination of the product p-nitroanilide (pNA) formed from a chromogenic substrate for elastase. Of the tested extracts, 41% inhibited pNA formation more than 60%, and 3% stimulated formation. Phoratoxin B and four new peptides, phoratoxins C-F, were isolated from Phoradendron tomentosum. In addition, the cardiac glycoside digitoxin was isolated from Digitalis purpurea. All these substances expressed cytotoxicity and a neutrophil challenging activity. Phoratoxins C-F were similar to earlier described phoratoxins A and B, which belong to the group of thionins. All the peptides were evaluated for cytotoxicity in a human cell line panel. Phoratoxin C was the most potent towards the cell lines (mean IC50: 160 nM), and was therefore investigated further on tumour cells from patients. Correlation analysis of the log IC50 values indicated a mechanism of action different from clinically used archetypal cytotoxic drugs. Phoratoxin C also showed selective toxicity to the solid tumours when compared to the haematological cancer types. The phoratoxin C was 18 times more potent towards the solid tumour samples from breast cancer cells (87 nM) compared to the tested haematological malignancies. The structure-activity relationship concerning cytotoxicity was evaluated for digitoxin and related cardiac glycosides. Digitoxin was shown to be potent, with the average IC50 37 nM being within the therapeutic concentration used for cardiac congestion (13-45 nM). Digitoxin expressed selective toxicity towards solid tumours from patients compared to haematological malignancies.

Les vésicules géantes unilamellaires (GUV) permettent d'étudier efficacement les interactions entre les lipides et les peptides. Dans ce manuscrit, il a été montré que les interactions attractives lipides-peptides sont supprimées par l'attachement de polypeptides de type élastine (ELP) sur des peptides riches en arginine et peuvent être modulées par l'auto-assemblage en micelles ainsi que par le nombre de groupements arginine dans la séquence des peptides capables de pénétrer les cellules. De plus, une nouvelle méthode pour former des GUV à partir de systèmes complexes en seulement quelques minutes a été développée. Cette méthode est basée sur le gonflement d'un film de PVA sous une bicouche lipidique. Elle supprime la dégradation des molécules pendant la formation des GUV de lipides synthétiques, tels que des glycolipides et des phospholipides portant des groupements amides, où les méthodes traditionnelles ne réussissent pas à produire des vésicules non endommagées.