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High-resolution probing of local conformational changes in proteins by the use of multiple labeling: Unfolding and self-assembly of human carbonic anhydrase II monitored by spin, fluorescent, and chemical reactivity probes
Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.ORCID-id: 0000-0001-5827-3587
Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi.
Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.ORCID-id: 0000-0002-7642-9263
Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
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2001 (Engelska)Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 80, nr 6, s. 2867-2885Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Two different spin labels, N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide (IPSL) and (1-oxyl-2.2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate (MTSSL), and two different fluorescent labels 5-((((2-iodoacetyl)amino)ethyl)amino)naphtalene-1 -sulfonic acid (IAEDANS) and 6-bromoacetyl-2-dimetylaminonaphtalene (BADAN), were attached to the introduced C79 in human carbonic anhydrase (HCA II) to probe local structural changes upon unfolding and aggregation, HCA II unfolds in a multi-step manner with an intermediate state populated between the native and unfolded states. The spin label IPSL and the fluorescent label IAEDANS reported on a substantial change in mobility and polarity at both unfolding transitions at a distance of 7.4-11.2 Angstrom from the backbone of position 79. The shorter and less flexible labels BADAN and MTSSL revealed less pronounced spectroscopic changes in the native-to-intermediate transition, 6.6-9.0 Angstrom from the backbone. At intermediate guanidine (Gu)-HCl concentrations the occurrence of soluble but irreversibly aggregated oligomeric protein was identified from refolding experiments. At similar to1 M Gu-HCl the aggregation was found to be essentially complete. The size and structure of the aggregates could be varied by changing the protein concentration. EPR measurements and line-shape simulations together with fluorescence lifetime and anisotropy measurements provided a picture of the self-assembled protein as a disordered protein structure with a representation of both compact as well as dynamic and polar environments at the site of the molecular labels. This suggests that a partially folded intermediate of HCA II self-assembles by both local unfolding and intermolecular docking of the intermediates vicinal to position 79. The aggregates were determined to be 40-90 Angstrom in diameter depending on the experimental conditions and spectroscopic technique used.

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2001. Vol. 80, nr 6, s. 2867-2885
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URN: urn:nbn:se:liu:diva-49242OAI: oai:DiVA.org:liu-49242DiVA, id: diva2:270138
Tillgänglig från: 2009-10-11 Skapad: 2009-10-11 Senast uppdaterad: 2018-04-25

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Hammarström, PerOwenius, RikardMårtensson, Lars-GöranCarlsson, Uno

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Hammarström, PerOwenius, RikardMårtensson, Lars-GöranCarlsson, Uno
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