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Volume 157, 2012
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Crowding effects on the small, fast-folding protein λ6–85

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Abstract

The microsecond folder λ6–85 is a small (9.2 kDa = 9200 amu) five helix bundle protein. We investigated the stability of λ6–85 in two different low-fluorescence crowding matrices: the large 70 kDa carbohydrate Ficoll 70, and the small 14 kDa thermophilic protein SubL. The same thermal stability of secondary structure was measured by circular dichroism in aqueous buffer, and at a crowding fraction φ = 15 ± 1% of Ficoll 70. Tryptophan fluorescence detection (probing a tertiary contact) yielded the same thermal stability in Ficoll, but 4 °C lower in aqueous buffer. Temperature-jump kinetics revealed that the relaxation rate, corrected for bulk viscosity, was very similar in Ficoll and in aqueous buffer. Thus viscosity, hydrodynamics and crowding seem to compensate one another. However, a new fast phase was observed in Ficoll, attributed to crowding-induced downhill folding. We also measured the stability of λ6–85 in φ = 14 ± 1% SubL, which acts as a smaller more rigid crowder. Significantly greater stabilization (7 to 13 °C depending on probe) was observed than in the Ficoll matrix. The results highlight the importance of crowding agent choice for studies of small, fast-folding proteins amenable to comparison with molecular dynamics simulations.

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Article information


Submitted
22 Jan 2012
Accepted
15 Feb 2012
First published
15 Feb 2012

Faraday Discuss., 2012,157, 451-462
Article type
Paper

Crowding effects on the small, fast-folding protein λ6–85

S. Denos, A. Dhar and M. Gruebele, Faraday Discuss., 2012, 157, 451
DOI: 10.1039/C2FD20009K

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