Salt effects on hydrophobic interaction and charge screening in the folding of a negatively charged peptide to a coiled coil (leucine zipper).
The stability of a coiled coil or leucine zipper is controlled by hydrophobic interactions and electrostatic forces between the constituent helices. We have designed a 30-residue peptide with the repeating seven-residue pattern of a coiled coil, (abcdefg)n, and with Glu in positions e and g of each heptad. The glutamate side chains prevented folding at pH values above 6 because of electrostatic repulsion across the helix dimer interface as well as within the individual helices. Protonation of the carboxylates changed the conformation from a random coil monomer to a coiled coil dimer. Folding at alkaline pH where the peptide had a net charge of -7e was promoted by the addition of salts. The nature of the charge screening cation was less important than that of the anion. The high salt concentrations (>1 M) necessary to induce folding indicated that the salt-induced folding resulted from alterations in the protein-water interaction. Folding was promoted by the kosmotropic anions sulfate and fluoride and to a lesser extent by the weak kosmotrope formate, whereas chloride and the strong chaotrope perchlorate were ineffective. Kosmotropes are excluded from the protein surface, which is preferentially hydrated, and this promotes folding by strengthening hydrophobic interactions at the coiled coil interface. Although charge neutralization also contributed to folding, it was effective only when the screening cation was partnered by a good kosmotropic anion. Folding conformed to a two-state transition from random coil monomer to coiled coil dimer and was enthalpy driven and characterized by a change in the heat capacity of unfolding of 3.9 +/- 1.2 kJ mol-1 K-1. The rate of folding was analyzed by fluorescence stopped-flow measurements. Folding occurred in a biphasic reaction in which the rapid formation of an initial dimer (kf = 2 x 10(7) M-1 s-1) was followed by an equally rapid concentration-independent rearrangement to the folded dimer (k > 100 s-1).
Jelesarov
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Biochemisches Institut der Universität Zürich, Switzerland.
Dürr
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Thomas
R M
RM
Bosshard
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HR
D000595:Amino Acid Sequence; D002412:Cations; D019281:Dimerization; D005459:Fluorides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016350:Leucine Zippers / Q000187:drug effects*; D018021:Lithium Chloride / Q000494:pharmacology; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D011189:Potassium Chloride / Q000494:pharmacology; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000187:drug effects*; D012492:Salts / Q000494:pharmacology*; D012965:Sodium Chloride / Q000494:pharmacology; D055672:Static Electricity; D013816:Thermodynamics; D014461:Ultracentrifugation; D014867:Water
D002412:Cations; D010455:Peptides; D012492:Salts; D015636:Magnesium Chloride; D014867:Water; D012965:Sodium Chloride; D011189:Potassium Chloride; D018021:Lithium Chloride; D005459:Fluorides