Boesen, Jane

Tryptophanhydroxylase (TPH) katalyserer det første og hastighedsbestemmende trin i biosyntesen af serotonin (5-hydroxytryptamin). Serotonin er involveret i mange fysiologiske funktioner, såsom appetit og søvnrytme, samt en lang række af psykiske sygdomme, som depression og obsessiv-kompulsiv lidelse (OCD). Karakterisering af TPH og bestemmelse af enzymets regulering og katalytiske mekanisme er derfor vigtig for forståelsen af serotonin.  I dette projekt er varianter af human TPH isoform 1 (hTPH1) and human TPH isoform 2 (hTPH2) blevet studeret. Det overordnede mål var at oprense fuldlængde hTPH1. På baggrund af tidligere resultater blev hTPH1 oprenset med anvendelse af detergent i oprensningsmetoden. Efter inkubering af hTPH1 prøven med 0.1 % of n-dodecyl-β-D-maltopyranosid (DDM) kan proteinet binde til anionbytterkolonnen. Den del af hTPH1 som binder til kolonnen eluerer over et stort område i anionbytningen, hvilket understreger at protein stadig findes på forskellige oligomere former. Dette ses også i gelfiltreringen. Varianter af både hTPH1 og hTPH2 indeholdende det regulatoriske domæne eller dele af dette blev fremstillet og testet for udtrykkelse i Escherichia coli samt opløselighed. Fra disse resultater blev det observeret at en ændring af aminosyre-sekvensen i det regulatoriske domæne, enten i form af punktmutation eller ved trunkering i den N-terminale ende, gør at proteinet er uopløseligt.  Det regulatoriske domæne af human TPH1 (rhTPH1) samt to fusionsproteiner af rhTPH1 fusioneret til henholdsvis det grønt-fluorescerende-protein (GFP) i den C-terminale ende og glutathione S-transferase (GST) i den N-terminale ende blev udtrykt i opløselig form. Fra oprensningsforsøg blev det observeret at en høj salt-koncentration er nødvendig for stabilisering af proteinet. GST-rhTPH1 varianten blev oprenset ved anvendelse af affinitetskromatografi efterfulgt af gelfiltrering til høj renhed og et udbytte på 40 mg/l kultur. Den oprensede GST-rhTPH1 variant eksisterer som en dimer i opløsning på grund af dimerisering af GST. GST blev succesfuldt kløvet fra fusionsproteinet med Faktor Xa og rhTPH1 adskilt fra GST efter kløvning.  Dette studie omhandler også karakterisering af tre hTPH varianter; Det katalytiske domæne af både hTPH1 (chTPH1) og hTPH2 (chTPH2) samt det katalytiske og tetrameriseringsdomænet af hTPH2 (cthTPH2). De kinetiske parametre for chTPH1 blev bestemt og sammenlignet med værdier for chTPH2. Der ses en stor forskel mellem de to isoformer og substratinhibering af tryptophan observeres for chTPH1, men ikke for chTPH2.  Massespektrometrisk analyse af chTPH1 viste at prøven indeholder to proteiner: chTPH1 samt et andet protein, som ikke kunne observeres i hverken oprensningen af proteinet eller ved elektroforese. Yderligere ses chTPH1 at være mættet med Fe, mens chTPH2 er heterogen med hensyn til Fe. chTPH2 binder tryptophan og tetrahydrobiopterin. Tandem-massespektrometrisk karakterisering af cthTPH2 viser at varianten er en tetramer.  Krystallisationsforsøg af chTPH1 blev udført til bestemmelse af krystallisationsbetingelser for chTPH1 med og uden ligander. Krystallionsbetingelserne blev fundet, men resulterede i meget små krystaller. Der blev opsamlet et datasæt til 4 Å af chTPH1 uden ligander og strukturen blev løst. Strukturen blev forfinet og sammenlignet med strukturen af det katalytiske domæne af hTPH1 krystalliseret med BH2. En strukturændring ses fra aminosyrerest 125 til 130. Dette er den første struktur af chTPH1 uden tilstedeværelsen af substrater eller inhibitorer.Tryptophan hydroxylase (TPH) catalyzes the first and rate-limiting step in the biosynthesis of the neurotransmitter and hormone serotonin (5-hydroxytryptamine). Serotonin is involved in many physiological functions, such as appetite and sleep rhythm, as well as a wide range of psychiatric disorders such as depression and obsessive-compulsive disorder (OCD). Characterization of TPH and elucidation of the enzymes regulation and catalytic mechanism is therefore vital to our understanding of the serotonin balance.  This study concerns variants of both human TPH isoform 1 (hTPH1) and human TPH isoform 2 (h PH2). The main goal was to purify full-length hTPH1. Based on earlier results, hTPH1 was purified using detergent in the purification methods. After incubation of the hTPH1 sample with 0.1 % of n-dodecyl-β-D-maltopyranoside (DDM) the protein binds to the anion exchange column and elutes over a large area in the anion exchange, indicating that the protein still exists in different oligomer forms. This was also observed in the gel filtration. Variants of both hTPH1 and hTPH2 containing the regulatory domain or parts of it were constructed and tested for expression in Escherichia coli as well as solubility. It was observed that changes in the amino acid sequence of the regulatory domain by point mutations or truncations in the N-terminal had a huge impact on the solubility of the protein and caused the protein to be insoluble.  The regulatory domain of human TPH1 (rhTPH1), and two fusion proteins of rhTPH1 fused to the green fluorescent protein (GFP) in the C-terminal and the glutathione S-transferase (GST) in the N-terminal, respectively, were expressed in a soluble form. The purification trials of the variants containing the regulatory domain showed that a high salt concentration was necessary to stabilize the variant. The GST-rhTPH1 variant could be purified using affinity chromatography followed by gel filtration with high purity and a yield of 40 mg/l culture. The purified GST-rhTPH1 exists as a dimer in solution due to dimerization of GST. The GST could be cleaved successfully from the fusion protein using Factor Xa and rhTPH1 was successfully purified from GST after cleavage.  Characterization was performed on the three hTPH variants: The catalytic domain of both hTPH1 (chTPH1) and hTPH2 (chTPH2) as well as the catalytic and tetramerization domain of hTPH2 (cthTPH2). The kinetic parameters of chTPH1 was determined and compared with parameters of chTPH2. Large differences were observed between the two isoforms and tryptophan inhibition was observed for chTPH1 but not for chTPH2.  Mass spectrometric analysis of chTPH1 shows that the sample contains two species: chTPH1 and another species, which could not be seen during purification or electrophoresis of the chTPH1 sample. Additionally, chTPH1 shows to be fully loaded with iron, whereas chTPH2 showed to be heterogeneous with respect to Fe. chTPH2 binds both tryptophan and tetrahydrobiopterin individually. Also characterization of cthTPH2 by tandem mass spectrometry shows that the variant is a tetramer.  Crystallization of chTPH1 was achieved both without substrate and with bound substrate (tryptophan and pterin) but resulted in very small crystals. A data set of the variant without bound substrate was collected to 4 Å and the structure was solved by molecular replacement. The structure was refined to an Rfree of 33.5 % and the overall structure is compared to the overall structure of the catalytic domain of hTPH1 co-crystallized with BH2. A structural change in the residues 125 to 130 is observed. This is the first structure of chTPH1 without any substrates or inhibitors.</p

Online Research Database In Technology

Cloning, expression, purification and characterization of tryptophan hydroxylase variants

http://orbit.dtu.dk/ws/files/10002832/Jane%20Boesen.pdf

Cloning, expression, purification and characterization of tryptophan hydroxylase variants

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Online Research Database In Technology