Bacillus stearothermophilus phosphofructokinase (BsPFK) is a homotetramer that is allosterically inhibited by phosphoenolpyruvate (PEP), which binds along one dimer-dimer interface. The substrate, fructose-6-phosphate (F6P), binds along the other dimer-dimer interface. The different functional forms BsPFK can take when in the presence of F6P and PEP can be described by the following diproportionation equilibrium: XE + EA <--> XEA + E where XE is the enzyme bound to PEP, EA is the enzyme bound to F6P, E represents the apo enzyme, and XEA is the ternary complex formed when both substrate and inhibitor are bound. Currently in the Protein Data Bank (PDB) there are two relevant forms of wild-type BsPFK, the EA form and the X'E form, which represents the enzyme bound to the PEP analog, phosphoglycolate (PGA). When comparing the EA and the X'E structures, a 7° rotation about the substrate-binding interface is observed and is termed the quaternary shift. The current study uses methyl TROSY NMR to examine the different liganded states of BsPFK, and for the first time structural data for the XEA species is shown. In addition, crystallography was used to obtain the first apo structure of BsPFK. To distinguish between changes associated with the quaternary shift and those associated with the intra-subunit tertiary changes, the variant D12A BsPFK was studied using kinetics, crystallography, and NMR. Crystal structures of apo and PEP bound forms of D12A BsPFK both indicate a shifted structure similar to the X'E form of wild-type. Kinetic studies of D12A BsPFK, when compared to wild-type, show a 50-fold diminished F6P binding affinity, 100-fold enhanced binding affinity, and a similar coupling constant. A conserved hydrogen bond between D12 and T156 takes place across the substrate binding interface in the EA form of BsPFK. The variant T156A BsPFK shows similar binding, coupling, and structural characteristics to D12A BsPFK. PEP still inhibits these variants of BsPFK despite the fact that the enzymes are in the quaternary shifted position prior to PEP binding. Therefore the quaternary shift of BsPFK primarily perturbs ligand binding but does not directly contribute to heterotropic allosteric inhibition.

Scytalidium thermophilum produces a novel phenol oxidase, which has turned out to be a bifunctional catalase-phenol oxidase (CATPO) during the course of this work, by other researchers of our group. Therefore, in the beginning of the studies, substrate specificity and inhibitor assays were conducted on the crude enzyme, followed by production, purification, cloning, expression, and mutagenesis and crystallography studies for further functional and structural analysis of CATPO. Accordingly, substrate specificity and inhibitory tests applied for crude enzyme characterisation presented the similarity of the phenol oxidase nature of CATPO essentially to catechol oxidase. Production studies were performed to investigate the effects of different factors including induction time, growth temperature, exogenous iron and hydrogen peroxide addition. In view of that, CATPO is constitutively produced in a growth associated manner. However, some phenolic compounds enhance its production. In this study, 15 phenolic compounds were tested for their ability to affect CATPO production. Of the phenolic compounds tested, catechol, resorcinol and vanillic acid caused repression of CATPO production. On the other hand, caffeic acid, myricetin and resveratrol enhanced CATPO production. As a biocatalyst, the efficiency of CATPO was examined and found to be a good candidate for getting pharmaceutically important drug intermediates. Its dual mechanism was analysed through side-directed mutagenesis. Two conserved residues (His101 and Val142) were mutated to discriminate catalase and phenol oxidase activities. Spectroscopic and mutagenesis studies exhibited the presence of heme d centre. Lastly, its structure was analysed by X-ray crystallography and found to have a tetrameric structure.

This thesis presents the crystal structures of 2', 5', cyclic and modified nucleosides / nucleotides. Chapter I gives a brief account of the structural studies on 2', 5' and modified nucleotides. It also presents a short, summary of unusual nucleic acids structures studies on hydration patterns and metal ion interactions Nomenclature and conventions used for describing the conformatioNa1 features are presented. FiNa1ly, the crystallographic suite of programs used for processing the intensity data, structure solution, refinement and generating various diagrams are mentioned. Chapter II describes the crystal structures of anhydrous and hydrated sodium salt of N6-methyl adenosine-S'-monophosphate. N6-AMP-A (anhydrous form) belongs to the trigoNa1 space group P3221 with unit cell dimensions a = b = 10.30 A and c= 25.03 A while N6-AMP-H (hydrated form) belongs to orthorhombic space group C222X with a= 6.910 A, b= 19.318 A, and c= 41.070 A. CuKα intensity data consisting of 1740 and 2740 observed reflections were collected on a CAD4 diffractometer. Both structures were solved using SHELXS97 and refined to R factors of 0.0336 and 0 0381 for anhydrous and hydrated forms respectively. In both structures the adenine bases are in the ant% conformation with respect to the ribose but their torsion angles XCN differ significantly by 78° The ribose moiety shows CS-endo puckering and the conformation about the C4/-C5/ bond is g+ and t in the anhydrous and hydrated structures respectively. The two Na+ ions, present m the hydrated form, coordinate with water oxygen atoms only. A notable feature of the Na+ ion coordination in the anhydrous form is the participation of N3 and N7 of the base besides macrochelation between base-ribose and base-phosphate moieties. Adenine bases in both forms stack at a separation of about 3.4 A between them N6-AMP molecules pack as if one set of bases intercalate between the other set in the hydrated structure while they form helix like pattern m the anhydrous structure Molecular dynamics calculations were carried out for both structures with a view to obtain greater insight into the effect of hydration on the conformation of the molecule. Stereochemically permissible models for poly-A using the N6-AMP-H coordinates were generated using the method developed by Srinivasan and Olson. Its features and possible biological relevance are discussed. Chapter III deals with the structure of sodium adenosine-5'-monosulfate trihydrate (5'-AMS). Intensity data for this modified nucleotide were collected at the Brookhaven NatioNa1 Laboratory, Synchrotron facility, USA. 5'-AMS belonged to the orthorhom bic space group P2!2!2i with unit cell parameters a= 20.698 A, b= 24.621 A and c= 25.925 A and eight molecules, eight Na+ ions and 23 water molecules in the asymmetric unit of the lattice. Never before a nucleotide structure having eight molecules in the asymmetric unit has been reported. Out of 84177 reflections collected using a radiation of A =0.92 A, 9108 independent reflections having Io>2a(Io) were considered observed. The structure was solved using the program Shake and Bake (SnB) and refined by, SHELXL97. The fiNa1 R factor for 1971 parameters was 0.0397. Adenine bases of all the eight 5'-AMS molecules are in anti conformation with respect to the ribose moiety with XCN angles varying from -150 to -177°. But the conformations of the ribose moieties and the sulfate groups about the C4/-C5/ bond are not the same for all the molecules. 5'-AMS molecules A, B and D show C2-exo-C3-endo mixed puckering while C has C£-exo puckering. The remaining four molecules E, F, G and H have C3-endo conformation. The conformation about the C4/-C5/ bond for molecules A, B, C and D is g~ while for E, F and G it is g+. Molecule H shows both g+ and g~ since the 05' atom is disordered. An important feature of the metal ion coordination is the bidentate formation by sodium ions Na3 and Na7 with the sulfate group of molecule C and ribose hydroxyl oxygen atoms of molecule D respectively. Another feature which deserves mention is the participation of Nl and N7 of the adenine base m metal coordination Adenine bases of molecules A, B, C and D form self pairs with those of H, G, F and E respectively through N6...N7 and N6...N1 hydrogen bonds. The 5'-AMS molecules pack as duplexes in the unit cell. A Stereochemically permissible model for poly-A with sugar sulfate backbone using the 5'-AMS coordinates were generated using the method developed by Srinivasan and Olson and its features are discussed. Crystal structures of two polymorphs of mixed sodium and potassium salts of cytidine-5'-monophosphate hexahydrate are discussed in Chapter IV. The two polymorphs of 5'-CMP were grown using methanol and DMF respectively m the crystallization experiments. MoKα intensity data for CMP-I were collected on a Rigaku AFC image plate system while that for CMP-II were collected on a Bruker CCD Smart system. Both belong to the monoclinic space group P2X with a= 8.869 A, b= 20 580 A, c= 23.179 A, β= 105.79° and a= 8.929 A, b= 22.257 A and c= 20.545 A, β= 90.02° for CMP-I and II respectively. The the unit cell volume of the two polymorphs differ by just 12 A3 as the unit cell parameters are same, although the b and c axes are interchanged m CMP-II and their β value differs by 16°. Both polymorphs of CMP have four nucleotide molecules in the asymmetric unit of their orthorhombic lattices. But the number of metal ions and solvents are not the same in the two structures. CMP-I has five sodium ions, three potassium ions, 23 water and two methanol molecules while CMP-II has two sodium ions, four potassium ions, 22 water and an unknown solvent molecule (assigned as dimethyl ether) in the asymmetric unit. This is the first nucleotide structure having two different alkali metal ions (Na+ and K4") in the crystal structure. Out of 36946 and 31293 reflections collected 12247 and 15476 independent reflections having IO>2<J(I0) were considered observed for CMP-I and II respectively. Both structures were solved by combination of heavy-atom and direct methods using DIRDIF96 and refined using SHELXL97 to R factors of 0.0819 and 0 0867 for CMP-I and II respectively In both forms all the four molecules have anti conformation about the glycosidic bond, CS-endo conformation for the ribose moiety and g+ conformation about the C4'-C5' bond but their metal coordination patterns are significantly different. K1 ion in CMP-I forms an intra molecular macrochelate between the ribose and adenine base while K2 and K3 ions form bidentates with the cytosine and phosphate group of molecules A and D respectively. Na1, Na3 and Na5 are all involved in bidentate interactions with the ribose of molecule C, ribose of molecule A and phosphate of molecule D respectively. In contrast, Na2 and Na4 coordinates with solvent atoms only and do not interact with the nucleotide atoms at all. K1 and K2 ions of CMP-II form bidentates with the cytosines of molecules C and D respectively while K2 and K4 form intra molecular macrochela-tion between the base and ribose of molecules C and B respectively. Na1 and Na2 form bidentates with the ribose of molecules C and D respectively Comparison of the two polymorphs of CMP reveals that despite several striking conformatioNa1 similarities there are also significant differences between them. It was noticed that molecules A, B, C and D of CMP-I corresponds to C, B, D and A of CMP-II. Out of eight metal ions (five Na+ and three K+ ions) present in CMP-I four of them (Kl, K2, K3 and Na3) are found to have partners (K4, Kl, K3 and Na1) in CMP-II within a distance of 0.75 A. One of the water molecules OW8 of CMP-I is replaced by a potassium ion K2 in CMP-II within a distance of 0.92 A. Out of 23 water molecules present in the structure 14 are common to both of them and only 8 are different while one is replaced by an ion. The four crystallographically independent 5'-CMP molecules are linked by metal ions Kl, K3, Na1, Na3, Na5 and Kl, K2, K4, Na1 ions forming a tetramers in CMP-I and CMP-II respectively. An interesting feature of CMP-I and CMP-II is the simultaneous display of base-base and base-ribose stacking patterns. The four nucleotide molecules in the asymmetric unit are related by several pseudo two-fold axis and the r.m.s. Deviations between them after applying the pseudo symmetry are 0.21 and 0.17 A for CMP-I and II respectively. The nucleotide molecules in CMP-I and II pack as infinite linear chains parallel to the b and c axis respectively which repeat along the c and b axis respectively. In between these nucleotide columns metal ions and water molecules are located forming channels between them. Chapter V deals with anhydrous cytidine-2/-phosphate and potassium uridine-5'-phosphate hexahydrate structures. 2'-CMP crystallizes m the orthorhombic space group P212121 with a= 6.698 A, b= 7.436 A and c= 25.291 A with one molecule in the asymmetric unit. MoKα intensity data were collected on a CCD SMART system consisting of 7647 reflection of which 1456 independent reflections having lo>2a(lo) were considered observed. The structure was solved and refined to an R factor of 0.0385 for 186 parameters using SHELXL97. The cytosine base is in the anti conformation with respect to the ribose with XCN = -141.1° similar to that in the hydrated structure. But it differs significantly from the syn conformation observed in several 2'-purine and 2'-5' dinucleotide structures containing purine-pyrimidine sequences. The ribose moiety shows Ctf-endo and the conformation about the C4/-C5/ bond is t with (f)α = 169.3° and <pO( = -72 7° The t conformation in the anhydrous form is different from the g+ conformation m the hydrated form of 2'-CMP 5'-UMP.K crystallizes in the monoclinic space group P2;i with a= 13 034 A, b= 8 916 A, c= 16 205 A and β=98 64° with two nucleotides, four K ions and ten water molecules in the asymmetric unit MoKα intensity data of 19261 were measured on a Bruker CCD system of which 6891 independent reflections having lo>2a(lo) were accepted as observed. The structure was solved and refined by full matrix least square methods to an R factor of 0.0324 for 609 parameters. Uracil bases of both nucleotide molecules are in the anti conformation with respect to the ribose with XCN= -129.4° and -132 7°. Uracil bases of both nucleotide molecules are protonated at N3 Both ribose moieties show C2’-endo puckering with C2' atom displaced by 0.57 and 0.59 A from the best plane constituted by the remaining atoms The phosphate group is in a staggered orientation and the conformation about the C4/-C5/ bond is g* with <j>00 = -67.1 and -62.7 and Øoc = 54.6 and 59.5 for molecules A and B respectively. Potassium ion K2 forms a bidentate by coordinating with ribose 02' and 03' atoms of molecule B and a macrochelate between the uracil base and ribose of molecule A by coordinating with 02 and 02' atoms. K4 also forms a bidentate by coordinating with ribose O2' and 03' atoms of molecule A. The two 5'-UMP molecules form a dimer by coordinating with K2 and K3 ions. They are related by a pseudo two-fold axis and the r.m.s. deviation between the coordinates is 0 12 A. Crystal structures of 8-Benzylamino cychc-3'-5'-monophosphate (8-Benz-cAMP) and 8-mercaptoguanosine (8-MERG) are presented in Chapter VI. 8-Benz-cAMP crystallizes in the monoclinic space group P2x with unit cell dimensions a= 7.989 A, b= 12 589 A, c= 11.773 A and β= 93.82°. MoKα data were collected on a CCD system yielded 4331 independent observed reflection with Io2cr(Io) out of 9733 reflections collected. The structure was solved and refined to a R factor of 0 0451 with 367 parameters. The adenine base is in the syn conformation with XCN= 84.7° as in few other 8-substituted cyclic purine nucleotides but different from the simple cyclic purine nucleotides. The phenyl moiety is in the trans conformation with respect to the base. The ribose moiety shows rare C4’-exo puckering with a deviation of 0.70 A from the best plane constituted by the remaining four atoms. The 05' atom is m the t conformation with respect to the ribose with cpα = -174.8° and <pα = -59.6° since only in this conformation 3' and 5' cyclization is possible. Hydrogen bonds Nl. .O1P and N6...O5' link two nucleotide molecules. Adenine bases stack on the phenyl ring from above and below. The only water molecule present in the structure form hydrogen bonds with the nucleotide atoms. 8-mercaptoguanosine crystallizes in the monoclinic space group C2 with unit cell dimensions a= 23.246 A, b=9.751 A, c= 6.406 A and b= 90.91°. MoKα intensity data collected on CAD diffractometer yielded 2683 independent observed reflections having I0>2<r(I0). The structure was solved using SHBLXS 97 and refined using SHELXL97 to a R factor of 0.0565. The guanine base is in the syn conformation with XCN= 64.1°. The ribose ring shows C2-endo puckering with C2' atom deviating by 0.62 A from the best plane. An interesting feature of this structure is the intra-molecular hydrogen bond between the base N3 and the ribose 05' atoms. The last chapter (VII) describes the crystal structures of three modified adenine nucleosides N6-benzyl adenosine (N6-BA), N6-cyclohexyl adenosine (N6-CA) and 5'-trityl adenosine (5'-TA). N6-BA belongs to the triclinic space group PI with a= 5.008 A, b= 8.921 A, c= 9.762 A and a = 111.73°, β= 90.37°, 7 = 91.42° while N6-CA and 5'-TA belong to the monoclinic space group P2i with a= 12.205 A, b=15.265 A, c= 15.095 A, P = 110.64° and a= 8.823 A, b= 15.613 A, c= 10.078 A and β = 115.01° with three and one molecules in their asymmetric units respectively. The three structures of N6-BA, N6-CA and 5'-TA were solved and refined to R factors of 0.0355, 0.0655 and 0.0262 using 1656, 7549, 2473 independent reflections and 244, 677, 360 parameters respectively using SHELX97. The adenine base of N6-BA is in the anti conformation with XCN= 168.9°. The benzyl moiety is in the distal geometry with respect to the imidazole ring. The furanose ring shows CSI-exo-CS'-endo mixed puckering. There are several 7r-7r interactions observed in this structure. In contrast to N6-BA all three molecules of N6-CA show syn conformation about the glycosidic bond with XCN= 47.0°, 54 8°, 49 V for molecules A, B and C respectively. The cyclohexyl moiety of all three molecules are in the chair conformation. The ribose moieties of all three molecules show C2-endo puckering with C2' atom deviating by 0 59, 0 54, 0.57 A for molecules A, B and C respectively. The adenine base of the 5'-TA is in the anti conformation with \Cs= 168.4° and the ribose moiety shows C2-endo puckering The three phenyl rings of the trityl group are in staggered orientation. Interesting tape formation via N6…02' and N7...O3' hydrogen bonds is observed in all three nuclosides.

Thesis (Ph. D.)--Institute of Paper Chemistry, 1969. / Includes bibliographical references (p. 119-122).

Ricin is a potent cytotoxin easily purified in large quantities. It presents a significant public health concern due to its potential use as a bioterrorism agent. For this reason, extensive efforts have been underway to develop antidotes against this deadly poison. The catalytic A subunit of the heterodimeric toxin has been biochemically and structurally well characterized, and is an attractive target for structure-based drug design. Aided by computer docking simulations, several ricin toxin A chain (RTA) inhibitors have been identified; the most promising leads belonging to the pterin family. To date, the most potent RTA inhibitors developed using this approach are only modest inhibitors with apparent IC50 values in the 10-4 M range, leaving significant room for improvement. This thesis discusses the development of a subset of inhibitors belonging to the pterin family in which amino acids have been utilized as building blocks. Inhibitors in this family have achieved a significant increase in potency, and have provided valuable structural information for further development. / text

The technological development and application of waste-to-resource strategy is significantly critical and crucial in both environmental and manufacturing industries, via which we do not only provide practical treatments to toxic waste materials but also translate them into usable products. It has been considered as a preferred method which should be applied in future wastewater treatment strategies. In this study, we investigated the process of incorporating cadmium oxide and nickel oxide into ceramic-based materials with the phases of CdAl4O7, and CdFe2O4. Such products are of less harmful effect to the natural environment and can also be of beneficial use with their good mechanical properties identified by nano-indentation. We proved the possibility and provided an opportunity to convert the waste from wastewater treatment process to a new material resource. XRD is preferred for characterizing solid mixtures to determine the relative abundances of crystalline phases during the reaction process. As a result, we can obtain the relative abundance information on the growth of the crystalline products, such as CdAl4O7, and CdFe2O4 according to the change of fabrication temperatures. In that case, the starting reaction temperature and the optimized temperature (at which the completed reaction could be achieved) could be revealed. In Cd-Al system, the starting temperature for CdAl4O7 formation is 900 °C, and the optimized formation temperature is around 1020 °C. On the other hand, for Cd-Fe system, such temperatures are of 700 °C and 850 °C correspondingly. In our research, it is shown that the ceramic-process is an effective strategy to stabilize the waste hazardous metals (cadmium and nickel) by materials such as aluminate, ferrite, and kaolin commonly used in ceramic industry. Through this method, the difficult-to-treat wasted metals would become reusable and applied in building and infrastructure projects. Products containing CdAl4O7, CdFe2O4 and NiAl2O4 have shown higher resistance to acidic leaching, comparing to CdO and NiO used as the starting materials to simulate the waste metal forms discharged from the industrial stream. Furthermore, similar measurements by alkaline attack on the sintered products (NiAl2O4 containing samples) were also studied. Besides the investigation to leaching behavior, the mechanical properties are also measured by nano-indentation in our work. The incorporation of metal waste into the fabrication of ceramic products is valuable due to the preferred stabilization mechanisms of crystal structures and the large volume of ceramic products needed by the construction industry. Furthermore, because the product safety and functionality should not be compromised, a fundamental understanding of the surface properties of metal containing phases should be further established, rather than relying solely on data from regulatory tests on bulk samples. Therefore, the results of this study demonstrate the superior mechanical properties of nickel spinel containing products, comparing to the cristobalite silica matrix, under severe acid attack. / published_or_final_version / Civil Engineering / Master / Master of Philosophy

Methionine synthases catalyze methyl transfer from 5-methyl-tetrahydrofolate (5-methyl-THF) to L-homocysteine (Hcy) in order to generate methionine (Met). Mammals, including humans, use a cobalamin dependent form, while fungi use a cobalamin independent protein called Met6p. The large structural differences between them make Met6p a potential anti-fungal drug target. Met6p is a 90 kDa protein with the active site located between two (βα)₈ barrels. The active site has a catalytic Zn²+ and binding sites for the two substrates, Hcy and folate. I present the crystal structures of three engineered variants of the Met6p enzyme from Candida albicans. I also solved Met6p in complex with several substrate and product analogs, including Hcy, Met, Gln, 5-methyl-THF-Glu₃ and Methotrexate-Glu₃ (MTX-Glu₃), and the bi-dentate ligand S-adenosyl homocysteine. Also described is a new fluorescence-based activity assay monitoring Hcy. Lastly, a high-throughput Differential Scanning Fluorimetry (DSF) assay was used to screen thousands of compounds in order to identify ligands which bind Met6p. My work details the mode of interaction of Hcy and folate with the Met6p protein. Several residues important to activity were discovered, like Asn 126 and Tyr 660, and proven to be important by site directed mutagenesis. Structural analysis revealed an important aspect of the mechanism. When Hcy binds to its pocket it makes strong ion pairs with the enzyme. In particular, 614 moves toward the substrate amine and triggers a rearrangement of active site loops; this draws the catalytic Zn²+ toward the Hcy thiol where a new ligand bond is formed, activating the thiol for methyl transfer. The work presented here lays the groundwork for structure based drug design and makes the development of Met6p specific bi-dentate ligands feasible. The fluorescence based activity assay I developed was successfully used to test the folate analog MTX-Glu₃, which inhibits with an IC₅₀ of ~4 mM. I also discovered our first bi-dentate ligand in the form of S-adenosyl homocysteine. / text

X-ray crystallography is a form of microscopy that allows the three-dimensional arrangement of atoms belonging to molecules within crystals to be determined. In this method, a crystal is illuminated with a beam of X-rays and the diffracted amplitudes resulting from the illumination are measured and computationally processed to enable the electron density of the unit molecule, or the unit cell, constituting the crystal to be calculated. The recent development of the X-ray free-electron laser (XFEL) provides new routes for determining molecular structures via its ability to generate intense but brief X-ray pulses. These new instruments enable diffraction measurements to be obtained from crystals that have a small number of unit cells, referred to as nanocrystals, and molecular structure determination via this technique is known as serial femtosecond nanocrystallography (SFX). This thesis is concerned with the characterisation of diffraction data obtained from SFX experiments and the techniques for reconstructing the electron density of the molecule from such data. The noise characteristics of diffraction measurements from nanocrystals is developed. Methods for directly inverting nanocrystal diffraction to obtain the electron density of the molecule are analysed and an approach to ameliorate the effect of noise is proposed and evaluated by simulation. A model for diffraction by nanocrystals that include the effects of different unit cell arrangements and incomplete unit cells on the crystal surface is also developed and explored by simulation. The diffraction by finite crystals is shown to be equal to the incoherent average over a set of unit cells that contain different molecular arrangements related to the symmetry of the crystal at hand. The problem of image reconstruction under this circumstance is investigated. The more general problem of reconstructing multiple, unrelated, objects from their averaged diffraction is also explored and uniqueness properties along with reconstruction algorithms developed. The problem of reconstructing multiple, related, unit cells is studied and preliminary results are obtained. These results show that iterative phase retrieval algorithms can in principle be adapted to reconstruct the electron density of a crystalline specimen from the data obtained in SFX and the retrieval of phases from the diffracted intensity averaged over multiple objects is feasible.

Transthyretin (TTR) is one of several proteins involved in amyloid disease in humans. Unknown conformational changes of the native state of TTR result in aggregation of TTR molecules into amyloid fibrils, which accumulate in extracellular tissues. This may result in different clinical symptoms, e.g. polyneuropathy or cardiomyopathy, depending on their site of accumulation. Our long-term goal is to identify structural changes associated with amyloid formation. For this work, structural characterization of TTR from other species than human may provide valuable information. The three-dimensional X-ray crystallographic structure of TTR from sea bream (Sparus aurata) was determined at 1.75 Å resolution. Human and sea bream TTR were found to be structurally very similar. However, interesting differences were present in the area at and around -strand D, which in fish forms an extended loop region. Interestingly, this area is believed to dissociate from the structure prior to amyloid formation, to allow -strands A and B to participate in polymerization. During evolution, TTR from different species have developed differences in preference to their natural ligands, the thyroid hormones 3,5,3’-triiodo-L-thyronine (T3) and 3,5,3’,5’-tetraiodo-L-thyronine (T4). While human TTR has higher affinity for T4, the opposite is true in lower vertebrates, e.g. fish and reptiles. We have determined two separate structures of sea bream TTR in complex with T3 and T4, both at 1.9 Å resolution. A significantly wider entrance and narrower thyroid hormone binding channel provide a structural explanation to the differences in thyroid hormone preference between human and piscine TTR. In a separate work, we identified a novel protein family with structural similarity to TTR, which we named the transthyretin-related protein (TRP) family. To attain information about this protein family, we cloned, expressed, purified and characterized TRP from Escherichia coli (EcTRP). Furthermore, we solved the structure of EcTRP to 1.65 Å resolution. As predicted, EcTRP and human TTR are structurally very similar. Interesting structural differences are found in the area corresponding to the thyroid hormone binding site in TTR, which due to its amino acid conservation within the TRP family we identified as a putative ligand-binding site in TRPs. The function of the TRP is not known, however, recent studies suggest that it might be involved in purine catabolism. It has been shown that partial acid denaturation of human TTR results in amyloid-fibril formation. Interestingly, we have shown that sea bream TTR also forms amyloid-like fibrils in vitro, even though it shares only 52% sequence identity to human TTR. Corresponding studies on EcTRP did not generate amyloid-like fibrils. EcTRP has 30% sequence identity to human TTR. The fact that two of the proteins form amyloid fibrils and one does not means that they can serve as a model system for the study of amyloid formation. Further studies on these three proteins are currently performed to attain more information about the mechanism of amyloid formation.

Calcium is a ubiquitous second messenger in cells that plays a vital role in the control of cellular and physiological processes as diverse as cell division, memory and learning, fertilization and muscle contraction. Opening of the sarcoplasmic reticulum (SR) Ca2+-release channel, the ryanodine receptor (RyR), in response to mechanical or chemical stimuli via the dihydropyridine receptor (DHPR) is a crucial step in the process of muscle excitation-contraction coupling. I have determined the first high-resolution structure of a folded domain of RyR1 (RyR1A). The structure adopts a β-trefoil fold that is similar to the homologous suppressor domain of the inositol 1,4,5-trisphosphate receptor (IP3R). I identified a loop region in RyR1A concentrated with malignant hyperthermia (MH)- and central core disease (CCD)-associated mutations that have been implicated in perturbing inter-domain interactions with downstream regions of RyR. More recently I have used nuclear magnetic resonance (NMR) spectroscopy to study the structure and dynamics of the cardiac isoform (RyR2) A domain and its mutants. I detected a dynamic α-helix that undergoes an α-helix to β-strand switch in the catecholaminergic polymorphic ventricular tachycardia (CPVT)-associated mutant, RyR2A Δ exon 3. This dynamic helix is localized at an interface with electron dense columns in the cryo-EM map of the tetrameric receptor that connect with the pore region, suggesting that this dynamic helix may also interact with downstream regions of RyR to gate the channel. My high-resolution structural studies in collaboration with others have shed light on the structural underpinnings of RyR function and dysfunction in human disease.