The work presented in this thesis describes the determination of structures of peptides and proteins at atomic resolution. Nuclear Magnetic Resonance (NMR) spectroscopy was used as the principal method of investigation. The thesis is divided into three parts. Part I of the thesis consists of chapters 1 to 4, and deals with structural studies of two novel conotoxins. Part II of the thesis consists of chapter 5 and deals with structural studies of Small MutS Related (Smr) domain from Helicobacter pylori MutS2. Part III of the thesis consists of Appendices A to D. Appendix A describes implementation of a novel pulse sequence for determination of disulfide connectivity using long-range 13 C–13 C scalar couplings across disulfide bonds. Appendices B, C and D contain supplementary infor- mation (acquisition parameters and chemical shifts) for the structural studies presented in parts I and II of the thesis. Part I: Structural studies of novel conotoxins from Conus monile Chapter 1 gives a brief overview of the conotoxins and their structural studies. The first half of the chapter describes biosynthesis, classification schemes, nomenclature, com- monly observed post-translational modifications and applications of conotoxins. The latter half of this chapter summarizes the challenges involved in the structural studies of conotoxins in light of the recent developments in integrated transcriptomic and venomic studies of conotoxins. The key homonuclear and heteronuclear NMR experiments that are employed for structural studies of conotoxins are summarized. Emphasis was laid on describing the spectral features and the structural information that can be gleaned from these experiments. Finally, the current mass spectrometric and NMR methods available for determination of disulfide connectivity are discussed Chapter 2 describes sample preparation and preliminary biophysical characteriza- tion of a conotoxin Mo3964 that contains a hitherto uncharacterized cysteine framework (C–CC–C–C–C). The sequence of Mo3964 was identified at the nucleic acid level as a cDNA clone. Analysis of the signal sequence revealed that the toxin belongs to the M-superfamily, while the cysteine framework bears more resemblance to O- and K- super- family of conotoxins. Structural studies were initiated to determine the disulfide connec- tivity, tertiary structure and biological activity. The gene corresponding to the mature toxin sequence was cloned in a bacterial expression vector pET21a(+) as a C-terminal tag to the cytochrome b5 fusion protein host system. The fusion protein was obtained by recombinant expression using the bacterial expression host E. coli BL21(DE3) and the mature toxin was obtained by either enzymatic or chemical cleavage of the fusion protein followed by size exclusion chromatography and reverse phase HPLC. Proton 1D NMR spectra of the purified peptide exhibited sharp lines and good spec- tral dispersion indicating that molecule was well folded. Formation of disulfide bonds in the mature toxin was ascertained by high resolution mass spectra of intact and chemically modified Mo3964. The peptide toxin exhibited remarkable stability to chemical denatu- ration and proteolytic digestion. Spectroscopic studies clearly showed that Mo3964 pos- sesses a very stable and well defined structure as long as its disulfide bonds are intact. Analytical size exclusion chromatography and Multi Angle Light Scattering (MALS) studies showed that Mo3964 exists in solution as monomer albeit with a non-globular structure. Electrophysiological studies showed that Mo3964 inhibits outward potassium currents in rat Dorsal Root Ganglion (DRG) neurons and increases the reversal potential of rat voltage gated sodium channel rNav 1.2 stably expressed on Chinese Hamster Ovary (CHO) cells at peptide concentrations as low as 10 nM. Chapter 3 describes the determination of disulfide connectivity and tertiary stricture of Mo3964. Initial attempts to determine disulfide connectivity using direct fragmenta- tion of the intact peptide in the mass spectrometer failed due to the relatively large size of the molecule and its resistance to endoproteases. Partial reduction alkylation based methods failed as the first stage of partial reduction gave rise to a mixture of various single disulfide bond reduced species which could not be separated from each other. Subsequently, information about the disulfide connectivity was obtained using a method that does not necessitate separation of such a mixture of single disulfide bond reduced species. This method involves partial reduction, cyanylation of the reduced cysteines and alkali mediated cleavage of the peptide backbone on the N-terminus of cyanylated cysteines. Structural studies were carried out using homonuclear and heteronuclear NMR meth- ods. The hydrogen bond network and hence topology of the molecule was determined with high accuracy using the long-range HNCO-COSY experiment that correlates hydrogen- bond donor-acceptor pairs. This experiment utilizes the three bond heteronuclear scalar coupling, i.e., the h3JN C O′ coupling across the hydrogen bonds. All these restraints proved crucial to the assignment of the disulfide connectivity in Mo3964, given its novel cysteine framework. The structure of Mo3964 was calculated using a total of 549 NOE distance restraints, 84 dihedral angle restraints and 28 hydrogen bond distance restraints. The tertiary structure was constructed from the disulfide connectivity pattern 1–3, 2–5 and 4–6, that is hitherto undescribed for the M–superfamily conotoxins. The ensemble of structures showed a backbone Root Mean Square Deviation of 0.68 ± 0.18 Å, with 87% and 13% of the backbone dihedral (φ, ψ) angles lying in the most favored and additional allowed regions of the Ramachandran map. The remarkable stability and anomalous spectral properties exhibited by Mo3964 could be rationalized using the disulfide connectivity and the tertiary structure. The tertiary structural fold has not been described for any of the known Conus peptides. Further, a search for structures similar to that of Mo3964 using the web server DALI returned no hits indicating that the peptide scaffold of Mo3964 has no structural homologues. Hence, the conotoxin Mo3964 represents a new bioactive peptide fold that is stabilized by disulfide bonds and adds to the existing repertoire of scaffolds that can be used to design stable bioactive peptide molecules. The structure of Mo3964 was submitted to the Protein Data Bank (PDB ID: 2MW7)[1]. Chapter 4 describes the structural studies of a 17 residue, single disulfide containing conopeptide Mo1853. The samples for structural studies were obtained either by chemical synthesis or by recombinant expression methods. Structural studies using homonuclear solution NMR methods revealed that Mo1853 exists as two equally populated cis and trans X–Pro conformers which are in slow exchange regime, compared to the chemical shift timescale. Sequence specific assignments were obtained for both the conformers by analysis of homonuclear 2D 1 H,1H–DQF–COSY,1H,1 H–TOCSY, 1H,1 H–NOESY and 1H,1 H–ROESY spectra. Temperature dependence of chemical shifts was measured and coalescence was observed for two amide protons at 318 K. At this temperature, the rate of exchange and the free energy of activation were determined to be 59 Hz and ≈ 67.2 kJ mol−1 respectively. The evidence for this conformational equilibrium was also observed as exchange correlation peaks in the 2D- NOESY and ROESY spectra. Tertiary structures of both the cis and trans conformers were determined using distance restraints, backbone dihedral angle restraints, the disulfide bond restraint and the cis or trans conformation of the X–Pro peptide bond. Tertiary structures of both the conformers consist of a 29-membered macro-cyclic ring formed by 9 amino acid residues which are cyclized by side chain to side chain disulfide bond. The conformation of the X–Pro peptide bond which is located within this macro-cyclic ring causes the cis structure to be compact and the trans structure to be in an extended form. Analysis of the tertiary structures indicated that the trans conformer is stabilized by hydrogen bonds while the cis conformer is likely to be stabilized by hydrophobic interactions. This was further corroborated by the fact that at lower temperatures, the hydrophobic interactions became weaker reducing the population of the cis conformer with respect to that of the trans conformer. Preliminary electrophysiological studies carried out on rat DRG neurons indicate that Mo1853 transiently reduces late outward potassium currents. Part II: Structural studies of Small MutS Related (Smr) domain from Helicobacter pylori MutS2 Chapter 5 presents the solution NMR studies of the Smr domain from MutS2 of H. pylori , henceforth called as HpSmr. In H. pylori , MutS2 is involved in suppression of homologous recombination and its Smr domain was shown to be necessary for this activity. As of date, in spite of the availability of structural information for the Smr domain, unambiguous identification of the residues involved in metal binding, DNA binding and catalysis remains elusive. Structural studies were carried out on two different constructs of HpSmr viz., HpSmr– (His)6 and GSHM–HpSmr, with and without the hexahistidine tag respectively. Se- quence specific assignments of HpSmr–(His)6 were obtained at two different sample pH conditions viz., pH 8.0 and pH 5.35 using the standard suite of triple resonance NMR experiments. Since, valines and leucines constitute about 25% of the total number of amino acid residues in HpSmr–(His)6 , stereospecific assignments were obtained for di- astereotopic methyl groups of these residues by preparing a fractionally 13C labeled sample of HpSmr–(His)6 . Solution structure of HpSmr–(His)6 at pH 8.0 was determined using 766 NOE restraints, 170 backbone dihedral angle restraints and 70 hydrogen bond distance restraints. The tertiary structure exhibits the canonical α/β sandwich fold ex- hibited by all the other known structures of Smr domains. Further, NMR studies and analytical gel filtration studies indicated the presence of pH dependent conformational exchange in HpSmr that involves strand to coil transition in the C-terminal β-strand. In order ascertain that the conformational equilibrium is not at an artifact caused by the C-terminal hexa-histidine-tag, HpSmr protein construct GSHM–HpSmr, which does not have the hexa-histidine-tag, was prepared. Conformational exchange was observed in this construct as well. The preliminary NMR evidence suggests that the conformational exchange is caused by pH dependent cis–trans isomerization of a semi-conserved Proline residue Pro66 . We have hypothesized that the pH dependent modulation of the activity of Smr domain of MutS2 can be advantageous to H. pylori . Such a regulation could help the bacteria to achieve optimal rate of homologous recombination in response to changes in pH, which is necessary for maintaining homeostasis and tiding over stress conditions. Part III: Appendix Appendix A describes an NMR pulse program LRCC_CH2 that was designed with the aim of determining disulfide connectivity using long-range 13C–13 C (C β –C β ′ ) couplings across the disulfide bond. This experiment is a modification of an earlier experiment pub- lished by Bax and co-workers designed to measure the side-chain χ3 dihedral angle in me- thionines. The experiment described here is optimized for the detection of 3 bond scalar coupled methylene carbons. The details of modifications introduced in LRCC_CH2, its product operator analysis, a representative spectrum acquired on [U-13C,15 N]–Mo3964, short-comings and future directions are described. The C programming code that was used to implement the pulse program is also included in the appendix. Appendices B, C and D contain the supplementary information (acquisition pa- rameters for the NMR experiments and chemical shifts) for the structural studies carried out on Mo3964, Mo1853 and HpSmr.

Design of complex protein folds requires complete understanding of the stereochemical principles that govern polypeptide chain folding. Extensive studies on design and synthesis of specific secondary structures like β-helices, β -sheets and hairpins have taught us that the unnatural amino acid aminoisobutyric acid (Aib) can be successfully employed for helix nucleation and tight turns of appropriate stereochemistry are facilitated by the use of DPro-Xxx sequences. Availability of such rigid secondary structure scaffolds therefore permits the design of synthetic peptides that can be used as models for investigation of tertiary interactions, primarily that of aromatic residues. Chapter 1 summarizes the present knowledge of peptide design using non-protein amino acids. The chapter also details the unique features of aromatic amino acids, especially tryptophan, and their employment as secondary structure stabilizing elements. Chapters 2-7 contain detailed descriptions of the work carried out on design, synthesis, and structural characterization of designed peptides containing aromatic amino acids. In Chapter 2, the use of aromatic pairs in strand segments of peptide hairpins has been discussed with the results clearly indicating that aromatic interactions at the non-hydrogen bonding position of peptide hairpins contribute to structure stability. In Chapter 3, accommodation of the Leu-Trp-Val segment in helical scaffolds the role of Trp residues in crystallization has been discussed. Chapter 4 outlines the influence of a large number of Trp residues on the preferred backbone conformation, with the studies clearly indicating a preference for helical scaffolds in small peptides. The role of Trp residues at turn regions of peptide hairpins has been discussed in Chapter 5, using examples from both synthetic peptides and from natural peptides containing Pro-Trp segments. The studies suggest that the Pro-Trp segments serve as helix nucleators and disrupt formation of peptide hairpins. The results of this study have been further extended to Conus monile peptides, discussed in Chapter 6. The studies also suggest the role of an aromatic-Pro segment on the cis-trans isomerization of the Xxx-Pro tertiary amide unit. Chapter 7 discusses the contribution of a Cys-His vs Tyr-His pair on strand segment stability in diproline nucleated peptide hairpins. Chapter 8 summarizes the key findings of the work. Chapter 9 lists the references cited in the thesis and the Appendix chapter provides details of experimental techniques used in the study.β

Elucidation of the primary structure of peptides and proteins de novo by mass spectrometry (MS) has become possible with the advent of tandem MS methods. The most widely used chemical method due to Edman (Edman & Begg, 1967) has shortcomings with regard to N- terminal blocked peptides, cyclic peptides and posttranslational modifications, for example phosphorylation (Metzger, 1994). However, mass spectrometric sequencing methods are increasingly becoming applicable for a variety of peptides and proteins, including N- and C- termini modified peptides and cyclic peptides (Jegorov et al., 2003; Sabareesh & Balaram, 2006; Sabareesh et al., 2007). Further, conventional and tandem mass spectrometry have proven useful in the detection of post-translational modifications (Hansson et al., 2004; Nair et al., 2006; Mandal et al., 2007). This thesis details mass spectrometric sequencing of acyclic and cyclic peptides, involving tandem MS methods carried out using both electrospray ionization (ESI) ion trap (Esquire 3000 plus, Bruker Daltonics) and matrix assisted laser desorption and ionization time-of-flight/time-of-flight (MALDI TOF/TOF) (Ultraflex TOF/TOF, Bruker Daltonics) instruments. The peptides are either chemically synthesized or isolated from diverse natural sources. Synthetically designed peptides possessing modified N- and C- termini and peptaibols from the soil fungus Trichoderma constitute the acyclic peptides. The cyclic peptides include backbone cyclized depsipeptides from the fungus Isaria and disulfide bonded peptides from the venom of marine cone snails. Chapter 1 gives an account of various concepts of mass spectrometry, tandem mass spectrometry and peptide fragmentation chemistry, providing necessary background information for the following chapters. Chapter 2 describes the fragmentation studies of [M + H]+ and [M + Na]+ adducts of six neutral peptides with blocked N- and C- termini investigated using an electrospray ion trap mass spectrometer. The N- terminus of these synthetically designed peptides is blocked with a tertiarybutyloxycarbonyl (Boc) group and the C- terminus is esterified. These peptides do not possess sidechains that are capable of complexation and hence the backbone amide units are the sole sites of protonation and metallation. The cleavage pattern of protonated adducts is strikingly different from that of sodium adducts. While the loss of the N- terminal blocking group happens quite readily in the case of MS/MS of [M + Na]+, the cleavage of C- terminal methoxy group seems to be a facile process in the case of MS/MS of [M + H]+. Fragmentation of the protonated adducts yields only bn ions, while yn and an type ions are predominantly formed from the fragmentation of sodium adducts. The an ions arising from the fragmentation of [M + Na]+ lack the N-terminal Boc group (termed as an*). MS/MS of [M + Na]+ species also yields bn ions of substantial lower intensities, that lack the N- terminal Boc group (bn*). Comparison of the fragmentation of [M + H]+ with [M + Na]+ of the peptides chosen in this study reveal that the combined use of both protonated and sodium adducts should prove useful in de novo sequencing of peptides that possess modified N- and C- termini, particularly naturally occurring neutral peptides, for example, peptaibols. Chapter 3 describes about the ESI-MS/MS investigation of an HPLC fraction from the soil fungus Trichoderma, which aided in identification of microheterogeneous trichotoxin peptaibols in that fraction. Dramatic differences were noted between the fragmentation spectra of [M + H]+ and [M + Na]+ species. While b-type ions were noted from the former, the latter yielded a-, b-and y- type ions (the same feature was noted in the cases presented in the previous chapter). Inspection of the isotope pattern of b-ions yielded from the dissociation of H+ species, clearly revealed the presence of three microheterogeneous trichotoxin sequences; two isobars (1718 Da), each possessing one Glu residue and another completely neutral peptide (1717 Da). The microheterogeneity is due to Gly ↔ Ala, Iva ↔ Aib and Gln ↔ Glu replacements and exchanges (Iva: DIva: R-Isovaline; Aib: α-aminoisobutyric acid). The MS/MS of [M + Na]+ adduct predominantly yielded product ions from the neutral peptaibol. Further, the fragmentation patterns of H+ and Na+ adducts of two N-acetyl peptide esters were found to be very similar to that of the neutral peptaibol component. The results presented in this chapter establish that under the electrospray ion trap conditions, the fragmentation patterns of the H+ and Na+ adducts of model peptides that possess modified N- (Boc and acetyl) and C- termini are indeed very similar to that of the neutral trichotoxin. Chapter 4 delineates the applicability of liquid chromatography coupled to conventional and tandem electrospray ionization mass spectrometry (LC-ESI-MS, LC-ESI-MS/MS, LC-ESI-MS3) for the screening of novel cyclic hexadepsipeptide metabolites directly from the crude hyphal extract of the fungus Isaria. The fungal strain was grown on a solid medium (potato carrot agar), which yields aerial hyphae growing erect from the basal mycelial colony (Ravindra et al., 2004). A total of ten microheterogeneous components were identified to belong to the isariin class of cyclodepsipeptides from the LC-ESI-MS and LC-ESI-MS/MS analysis of the crude hyphal extract. Out of ten, six are determined to be new and the remaining four are previously reported isariins A-D. The primary structures of isariins A-D were from the fungi Isaria cretacea and Isaria felina (Vining & Taber 1962; Deffieux et al., 1981) and the fungal strain used in this study resembles Isaria felina (Sabareesh et al., 2007). Isariins are backbone cyclized hexadepsipeptides composed of a D-β-hydroxy acid possessing a hydrocarbon sidechain and five α-amino acids; one of the α-amino acids is a D-amino acid (Vining & Taber 1962; Deffieux et al., 1981). The detection of fragment ions due to loss of CO concomitant with the loss of H2O from the protonated precursor ion ([M + H]+) ascertained the cyclic depsipeptide nature of both the known and the new components. The fragmentation behavior of the [M + H]+ of known isariins facilitated sequence determination of the new components. Therefore, the configuration of the amino acids and the β-hydroxy acid of the new components is assumed to be same as that of the reported peptides. The microheterogeneity of the ten sequences is due to changes in the D-β-hydroxy acid (residue 1) and the adjoining α-amino acid (residue 6), whose carbonyl is linked to the hydroxyl function by an ester linkage. The number of methylene units ((-CH2)n) in the hydrocarbon sidechain of the residue 1 differs between 2 and 8 and the variability of the residue 6 is limited to Ala/Val. The ester oxygen atom was chosen as the preferable site of protonation causing ring-opening, based on the observed distribution of the fragment ions. Chapter 5 demonstrates the utility of the LC-ESI-MS and LC-ESI-MS/MS methods in the identification and characterization of six microheterogeneous backbone cyclized hexadepsipeptides, isaridins, directly from the crude hyphal extract of the fungus Isaria. Among the six components, four were found to be novel. The other two peptides, isaridins A and B were identified earlier from this laboratory (Ravindra et al., 2004). The isaridins are characterized by the presence of unusual amino acids such as N-methylated residues, β-methylproline (β-MePro) and hydroxyleucine (HyLeu) (Ravindra et al., 2004). The cyclic nature of both the known and the new peptides were confirmed from the observation of peaks due to loss of CO and H2O from the protonated precursor ion ([M + H]+). However, unlike isariins (Chapter 4), the intensity of the peak corresponding to [M + H - H2O]+ was noted to be of very low intensity, in the case of isaridins. Detection of product ion peak due to [M + H - CO2]+ suggests an additional dissociation pathway involving cleavage at the depsipeptide linkage and is supportive of the cyclic depsipeptide nature (Eckart, 1994). The sequencing of the newly detected components was enabled by understanding the fragmentation mechanism of the known isaridins. The tertiary amide nitrogens of the N-methylated residues were regarded as the preferable sites of protonation leading to ring-opening, as noted from the fragmentation spectra. The microheterogeneity in the sequences was identified using the diagnostic product ions obtained from the protonated precursor of the known isaridins. The microheterogeneity can be attributed to the variations of two residues; Pro ↔ β-MePro and N-MePhe ↔ N-MeLxx (Lxx: Leu, Ile, alloIle). The recently reported ‘isarfelins’ from the fungus Isaria felina (Guo et al., 2005) were reassigned as ‘isaridins’. The reassignment was based on very similar fragmentation profiles observed for the [M + Na]+ adduct of isaridins and isarfelins; further, the fungal strain used in this study resembles Isaria felina (Sabareesh et al., 2007). Chapter 6 presents mass spectrometric sequencing of disulfide bonded peptides from marine cone snails (conopeptides), using the MALDI LIFT MS/MS method. Lo959, a single disulfide bonded octapeptide isolated from Conus loroisii, was identified to belong to the class of contryphans (Sabareesh et al., 2006). Contryphans are small single disulfide bonded conopeptides, whose length is in the range of 7-11 residues and are rich in tryptophan. A significant feature of the contryphans is the presence of conserved DTrp (DW) at the 3rd residue within the disulfide loop (Sabareesh et al., 2006). Lo959 displays an unusual behavior under reverse phase chromatographic conditions, typical of the DW containing contryphans (Jacobsen et al., 1998). It undergoes slow conformational interconversion on the chromatographic time scale exhibiting two distinct peaks. The presence of DW at the 4th position in Lo959 was established by comparing the chromatographic profiles of natural peptide with that of two chemically synthesized peptides, one containing LW (4) and another possessing DW (4). De novo sequencing of the two peptides Ar1446 and Ar1430 from Conus araneosus established that they belonged to M-superfamily of conotoxins, in particular m-2 branch. M-superfamily conotoxins are three-disulfide bonded peptides characterized by the consensus cysteine framework, CC…C…C…CC (Corpuz et al., 2005). Ar1446 and Ar1430 are fourteen residue long peptides, each possessing three disulfide bonds. The peptides have the cysteine scaffold typical of the M-superfamily, as shown above. Specifically, the peptides belong to m-2 branch of M-superfamily, where the fourth and fifth cysteines are separated by two residues (Corpuz et al., 2005). The sequences of the peptides were derived following chemical and enzymatic modifications. The carboxamidomethylation reaction established the presence of three disulfide bonds. Indeed, the sequences were deduced from the MALDI LIFT MS/MS of [M + H]+ of the tryptic peptides. The sequences of the two peptides are almost identical and they differ only at residue 12; hydroxyproline in Ar1446, proline in Ar1430.

The venom of cone snails (predatory marine molluscs of the genus Conus) has yielded a rich source of novel neuroactive peptides or “conotoxins”. Conotoxins are bioactive peptides found in the venom duct of Conus spp. Like other neuropeptides, conotoxins are expressed as propeptides that undergo posttranslational proteolytic processing. Peptides derived from propeptides are typically cleaved at a pair of dibasic residues (Lys-Arg, Arg-Arg, Lys-Lys or Arg-Lys) by proteases found in secretory vesicles. However, many precursor peptides contain multiple sets of basic residues, suggesting that highly substrate specific or differentially expressed proteases can determine processing outcomes. As many of the substrate-specific proteases remain unidentified, predicting new bioactive peptides from cDNA sequences is presently difficult, if not impossible. In order to understand more about the substrate specificity of conotoxin substrate-specific proteases a characterisation study of one such endoprotease isolated from the venom duct of Conus textile was undertaken. The C. textile mollusc was chosen as a good source from which to isolate the endoprotease for two reasons; firstly, these cone shells are found in great abundance on the Great Barrier Reef (Queensland, Australia) and are readily obtainable and secondly, a number of conotoxin precursors and their cleavage products have been previously identified in the venom duct. In order to purify the endoprotease an activity-guided fractionation protocol that included a para-nitroanilide (p-NA) substrate assay was developed. The p-NA substrate mimicked the cleavage site of the conotoxin TxVIA, a member of the C. textile O-superfamily of toxins. The protocol included a number of chromatographic techniques including ion exchange, size-exclusion and reverse-phased HPLC and resulted in isolation of an active protease, termed Tex31, to >95% purity. The purification of microgram quantities of Tex31 made it possible to characterise the proteolytic nature of Tex31 and to further characterise the O-superfamily conopeptide propeptide cleavage site specificity. Specificity experiments showed Tex31 requires a minimum of four residues including a leucine in the P4 position (LNKR↓) for efficient substrate processing. The complete sequence of Tex31 was determined from cDNA. A BLAST search revealed Tex31 to have high amino acid sequence similarity to the CAP (abbreviated from CRISP (Cysteine-rich secretory protein), Antigen 5 and PR-1 (pathogenesis-related protein)) superfamily and most closely related to the CRISP family of mammalian and venom proteins that, like Tex31, have a cysteine-rich C-terminal domain. The CAP superfamily is widely distributed in the animal, plant and fungal kingdoms, and is implicated in processes as diverse as human brain tumour growth and plant pathogenesis. This is the first report of a biological role for the N-terminal domain of CAP proteins. A homology model of Tex31 constructed from two PR-1 proteins, Antigen 5 and P14a, revealed the highly conserved and likely catalytic residues, His78, Ser99 and Glu115. These three amino acids fall within a structurally conserved N-terminal domain found in all CAP proteins. It is possible that other CAP proteins are also substrate-specific proteases. With no homology to any known proteases, Tex31 may belong to a new class of protease. The sequence alignment of five Tex31-like proteins cloned from C. marmoreus, C. litteratus, C. arentus, C. planboris, and C. omaria show very high sequence similarity to Tex31 (~80%), but only one weakly conserved serine residue was identified when the conserved residues of the new Tex31-like protein sequences were aligned with members of the CAP superfamily. Future work to identify members of catalytic diad or triad, e.g. by site-directed mutagenesis, will rely on the expression of active recombinant Tex31. In this study neither Escherichia coli nor Pichia pastoris expression systems yielded active recombinant Tex31 protein, possibly due to the number of cysteine residues hindering the expression of correctly folded active Tex31. This study has shown Tex31 to be highly sequence specific in its cleavage site and it is likely that this high substrate specificity has confounded previous attempts to identify the proteolytic nature of other CAP proteins. With the proteolytic nature of one member of the CAP protein family confirmed, it is hoped this important discovery may lead the way to discovering the role of other CAP family members.

This thesis deals with the analysis of natural peptide libraries using mass spectrometry. In the course of the study, both ribosomal and non-ribosomal classes of peptides have been investigated. Microheterogeneity, post-translational modifications (PTM), isobaric amino acids and disulfide crosslinks present critical challenges in routine mass spectral structure determination of natural peptides. These problems form the core of this thesis. Chapter 2 describes an approach where chemical derivatization, in unison with high resolution LC-MSn experiments, resulted in deconvolution of a microheterogenous peptide library of B. subtilis K1. Chapter 3 describes an approach for distinction between isobaric amino acids (Leu/Ile/Hyp), by the use of combined ETD-CID fragmentation, through characteristic side chain losses. Chapters 4-6 address a long standing problem in structure elucidation of peptide toxins; the determination of disulfide connectivity. Through the use of direct mass spectral CID fragmentation, a methodology has been proposed for determination of the S-S pairing schemes in polypeptides. Further, an algorithm DisConnect has been developed for a rapid and robust solution to the problem. This general approach is applicable to both peptides and proteins, irrespective of the size and the number of disulfide bonds present. The method has been successfully applied to a large number of peptide toxins from marine cone snails, conotoxins, synthetic foldamers and proteins. Chapter 7 describes an attempt to integrate next generation sequencing (NGS) data with mass spectrometric analysis of the crude venom. This approach couples rapidly generated cDNA sequences, with high-throughput LC-ESI-MS/MS analysis, which provides mass spectral fragmentation information. An algorithm has been developed that allows the construction of a putative conus peptide database from the NGS data, followed by a protocol that permits rapid annotation of tandem MS data. The approach is exemplified by an analysis of the peptide components present in the venom of Conus amadis, yielding 225 chemically unique sequences, with identification of more than 150 sites of PTMs. In summary, this thesis presents different methodologies that address the existing limitations of de novo mass spectral structure determination of natural peptides and presents new methodologies that permit for rapid and efficient analysis of complex mixtures.