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Cutting Off Functional Loops from Homodimeric Enzyme Superoxide Dismutase 1 (SOD1) Leaves Monomeric beta-Barrels
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 38, 33070-33083 p.Article in journal (Refereed) Published
Abstract [en]

Demetallation of the homodimeric enzyme Cu/Zn-superoxide dismutase (SOD1) is known to unleash pronounced dynamic motions in the long active-site loops that comprise almost a third of the folded structure. The resulting apo species, which shows increased propensity to aggregate, stands out as the prime disease precursor in amyotrophic lateral sclerosis (ALS). Even so, the detailed structural properties of the apoSOD1 framework have remained elusive and controversial. In this study, we examine the structural interplay between the central apoSOD1 barrel and the active-site loops by simply cutting them off; loops IV and VII were substituted with short Gly-Ala-Gly linkers. The results show that loop removal breaks the dimer interface and leads to soluble, monomeric beta-barrels with high structural integrity. NMR-detected nuclear Overhauser effects are found between all of the constituent beta-strands, confirming ordered interactions across the whole barrel. Moreover, the breathing motions of the SOD1 barrel are overall insensitive to loop removal and yield hydrogen/deuterium protection factors typical for cooperatively folded proteins (i.e. the active-site loops act as a bolt-on domain with little dynamic influence on its structural foundation). The sole exceptions are the relatively low protection factors in beta-strand 5 and the turn around Gly-93, a hot spot for ALS-provoking mutations, which decrease even further upon loop removal. Taken together, these data suggest that the cytotoxic function of apoSOD1 does not emerge from its folded ground state but from a high energy intermediate or even from the denatured ensemble.

Place, publisher, year, edition, pages
2011. Vol. 286, no 38, 33070-33083 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-66524DOI: 10.1074/jbc.M111.251223ISI: 000294968800026OAI: oai:DiVA.org:su-66524DiVA: diva2:469835
Note

authorCount :4;

Available from: 2011-12-27 Created: 2011-12-20 Last updated: 2017-12-08Bibliographically approved
In thesis
1. Protein folding without loops and charges
Open this publication in new window or tab >>Protein folding without loops and charges
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Going down the folding funnel, proteins may sample a wide variety of conformations, some being outright detrimental to the organism. Yet, the vast majority of polypeptide molecules avoid such pitfalls. Not only do they reach the native minimum of the energy landscape; they do so via blazingly fast, biased, routes. This specificity and speed is remarkable, as the surrounding solution is filled to the brim with other molecules that could potentially interact with the protein and in doing so stabilise non-native, potentially toxic, conformations. How such incidents are avoided while maintaining native structure and function is not understood. 

This doctoral thesis argues that protein structure and function can be separated in the folding code of natural protein sequences by use of multiple partly uncoupled factors that act in a concerted fashion. More specifically, we demonstrate that: i) Evolutionarily conserved functional and regulatory elements can be excised from a present day protein, leaving behind an independently folded protein scaffold. This suggests that the dichotomy between functional and structural elements can be preserved during the course of protein evolution. ii) The ubiquitous charges on soluble protein surfaces are not required for protein folding in biologically relevant timescales, but are critical to intermolecular interaction. Monomer folding can be driven by hydrophobicity and hydrogen bonding alone, while functional and structural intermolecular interaction depends on the relative positions of charges that are not required for the native bias inherent to the folding mechanism. It is possible that such uncoupling reduces the probability of evolutionary clashes between fold and function. Without such a balancing mechanism, functional evolution might pull the carpet from under the feet of structural integrity, and vice versa. These findings have implications for both de novo protein design and the molecular mechanisms behind diseases caused by protein misfolding.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2012. 70 p.
Keyword
protein folding, folding cooperativity, protein aggregation, protein charges, protein engineering
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-80512 (URN)978-91-7447-545-6 (ISBN)
Public defence
2012-10-26, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Available from: 2012-10-04 Created: 2012-09-24 Last updated: 2012-10-01Bibliographically approved

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