Abstract:
:Examination of the first crystal structures of proteins from a halophilic organism suggests that an abundance of acidic residues distributed over the protein surface is a key determinant of adaptation to high-salt conditions. Although one extant theory suggests that acidic residues are favored because of their superior water-binding capacity, it is clear that extensive repulsive electrostatic interactions will also be present in such proteins at physiological pH. To investigate the magnitude and importance of such electrostatic interactions, we conducted a theoretical analysis of their contributions to the salt and pH-dependence of stability of two halophilic proteins. Our approach centers on use of the Poisson-Boltzmann equation of classical electrostatics, applied at an atomic level of detail to crystal structures of the proteins. We first show that in using the method, it is important to account for the fact that the dielectric constant of water decreases at high salt concentrations, in order to reproduce experimental changes in pKa values of small acids and bases. We then conduct a comparison of salt and pH effects on the stability of 2Fe-2S ferredoxins from the halophile Haloarcula marismortui and the non-halophile anabaena. In both proteins, substantial upward shifts in pKa accompany protein folding, though shifts are considerably larger, on average, in the halophile. Upward shifts for basic residues occur because of favorable salt-bridge interactions, whilst upward shifts for acidic residues result from unfavorable electrostatic interactions with other acidic groups. Our calculations suggest that at pH 7 the stability of the halophilic protein is decreased by 18.2 kcal/mol on lowering the salt concentration from 5 M to 100 mM, a result that is in line with the fact that halophilic proteins generally unfold at low salt concentrations. For comparison, the non-halophilic ferredoxin is calculated to be destabilized by only 5.1 kcal/mol over the same range. Analysis of the pH stability curve suggests that lowering the pH should increase the intrinsic stability of the halophilic protein at low salt concentrations, although in practice this is not observed because of aggregation effects. We report the results of a similar analysis carried out on the tetrameric malate dehydrogenase from H. marismortui. In this case, we investigated the salt and pH dependence of the various monomer-monomer interactions present in the tetramer. All monomer-monomer interactions are found to make substantial contributions to the salt-dependence of stability of the tetramer. Excellent agreement is obtained between our calculated results for the stability of the tetramer and experimental results. In particular, the finding that at 4 M NaCl, the tetramer is stable only between pH 4.8 and 10 is accurately reproduced. Taken together, our results suggest that repulsive electrostatic interactions between acidic residues are a major factor in the destabilization of halophilic proteins in low-salt conditions, and that these interactions remain destabilizing even at high salt concentrations. As a consequence, the role of acidic residues in halophilic proteins may be more to prevent aggregation than to make a positive contribution to intrinsic protein stability.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Elcock AH,McCammon JAdoi
10.1006/jmbi.1998.1904subject
Has Abstractpub_date
1998-07-24 00:00:00pages
731-48issue
4eissn
0022-2836issn
1089-8638pii
S0022-2836(98)91904-0journal_volume
280pub_type
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