Frankær, Christian Grundahl

Nærværende ph.d.-afhandling omfatter arbejdet med at kombinere komplementære røntgenmetoder til at undersøge strukturen af proteiner og biomaterialer og indeholder tre forskellige projekter: (i) Karakterisering af proteinpulvre med røntgenpulverdiffraktion (XRPD). (ii) Kombinationen af røntgenkrystallografi og røntgenabsorptionsspektroskopi (XAS) anvendt på et studiumaf forskellige konformationer af insulinhexamere. (iii) Strukturerne af strontiumranelat polymorfer samt fordelingen af strontium i knoglevæv. En procedure blev udviklet til hurtigt at kunne identificere og verificere proteinpulvre med XRPD, og blev afprøvet på mikrokrystaller af lysozym og insulin. Forskellige proteinkrystalformer blev identificeret ved at sammenligne eksperimentelt opnåede pulverdiffraktogrammer med diffraktogrammer beregnet ud fra PDB-koordinater. Korrektion for uordnet solvent blev identificeret somden vigtigste faktor til at opnå god overensstemmelse mellem beregnede og eksperimentelle diffraktogrammer, men også baggrundskorrektion samt optimering af enhedcelleparametre anses for vigtige. En prøveholder blev designet til opsamling af pulverdiffraktionsdata på standard pulverdiffraktometre. Pinholes integreret i prøveholderdesignet bevirkede reduktion af baggrunden, og krystalformer af veldiffrakterende proteinpulvre blev identificeret fra blot 7 μl prøve. Prøveholderen var designet til også at kunne anvendes til XAS-målinger. Krystalstrukturerne af tre konformationer af bovin insulinhexameremed zink (T6, T3R3 og R6) blev løst med røntgendiffraktion (XRD) fra énkrystaller til opløsninger på hhv. 1.40 Å, 1.30 Å og 1.80 Å. Koordinationen af zink i de enkelte konformationer blev undersøgtmed XAS, hvilket både inkluderede EXAFS (extended X-ray absorption fine structure) og XANES (X-ray absorption near edge structure). XAS-spektre blev målt på Zn K-kanten på insulinmikrokrystallermed EXAFS-signal ud til k = 14 Å−1. Ved at betragte hexameren som en dimer af trimere, hvor hver trimer indeholder en Zn2+-ion, antog zink tetraedrisk koordinationsgeometri i alle R3-trimere, og oktaedrisk koordination i T3-trimerene. Koordinationsafstande til zink blev forfinet vha. EXAFS med standardafvigelser under 0.01 Å for den inderste koordinationssfære. I modsætning til de zinkafstande, der forekommer i krystalstrukturerne til middel opløsning, stemte XAS resultaterne bedre overens med zinkafstande observeret blandt småmolekylstrukturermed lignende koordinationsgeometrier, såvel somblandt andre insulinstrukturer til høj opløsning. Eftersomstrålingsdoserne for XRD-forsøgene var signifikant højere i forhold til doserne for XAS-forsøgene, var énkrystallerne udsatte for større strålingsskade, hvilket påvirkede særligt koordinationen af zink i T3-trimerene. Endvidere blev XANES-spektre af zinkcentrene i T6 og R6 insulin beregnet vha. FDM (finite difference methods), og bindingsafstande samt -vinkler blev optimeret vha. en kvantitativ analyse af XANES-spektrene. T6 insulin blev endvidere krystalliseret med jern, nikkel og kobber, og deres krystalstrukturer blev løst til opløsninger på hhv. 1.90 Å, 1.50 Å and 1.45 Å. Eftersom affiniteten overfor jern er lav, blev jerninsulin krystalliseret under tilstedeværelse af små mængder af zink. Dermed optog Fe2+ og Zn2+ hhv. hver af de to metalcentre i krystalstrukturen, og med hhv. tetraedrisk og oktaedrisk koordinationsgeometri. Metalcentrene in nikkel og kobberinsulin blev undersøgt med XAS. Koordinationsafstande blev forfinet vha. EXAFS, som viste en meget regulær oktaedrisk koordination af Ni2+, hvilket yderligere blev bekræftet vha. beregnede XANES-spektre. Kobbers koordinationsgeometri var flertydigt bestemt i krystalstrukturen af kobberinsulin. XANES-spektret påviste tilstedeværelsen af kobber i oxidationstrin +I, hvilket var forårsaget af en fotoreduktion af Cu2+ i løbet af XASmålingerne. Fotoreduktion blev ikke observeret for nikkel- og zinkinsulin. En efterfølgende EXAFS-analyse påviste tetraedrisk koordination af kobber, hvilket yderligere blev bekræftet vha. ab initio Hartree-Fock beregninger samt beregnede XANES-spektre. Strontium ranelat, som er et lægemiddel brugt til behandling af knogleskørhed, blev syntetiseret og krystalliseret, hvorefter krystalstrukturen af nonahydratet blev løst. Krystallerne var yderst følsomme overfor luftfugtighed og temperatur, og gik let i stykker under dehydrering. Fire andre hydrater blev observeret under dehydreringen vha. in situ XRPD. Disse hydrater blev identificeret som heptahydrat, pentahydrat, trihydrat samt dihydrat, ved at estimere vandindholdet ud fra en termogravimetrisk analyse. Knoglevæv fra hunde behandlet med strontiummalonat blev undersøgt med XAS. En ny måde at gribe dataanalysen af absorptionsspektrene an på, resulterede i en model, hvor fra den relative fordeling af strontium i de forskellige knoglebestanddele kunne estimeres. Cirka 35–45 % af det tilstedeværende strontium var inkorporeret i alciumhydroxyapatit under udskiftning af nogle af calciumionerne med strontium, og mindst 30 % optog uordnede sites som strukturelt ligner dem for Sr2+-ioner i vandig opløsning. Den resterende mængde strontium var absorberet i kollagen. Den relative fordeling af strontium i de forskellige knoglebestanddele forekom at være uafhængig af behandlingstid samt dosis.This thesis presents thework on combining complementary X-rays techniques for studying the structures of proteins and other biomaterials, and consists of three different projects: (i) Characterization of protein powders with X-ray powder diffraction (XRPD). (ii) The combination of X-ray crystallography and X-ray absorption spectroscopy (XAS) applied to studying different hexameric insulin conformations. (iii) The structures of polymorphs of strontium ranelate and the distribution of strontium in bone tissue. A procedure for fast identification and verification of protein powders using XRPD was developed and tested on micro-crystals of lysozyme and insulin. Different protein crystal forms were identified by comparing experimental powder diffraction patternswith patterns calculated from PDB coordinates. The key factor to bring the calculated patterns in agreement with the observed patterns was correction for disordered bulk-solvent, but also correction for background and optimization of unit cell parameters have to be taken into account. A sample holder was designed for collecting powder diffraction data on a standard laboratory X-ray powder diffractometer. The background was reduced by use of pinholes integrated in the sample holder design, and crystal forms of well diffracting protein powders were safely identified from only 7 μl of sample. The sample holder was furthermore adaptable for XAS experiments. The crystal structures of three conformations of hexameric bovine zinc insulin (T6, T3R3 and R6) were solved by single crystal X-ray diffraction (XRD) to 1.40 Å, 1.30 Å and 1.80 Å resolution, respectively. The zinc coordination in each conformation was studied by XAS including both extended X-ray absorption fine structure (EXAFS) spectroscopy and X-ray absorption near edge structure (XANES) spectroscopy. High quality XAS spectra were collected at the Zn K-edge on insulin micro-crystals with EXAFS signal up to 14 Å−1 in k-space. Regarding the hexamer as a dimer of trimers where each trimer contains a Zn2+ ion, zinc adopted tetrahedral coordination in all R3-trimers, and octahedral coordination in T3-trimers. The coordination distances to zinc were refined from EXAFS with standard deviations below 0.01 Å for the first coordination sphere. In contrast to the distances determined from the medium resolution crystal structures, the XAS results were in good agreement with distances found in small molecules with similar coordination geometries, as well as in other high resolution insulin structures. As the radiation doses for the XRD experiments were significantly higher compared to that of XAS experiments, the single crystals were exposed to a higher degree of radiation damage affecting the zinc coordination in the T3-sites, in particular. Furthermore, XANES spectra for the zinc sites in T6 and R6 insulin were successfully calculated using finite difference methods, and the bond distances and angles were optimized from a quantitative XANES analysis. T6 insulin was furthermore crystallized with iron, nickel and copper, and their XRD crystal structures were solved to 1.90 Å, 1.50 Å and 1.45 Å resolution, respectively. As the affinity to iron is low, iron insulin crystals were grown in presence of small amounts of zinc. The two metal sites in the XRD structure thus contained respectively one Fe2+ and one Zn2+ ion, with respectively tetrahedral and octahedral coordination geometry. The metal sites in nickel and copper insulin were studied by XAS. Coordination distances were refined from EXAFS showing a very regular octahedral coordination of Ni2+, which was further verified by calculated XANES spectra. The coordination geometry of copper was, however, ambiguously determined in the XRD structure of copper insulin. The XANES spectrum revealed the presence of copper in oxidation state +I, which was caused by photoreduction of Cu2+ during the XAS experiments. Photoreductionwas not observed for nickel and zinc insulin. EXAFS analysis showed a tetrahedral coordination of copper, which was further supported by ab initio Hartree-Fock calculations, as well as calculated XANES spectra. Strontium ranelate, a pharmaceutical used in the treatment of osteoporosis, was synthesized and crystallized and the crystal structure of the nonahydrate was solved. The crystalswere highly sensitive to humidity and temperature and easily deteriorate upon dehydration. Four other hydrates were observed by in situ XRPD upon dehydration. These were identified as heptahydrate, pentahydrate, trihydrate and dihydrate by estimation of the water content by thermogravimetric analysis.  Bone tissue from dogs treated with strontiummalonate was studied using XAS. A new approach for analysing the X-ray absorption spectra resulted in a compositional model, from which the relative distribution of strontium in the different bone composites was estimated. Approximately 35–45 % of the strontium present was incorporated into calcium hydroxyapatite by substitution of some of the calcium ions, and at least 30 % was located at sites with a high structural disorder similar to Sr2+ in solution. The remaining strontium was absorbed in collagen. The relative distribution of strontium among the different composites seemed to be independent of treatment period and dose level.</p

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Characterization of Metalloproteins and Biomaterials by X-ray Absorption Spectroscopy and X-ray Diffraction

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Characterization of Metalloproteins and Biomaterials by X-ray Absorption Spectroscopy and X-ray Diffraction

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