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Stabilization of tryptophan hydroxylase 2 by l‐phenylalanine‐induced dimerization
Tryptophan hydroxylase 2 (TPH2) catalyses the initial and rate‐limiting step in the biosynthesis of serotonin, which is associated with a variety of disorders such as depression, obsessive compulsive disorder, and schizophrenia. Full‐length TPH2 is poorly characterized due to low purification quanti...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5055035/ https://www.ncbi.nlm.nih.gov/pubmed/27761358 http://dx.doi.org/10.1002/2211-5463.12100 |
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author | Tidemand, Kasper D. Christensen, Hans E. M. Hoeck, Niclas Harris, Pernille Boesen, Jane Peters, Günther H. |
author_facet | Tidemand, Kasper D. Christensen, Hans E. M. Hoeck, Niclas Harris, Pernille Boesen, Jane Peters, Günther H. |
author_sort | Tidemand, Kasper D. |
collection | PubMed |
description | Tryptophan hydroxylase 2 (TPH2) catalyses the initial and rate‐limiting step in the biosynthesis of serotonin, which is associated with a variety of disorders such as depression, obsessive compulsive disorder, and schizophrenia. Full‐length TPH2 is poorly characterized due to low purification quantities caused by its inherent instability. Three truncated variants of human TPH2 (rch TPH2; regulatory and catalytic domain, NΔ47‐rch TPH2; truncation of 47 residues in the N terminus of rch TPH2, and ch TPH2; catalytic domain) were expressed, purified, and examined for changes in transition temperature, inactivation rate, and oligomeric state. ch TPH2 displayed 14‐ and 11‐fold higher half‐lives compared to rch TPH2 and NΔ47‐rch TPH2, respectively. Differential scanning calorimetry experiments demonstrated that this is caused by premature unfolding of the less stable regulatory domain. By differential scanning fluorimetry, the unfolding transitions of rch TPH2 and NΔ47‐rch TPH2 are found to shift from polyphasic to apparent two‐state by the addition of l‐Trp or l‐Phe. Analytical gel filtration revealed that rch TPH2 and NΔ47‐rch TPH2 reside in a monomer–dimer equilibrium which is significantly shifted toward dimer in the presence of l‐Phe. The dimerizing effect induced by l‐Phe is accompanied by a stabilizing effect, which resulted in a threefold increase in half‐lives of rch TPH2 and NΔ47‐rch TPH2. Addition of l‐Phe to the purification buffer significantly increases the purification yields, which will facilitate characterization of hTPH2. |
format | Online Article Text |
id | pubmed-5055035 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-50550352016-10-19 Stabilization of tryptophan hydroxylase 2 by l‐phenylalanine‐induced dimerization Tidemand, Kasper D. Christensen, Hans E. M. Hoeck, Niclas Harris, Pernille Boesen, Jane Peters, Günther H. FEBS Open Bio Research Articles Tryptophan hydroxylase 2 (TPH2) catalyses the initial and rate‐limiting step in the biosynthesis of serotonin, which is associated with a variety of disorders such as depression, obsessive compulsive disorder, and schizophrenia. Full‐length TPH2 is poorly characterized due to low purification quantities caused by its inherent instability. Three truncated variants of human TPH2 (rch TPH2; regulatory and catalytic domain, NΔ47‐rch TPH2; truncation of 47 residues in the N terminus of rch TPH2, and ch TPH2; catalytic domain) were expressed, purified, and examined for changes in transition temperature, inactivation rate, and oligomeric state. ch TPH2 displayed 14‐ and 11‐fold higher half‐lives compared to rch TPH2 and NΔ47‐rch TPH2, respectively. Differential scanning calorimetry experiments demonstrated that this is caused by premature unfolding of the less stable regulatory domain. By differential scanning fluorimetry, the unfolding transitions of rch TPH2 and NΔ47‐rch TPH2 are found to shift from polyphasic to apparent two‐state by the addition of l‐Trp or l‐Phe. Analytical gel filtration revealed that rch TPH2 and NΔ47‐rch TPH2 reside in a monomer–dimer equilibrium which is significantly shifted toward dimer in the presence of l‐Phe. The dimerizing effect induced by l‐Phe is accompanied by a stabilizing effect, which resulted in a threefold increase in half‐lives of rch TPH2 and NΔ47‐rch TPH2. Addition of l‐Phe to the purification buffer significantly increases the purification yields, which will facilitate characterization of hTPH2. John Wiley and Sons Inc. 2016-08-22 /pmc/articles/PMC5055035/ /pubmed/27761358 http://dx.doi.org/10.1002/2211-5463.12100 Text en © 2016 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution (http://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Articles Tidemand, Kasper D. Christensen, Hans E. M. Hoeck, Niclas Harris, Pernille Boesen, Jane Peters, Günther H. Stabilization of tryptophan hydroxylase 2 by l‐phenylalanine‐induced dimerization |
title | Stabilization of tryptophan hydroxylase 2 by l‐phenylalanine‐induced dimerization |
title_full | Stabilization of tryptophan hydroxylase 2 by l‐phenylalanine‐induced dimerization |
title_fullStr | Stabilization of tryptophan hydroxylase 2 by l‐phenylalanine‐induced dimerization |
title_full_unstemmed | Stabilization of tryptophan hydroxylase 2 by l‐phenylalanine‐induced dimerization |
title_short | Stabilization of tryptophan hydroxylase 2 by l‐phenylalanine‐induced dimerization |
title_sort | stabilization of tryptophan hydroxylase 2 by l‐phenylalanine‐induced dimerization |
topic | Research Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5055035/ https://www.ncbi.nlm.nih.gov/pubmed/27761358 http://dx.doi.org/10.1002/2211-5463.12100 |
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