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Direct Observation of Protein Unfolded State Compaction in the Presence of Macromolecular Crowding
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.
2013 (Engelska)Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, nr 3, s. 694-704Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Proteins fold and function in cellular environments that are crowded with other macromolecules. As a consequence of excluded volume effects, compact folded states of proteins should be indirectly stabilized due to destabilization of extended unfolded conformations. Here, we assess the role of excluded volume in terms of protein stability, structural dimensions and folding dynamics using a sugar-based crowding agent, dextran 20, and the small ribosomal protein S16 as a model system. To specifically address dimensions, we labeled the protein with BODIPY at two positions and measured Trp-BODIPY distances under different conditions. As expected, we found that dextran 20 (200 mg/ml) stabilized the variants against urea-induced unfolding. At conditions where the protein is unfolded, Förster resonance energy transfer measurements reveal that in the presence of dextran, the unfolded ensemble is more compact and there is residual structure left as probed by far-ultraviolet circular dichroism. In the presence of a crowding agent, folding rates are faster in the two-state regime, and at low denaturant concentrations, a kinetic intermediate is favored. Our study provides direct evidence for protein unfolded-state compaction in the presence of macromolecular crowding along with its energetic and kinetic consequences.

Ort, förlag, år, upplaga, sidor
Cell Press , 2013. Vol. 104, nr 3, s. 694-704
Nationell ämneskategori
Biofysik
Identifikatorer
URN: urn:nbn:se:umu:diva-65815DOI: 10.1016/j.bpj.2012.12.020ISI: 000314619900020OAI: oai:DiVA.org:umu-65815DiVA, id: diva2:604727
Tillgänglig från: 2013-02-12 Skapad: 2013-02-12 Senast uppdaterad: 2018-06-08Bibliografiskt granskad
Ingår i avhandling
1. Electronic Energy Migration/Transfer as a Tool to Explore Biomacromolecular Structures
Öppna denna publikation i ny flik eller fönster >>Electronic Energy Migration/Transfer as a Tool to Explore Biomacromolecular Structures
2014 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Fluorescence-based techniques are widely used in bioscience, offering a high sensitivity and versatility. In this work, fluorescence electronic energy migration/ transfer is applied to measure intramolecular distances in two types of systems and under various conditions.

The main part of the thesis utilizes the process of donor-acceptor energy transfer to probe distances within the ribosomal protein S16. Proteins are essential to all organisms. Therefore, it is of great interest to study protein structure and function in order to understand and prevent protein malfunction. Moreover, it is also important to try to study the proteins in an environment which resembles its natural habitat. Here two protein homologs were investigated; S16Thermo and S16Meso, isolated from a hyperthemophilic bacterium and a mesophilic bacterium, respectively. It was concluded that the chemically induced unfolded state ensemble of S16Thermo is more compact than the corresponding ensemble of S16Meso. This unfolded state compaction may be one reason for the increased thermal stability of S16Thermo as compared to S16Meso.

The unfolded state of S16 was also studied under highly crowded conditions, mimicking the environment found in cells. It appears that a high degree of crowding, induced by 200 mg/mL dextran 20, forces the unfolded state ensemble of S16Thermo to become even more compact. Further, intramolecular distances in the folded state of five S16 mutants were investigated upon increasing amounts of dextran 20. We found that the probed distances in S16Thermo are unaffected by increasing degree of crowding. However, S16Meso shows decreasing intramolecular distances for all three studied variants, up to 100 mg/mL dextran. At higher concentrations, the change in distance becomes anisotropic. This suggests that marginally stable proteins like s16Meso may respond to macromolecular crowding by fine-tuning its structure. More stable proteins like S16Thermo however, show no structural change upon increasing degree of crowding.

We also investigated the possibility of local specific interactions between the protein and crowding agent, by means of fluorescence quenching experiments. Upon increasing amounts of a tyrosine labelled dextran, a diverse pattern of fluorescence quantum yield and lifetime suggests that specific, local protein-crowder interactions may occur.

In a second studied system, electronic energy migration between two donor-groups, separated by a rigid steroid, was studied by two-photon excitation depolarization experiments. Data were analysed by using recent advances, based on the extended Förster theory, which yield a reasonable value of the distance between the two interacting donor-groups. To the best of our knowledge, this is the first quantitative analysis of energy migration data, obtained from two-photon excited fluorescence. 

Ort, förlag, år, upplaga, sidor
Umeå: Umeå Universitet, 2014. s. 55
Nyckelord
Fluorescence, electronic energy transfer, two-photon excitation, small ribosomal protein S16, macromolecular crowding, dextran 20
Nationell ämneskategori
Fysikalisk kemi
Forskningsämne
fysikalisk kemi
Identifikatorer
urn:nbn:se:umu:diva-86794 (URN)978-91-7601-021-1 (ISBN)
Disputation
2014-04-04, KBC-huset, KB3B1, Umeå universitet, Linnaeus Väg, Umeå, 09:00 (Engelska)
Opponent
Handledare
Forskningsfinansiär
Vetenskapsrådet, 550021104
Tillgänglig från: 2014-03-12 Skapad: 2014-03-11 Senast uppdaterad: 2018-06-08Bibliografiskt granskad

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Mikaelsson, ThereseÅdén, JörgenJohansson, Lennart B-ÅWittung-Stafshede, Pernilla

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