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Small pH and Salt Variations Radically Alter the Thermal Stability of Metal-Binding Domains in the Copper Transporter, Wilson Disease Protein
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Computational Life Science Center (CLiC), Umeå University,.
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2013 (Engelska)Ingår i: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 117, nr 42, s. 13038-13050Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Although strictly regulated, pH and solute concentrations in cells may exhibit temporal and spatial fluctuations. Here we study the effect of such changes on the stability, structure, and dynamics in vitro and in silico of a two-domain construct (WD56) of the fifth and sixth metal-binding domains of the copper transport protein, ATP7B (Wilson disease protein). We find that the thermal stability of WD56 is increased by 40 °C when increasing the pH from 5.0 to 7.5. In contrast, addition of salt at pH 7.2 decreases WD56 stability by up to 30 °C. In agreement with domain-domain coupling, fractional copper loading increases the stability of both domains. HSQC chemical shift changes demonstrate that, upon lowering the pH from 7.2 to 6, both His in WD6 as well as the second Cys of the copper site in each domain become protonated. MD simulations reveal increased domain-domain fluctuations at pH 6 and in the presence of high salt concentration, as compared to at pH 7 and low salt concentration. Thus, the surface charge distribution at high pH contributes favorably to overall WD56 stability. By introducing more positive charges by lowering the pH, or by diminishing charge-charge interactions by salt, fluctuations among the domains are increased and thereby overall stability is reduced. Copper transfer activity also depends on pH: delivery of copper from chaperone Atox1 to WD56 is more efficient at pH 7.2 than at pH 6 by a factor of 30. It appears that WD56 is an example where the free energy landscapes for folding and function are linked via structural stability.

Ort, förlag, år, upplaga, sidor
American Chemical Society (ACS), 2013. Vol. 117, nr 42, s. 13038-13050
Nationell ämneskategori
Kemi
Identifikatorer
URN: urn:nbn:se:umu:diva-71579DOI: 10.1021/jp402415yISI: 000326259800038PubMedID: 23675861OAI: oai:DiVA.org:umu-71579DiVA, id: diva2:625128
Tillgänglig från: 2013-06-04 Skapad: 2013-06-04 Senast uppdaterad: 2018-06-08Bibliografiskt granskad
Ingår i avhandling
1. Human copper ion transfer: from metal chaperone to target transporter domain
Öppna denna publikation i ny flik eller fönster >>Human copper ion transfer: from metal chaperone to target transporter domain
2015 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Many processes in living systems occur through transient interactions among proteins. Those interactions are often weak and are driven by small changes in free energy. Due to the short-living nature of these interactions, our knowledge about driving forces, dynamics and structures of these types of protein-protein heterocomplexes are though limited. This is especially important for cellular copper (Cu) trafficking:

Copper ions are essential for all eukaryotes and most bacteria. As a cofactor in many enzymes, copper is especially vital in respiration or detoxification. Since the same features that make copper useful also make it toxic, it needs to be controlled tightly. Additionally, in the reducing environment of the cytosol, Cu is present as insoluble Cu(I). To circumvent both toxicity and solubility issues, a system has evolved where copper is comforted by certain copper binding proteins, so-called Cu-chaperones. They transiently interact with each other to distribute the Cu atoms in a cell. In humans, one of them is Atox1. It binds copper with a binding site containing two thiol residues and transfers it to other binding sites, mostly those of a copper pump, ATP7B (also known as Wilsons disease protein).

My work was aimed at understanding copper-mediated protein-protein interactions on a molecular and mechanistic level. Which amino acids interact with the metal? Which forces drive the transfer from one protein to the other? Using biophysical and biochemical methods such as chromatography and calorimetry on wild type and point-mutated proteins in vitro, we found that the copper is transferred via a dynamic intermediate complex that keeps the system flexible while shielding the copper against other interactions.

Although similar transfer interactions can be observed in other organisms, and many conclusions in the copper field are drawn from bacterial and yeast analogs, we believe that it is important to investigate human proteins, too. Not only is their regulation different, but also only in humans we find the diseases linked to the proteins: Copper level regulation diseases are to be named first, but atypical copper levels have also been linked to tumors and amyloid dispositions. In summary, my observations and conclusions are of basic research character and can be of importance for both general copper and human medicinal research.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå Universitet, 2015. s. 96
Nyckelord
copper homeostasis, copper chaperone, Atox1, ATP7B, Wilson disease protein, metal transport, size exclusion chromatography, thermodynamics, isothermal calorimetry
Nationell ämneskategori
Oorganisk kemi Biofysik Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:umu:diva-100511 (URN)978-91-7601-203-1 (ISBN)
Disputation
2015-03-27, Lilla Hörsalen, KBC KB3A9, Umeå Universitet, Umeå, 10:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2015-03-06 Skapad: 2015-03-03 Senast uppdaterad: 2018-06-07Bibliografiskt granskad

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Nilsson, LinaÅdén, JörgenNiemiec, Moritz S.Nam, KwanghoWittung-Stafshede, Pernilla

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Nilsson, LinaÅdén, JörgenNiemiec, Moritz S.Nam, KwanghoWittung-Stafshede, Pernilla
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Journal of Physical Chemistry B
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