Yes / We describe a method for performing time-resolved X-ray crystallographic experiments based on the Hadamard transform, in which time resolution is defined by the underlying periodicity of the probe pulse sequence, and signal/noise is greatly improved over that for the fastest pump-probe experiments depending on a single pulse. This approach should be applicable on standard synchrotron beamlines and will enable high-resolution measurements of protein and small-molecule structural dynamics. It is also applicable to other time-resolved measurements where a probe can be encoded, such as pump-probe spectroscopy. / Wellcome Trust 4-year PhD program “The Molecular Basis of Biological Mechanisms” 089312/Z/09/Z. This work was also supported by the EPSRC Award “Dynamic Structural Science at the Research Complex at Harwell” EP/I01974X/1 and by BBSRC Award BB/H001905/1.

The three-dimensional x-ray structure of 2,2'-bipyridylglycinatochloro copper(II) dihydrate has been fully refined to a final R factor of 0.081. The bipyridyl and glycine ligands are arranged about the central copper atom in a square planar configuration while the chlorine atom is 2.635 angstroms above this plane directly over the copper atom. This unusually long distance is explained by the positioning of a glycine group on the opposite side of the square plane, resulting in a distorted octahedral arrangement. Also, the chlorine atom is linked to three oxygen atoms via hydrogen bonding, thus stabilizing the distorted octahedral complex.

abstract: Membrane proteins are a vital part of cellular structure. They are directly involved in many important cellular functions, such as uptake, signaling, respiration, and photosynthesis, among others. Despite their importance, however, less than 500 unique membrane protein structures have been determined to date. This is due to several difficulties with macromolecular crystallography, primarily the difficulty of growing large, well-ordered protein crystals. Since the first proof of concept for femtosecond nanocrystallography showing that diffraction patterns can be collected on extremely small crystals, thus negating the need to grow larger crystals, there have been many exciting advancements in the field. The technique has been proven to show high spatial resolution, thus making it a viable method for structural biology. However, due to the ultrafast nature of the technique, which allows for a lack of radiation damage in imaging, even more interesting experiments are possible, and the first temporal and spatial images of an undamaged structure could be acquired. This concept was denoted as time-resolved femtosecond nanocrystallography. This dissertation presents on the first time-resolved data set of Photosystem II where structural changes can actually be seen without radiation damage. In order to accomplish this, new crystallization techniques had to be developed so that enough crystals could be made for the liquid jet to deliver a fully hydrated stream of crystals to the high-powered X-ray source. These changes are still in the preliminary stages due to the slightly lower resolution data obtained, but they are still a promising show of the power of this new technique. With further optimization of crystal growth methods and quality, injection technique, and continued development of data analysis software, it is only a matter of time before the ability to make movies of molecules in motion from X-ray diffraction snapshots in time exists. The work presented here is the first step in that process. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2014

Clustered regularly interspaced short palindromic repeats (CRISPRs) act to prevent viral infection and horizontal gene transfer in prokaryotes. The genomic CRISPR array contains short sequences (“spacers”) that are derived from foreign genetic elements. The CRISPR array is transcribed and processed into CRISPR RNAs (crRNAs) used in the sequence-specific degradation of foreign nucleic acids. This process is called interference and is mediated by CRISPR-associated (Cas) proteins. This thesis has focused on the structural and functional characterisation of four Cas proteins from the CRISPR/Cas system of Sulfolobus solfataricus. The crystal structure of Cmr7 (Sso1725), a Sulfolobales-specific subunit of the ssRNA-degrading CMR complex, allowed for the identification of a putative protein-binding site, though no specific function could be ascribed to the protein. Cas6 (Sso1437) is the enzyme responsible for crRNA maturation and the characterisation of this protein allowed for the molecular rationalisation of its atypical RNA cleavage mechanism. Csa5 and Cas8a2 are subunits of the aCascade complex that targets dsDNA. Csa5 (Sso1398) was shown to have a putative role in R-loop stabilisation during interference while the role of Cas8a2 (Sso1401) was not determined. The structures of these two proteins were used to define relationships between the subunits of interference complexes from various CRISPR/Cas systems. A second aspect of this work has been the expression and purification of eukaryotic ion channels for structural studies. The acid sensing ion channel (ASIC) and FMRFamide-gated sodium channel (FaNaC) are gated ion channels with unknown mechanisms of channel activation. These ion channels must be expressed in eukaryotic systems and so human embryonic kidney (HEK) cells and baculovirus-insect cell expression systems were developed to express ASIC and FaNaC constructs. The expression and purification protocols have been optimised to allow for the preparation of soluble protein that will in future be used for crystallography and electron paramagnetic resonance (EPR) studies.

Viruses are a diverse genera of organisms adapted to thrive in many different hosts from prokaryotic to eukaryotic. We present here the structure of bacteriophage PRR1 virus-like particle (VLP), belonging to Leviviridae family. Our structure reveals calcium ions in the VLP. Metal ions are rare in the VLP among the Leviviridae and the calcium ions were found to affect VLP stability. Gene expression in Leviviridae is controlled by a specific interaction between the viral coat protein that assembles to create the VLP, and the genomic RNA. This interaction has been thoroughly studied for the levivirus MS2 but other structural data are scarce. We have solved the structure of PRR1 VLP in complex with its RNA operator stem-loop. Binding of the stem-loop in PRR1 shows similarities to MS2 but also a different arrangement of the nucleotides, in the area of the loop that we could interpret, compared to MS2. The structures of PRR1 increase our knowledge about translational control in Leviviridae and add new information about particle stability within this family. The other virus we investigated is the more infamous human pathogen, the HIV. Because of the high mutation rate of HIV new drugs are needed on a continuous basis. We describe here the structure of two new protease inhibitors bound to the HIV-1 protease and compare them with two previously published inhibitors. Due to an extended P1´site the new compounds are able to exploit a new interaction to Phe53 in the protease structure. / <p>Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 724</p>

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.

Indiana University-Purdue University Indianapolis (IUPUI) / The human genome encodes 19 members of the aldehyde dehydrogenase (ALDH) superfamily, critical enzymes involved in the metabolism of aldehyde substrates. A major function of the ALDH1A subfamily is the oxidation of retinaldehyde to retinoic acid, a key regulator of numerous cell growth and differentiation pathways. ALDH1A1 has been identified as a biomarker for both normal stem cells and cancer stem cells. Small molecule probes are needed to better understand the role of this enzyme in both normal and disease states. However, there are no commercially available, small molecules that selectively inhibit ALDH1A1. Our goal is to identify and characterize small molecule inhibitors of ALDH1A1 as chemical tools and as potential therapeutics. To better understand the basis for selective inhibition of ALDH1A1, we characterized N,N-diethylaminobenzaldehyde (DEAB), which is a commonly used inhibitor of ALDH1A1 and purported to be selective. DEAB serves as the negative control for the Aldefluor assay widely utilized to identify stem cells. Rather than being a selective inhibitor for ALDH1A1, we found that DEAB is a slow substrate for multiple ALDH isoenzymes, and depending on the rate of turnover, DEAB behaves as either a traditional substrate or as an inhibitor. Due to its very slow turnover, DEAB is a potent inhibitor of ALDH1A1 with respect to propionaldehyde oxidation, but it is not a good candidate for the development of selective ALDH1A1 inhibitors because of its promiscuity. Next, to discover novel selective inhibitors, we used an in vitro, high-throughput screen of 64,000 compounds to identify 256 hits that either activate or inhibit ALDH1A1 activity. We have characterized two structural classes of compounds, CM026 and CM037, using enzyme kinetics and X-ray crystallographic structural data. Both classes contained potent and selective inhibitors for ALDH1A1. Structural studies of ALDH1A1 with CM026 showed that CM026 binds at the active site, and its selectivity is achieved by a single residue substitution. Importantly, CM037 selectively inhibits proliferation of ALDH+ ovarian cancer cells. The discovery of these two selective classes of ALDH1A1 inhibitors may be useful in delineating the role of ALDH1A1 in biological processes and may seed the development of new chemotherapeutic agents.

The vascular system is critical for maintaining homeostasis in all vertebrates. Structural studies of Neuropilin (Nrp), an essential angiogenic receptor, have defined its role in regulating angiogenesis, the formation of new vessels from the existing vasculature. Utilizing biochemical and biophysical tools we describe the ability of Nrp to function as a co-receptor for the VEGFR receptor tyrosine kinase. Two families of Nrp-1 ligands, Vascular Endothelial Growth Factor A (VEGF-A) and Semaphorin3F (Sema3F), physically compete for binding to the Nrp-1 b1 domain, and have opposite roles. VEGF-A is a potent pro-angiogenic cytokine while Sema3F is an angiogenesis inhibitor. Using coupled structural and functional studies, we have discovered the basis for potent competitive binding of Sema3F to Nrp1 requires engagement of two distinct binding sites. These data provide a basis for understanding the rational design of novel high affinity Nrp-1 inhibitor.

Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: Please refer to fulltext for abstract / AFRIKAANSE OPSOMMING: Sien volteks vir opsomming

Lactobacillus casei BL23 expresses two enzymes encoded by the genes iolG1 and iolG2. They have been putatively assigned as myo-inositol dehydrogenases by sequence comparison. The enzyme catalyzes the reversible conversion of myo-inositol to scyllo-inosose and the concurrent reduction of NAD+ to NADH. iolG1 was subsequently determined to be a myo-inositol dehydrogenase but iolG2 was determined to be a scyllo-inositol dehydrogenase. Sequence analysis and kinetics by themselves did not provide insight as to why the enzymes are functionally different. This manuscript provides a structural rationalization for the differences in stereoisomer selectivity by X- ray crystal structure analysis and comparison. High resolution apo, binary, and ternary crystal structures for iolG1 and iolG2 wild type enzymes were determined. For iolG1 the ternary structures were determined for myo-inositol and d-chiro-inositol and for iolG2 the scyllo-inositol bound structure was determined. The high resolution structure information revealed the composition of their respective active sites and showed that subtle differences in critical amino acids for each enzyme define the orientation of the inositol stereoisomer for inline transfer of a hydride to NAD+. Mutagenesis studies of a closely related myo-inositol dehydrogenase from Bacillus subtilis were carried out. The wild type structure for BsIDH had already been determined and characterized. A portion of the results in this manuscript briefly explore structures of dehydrogenase mutants which validate the structural role of residues involved in cofactor selectivity