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Structural determinants of protein stabilization by solutes - the importance of the hairpin loop in rubredoxins

Structural determinants of protein stabilization by solutes - the importance of the hairpin loop in rubredoxins
Structural determinants of protein stabilization by solutes - the importance of the hairpin loop in rubredoxins
Despite their high sequence homology, rubredoxins from Desulfovibrio gigas and D. desulfuricans are stabilized to very different extents by compatible solutes such as diglycerol phosphate, the major osmolyte in the hyperthermophilic archaeon Archaeoglobus fulgidus [Lamosa P, Burke A, Peist R, Huber R, Liu M Y, Silva G, Rodrigues-Pousada C, LeGall J, Maycock C and Santos H (2000) Appl Environ Microbiol 66, 1974-1979]. The principal structural difference between these two proteins is the absence of the hairpin loop in the rubredoxin from D. desulfuricans. Therefore, mutants of D. gigas rubredoxin bearing deletions in the loop region were constructed to investigate the importance of this structural feature on protein intrinsic stability, as well as on its capacity to undergo stabilization by compatible solutes. The three-dimensional structure of the mutant bearing the largest deletion, Delta17\29, was determined by H-1-NMR, demonstrating that, despite the drastic deletion, the main structural features were preserved. The dependence of the NH chemical shifts on temperature and solute concentration (diglycerol phosphate or mannosylglycerate) provide evidence of subtle conformational changes induced by the solute. The kinetic stability (as assessed from the absorption decay at 494 nm) of six mutant rubredoxins was determined at 90 degreesC and the stabilizing effect exerted by both solutes was assessed. The extent of protection conferred by each solute was highly dependent on the specific mutant examined: while the half-life for iron release in the wild-type D. gigas rubredoxin increased threefold in the presence of 0.1 M diglycerol phosphate, mutant Delta23\29 was destabilized. This study provides evidence for solute-induced compaction of the protein structure and occurrence of weak, specific interactions with the protein surface. The relevance of these findings to our understanding of the molecular basis for protein stabilization is discussed.
compatible solutes, hairpin structure, nmr rubredoxin, thermostability clostridium-pasteurianum rubredoxin, desulfovibrio-gigas rubredoxin, pyrococcus-furiosus, diglycerol phosphate, angstrom resolution, thermotoga-maritima, thermal-stability, crystal-structure, nmr, hyperthermophile
1742-464X
999-1011
Pais, Tiago M.
a68c93b5-3ded-4f9e-be41-a3a9f0862568
Lamosa, Pedro
9c2fdf0a-8bc3-4ed6-b295-f26bc448bfd7
Dos Santos, Wagner
73c7a82e-6475-4c53-9c28-042603259492
LeGall, Jean
8b9fe4c9-d915-4c71-a05d-db0a771ed393
Turner, David L.
6c4c1ccb-a6f2-47cf-944e-e69269198a29
Santos, Helena
1c7cb520-ed15-41e7-b381-467ca075ed30
Pais, Tiago M.
a68c93b5-3ded-4f9e-be41-a3a9f0862568
Lamosa, Pedro
9c2fdf0a-8bc3-4ed6-b295-f26bc448bfd7
Dos Santos, Wagner
73c7a82e-6475-4c53-9c28-042603259492
LeGall, Jean
8b9fe4c9-d915-4c71-a05d-db0a771ed393
Turner, David L.
6c4c1ccb-a6f2-47cf-944e-e69269198a29
Santos, Helena
1c7cb520-ed15-41e7-b381-467ca075ed30

Pais, Tiago M., Lamosa, Pedro, Dos Santos, Wagner, LeGall, Jean, Turner, David L. and Santos, Helena (2005) Structural determinants of protein stabilization by solutes - the importance of the hairpin loop in rubredoxins. Febs Journal, 272 (4), 999-1011. (doi:10.1111/j.1742-4658.2004.04534.x).

Record type: Article

Abstract

Despite their high sequence homology, rubredoxins from Desulfovibrio gigas and D. desulfuricans are stabilized to very different extents by compatible solutes such as diglycerol phosphate, the major osmolyte in the hyperthermophilic archaeon Archaeoglobus fulgidus [Lamosa P, Burke A, Peist R, Huber R, Liu M Y, Silva G, Rodrigues-Pousada C, LeGall J, Maycock C and Santos H (2000) Appl Environ Microbiol 66, 1974-1979]. The principal structural difference between these two proteins is the absence of the hairpin loop in the rubredoxin from D. desulfuricans. Therefore, mutants of D. gigas rubredoxin bearing deletions in the loop region were constructed to investigate the importance of this structural feature on protein intrinsic stability, as well as on its capacity to undergo stabilization by compatible solutes. The three-dimensional structure of the mutant bearing the largest deletion, Delta17\29, was determined by H-1-NMR, demonstrating that, despite the drastic deletion, the main structural features were preserved. The dependence of the NH chemical shifts on temperature and solute concentration (diglycerol phosphate or mannosylglycerate) provide evidence of subtle conformational changes induced by the solute. The kinetic stability (as assessed from the absorption decay at 494 nm) of six mutant rubredoxins was determined at 90 degreesC and the stabilizing effect exerted by both solutes was assessed. The extent of protection conferred by each solute was highly dependent on the specific mutant examined: while the half-life for iron release in the wild-type D. gigas rubredoxin increased threefold in the presence of 0.1 M diglycerol phosphate, mutant Delta23\29 was destabilized. This study provides evidence for solute-induced compaction of the protein structure and occurrence of weak, specific interactions with the protein surface. The relevance of these findings to our understanding of the molecular basis for protein stabilization is discussed.

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More information

Published date: 1 February 2005
Keywords: compatible solutes, hairpin structure, nmr rubredoxin, thermostability clostridium-pasteurianum rubredoxin, desulfovibrio-gigas rubredoxin, pyrococcus-furiosus, diglycerol phosphate, angstrom resolution, thermotoga-maritima, thermal-stability, crystal-structure, nmr, hyperthermophile

Identifiers

Local EPrints ID: 20879
URI: http://eprints.soton.ac.uk/id/eprint/20879
ISSN: 1742-464X
PURE UUID: 7ebbab4b-e88c-4982-85bd-5c576925d293

Catalogue record

Date deposited: 02 Mar 2006
Last modified: 15 Jul 2019 19:25

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