The role of protein surface charges in ion binding

Linse, Sara; Brodin, Peter; Johansson, Charlotta; Thulin, Eva; Grundström, Thomas; Forsén, Sture

doi:10.1038/335651a0

Cited by 140 publications

(117 citation statements)

References 10 publications

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“…A similar effect with divalent cations (and negative charges) has been noted in other protein systems (Voordouw et al, 1976;Filimonov et al, 1978;Bashford et al, 1986;Linse et al, 1988;Pantoliano et al, 1988). The effect of phosphate above its second pK, is expected to be essentially the same as sulfate.…”

Section: Effect Of Phosphatesupporting

confidence: 58%

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding

Wilcox

Eisenberg

1992

Protein Science

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The tetramerization of melittin, a 26-amino acid peptide from Apis mellifera bee venom, has been studied as a model for protein folding. Melittin converts from a monomeric random coil to an a-helical tetramer as the pH is raised from 4.0 to 9.5, as ionic strength is increased, as temperature is raised or lowered from about 37 "C, or as phosphate is added. The thermodynamics of this tetramerization (termed "folding") are explored using circular dichroism. The melittin tetramer has two pK, values of 7.5 and 8. [7][8][9][10][11][12][13][14][15][16][17][18] agree with experimental titration data. Greater electrostatic repulsion of these protonated groups destabilizes the tetramer by 3.6 kcal/mol at pH 4.0 compared to pH 9.5. Increasing the concentration of NaCl in the solution from 0 to 0.5 M stabilizes the tetramer by 5-6 kcal/mol at pH 4.0. The effect of NaCl is modeled with a ligand-binding approach. The melittin tetramer is found to have a temperature of maximum stability ranging from 35.5 to 43 "C depending on the pH, unfolding above and below that temperature. AC; for folding ranges from -0.085 to -0.102 cal g" K-', comparable to that of other small globular proteins (Privalov, P.L., 1979, Adv. Protein Chem. 33, 167-241). A H o and ASo are found to decrease with temperature, presumably due to the hydrophobic effect (Kauzmann, w., 1959, Adv. Protein Chem.14, 1-63). Phosphate is found to perturb the equilibrium substantially with a maximal effect at 150 mM, stabilizing the tetramer at pH 7.4 and 25 "C by 4.6 kcal/mol. The enthalpy change due to addition of phosphate (-7.5 kcal/mol at 25 "C) can be accounted for by simple dielectric screening. Both circular dichroism and crystallographic results suggest that phosphate may bind Lys 23 at the ends of the elongated tetramer. These detailed measurements give insight into the relative importance of various forces for the stability of melittin in the folded form and may provide an experimental standard for future tests of computational energetics on this simple protein system. Abbreviations: CD, circular dichroism; far UV, 260-185 nm; near UV, 330-245 nm; AzgO, light absorption at 280 nm; AC:, the standard change in heat capacity from the unfolded to the folded form; At = AL -AR, where AL and AR are the absorbance of left and right circularly polarized light, respectively; AcZU, Ac at 222 nm; AtZB2, At at 282 nm; Th, a temperature at which AHo(T) = 0; T,, a temperature at gates to form an a-helical tetramer. The amount of tetwhich ASo( T ) = 0; TES, N-tris(hydroxyrnethyl)methyl-2-aminoethane sulfonic acid. Keywords

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Section: Effect Of Phosphatesupporting

confidence: 58%

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding

Wilcox

Eisenberg

1992

Protein Science

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“…Electrostatic interactions also play a role in the calcium binding properties as has been demonstrated in the case of the intact protein [IO,1 11. This is also true for the fragments.…”

Section: Discussionmentioning

confidence: 92%

Dissection of Calbindin D_9k into two Ca²⁺‐binding subdomains by a combination of mutagenesis and chemical cleavage

et al. 1992

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Calbindin D,, is a 7kesiduc globular protein made up of two Ca" binding subdomains of the EF-hand type. In order to examine the subdomains independently. a method was devised to selectively cleave the loop between them. Using site-directed mutagenesis, a unique mcthioninc was substituted for Pro"' in the loop. thus nllowiny cleavage usingcyanogen bromide. Agarose gel clectrophoresis shows that the framents have a high affinity for one another, although less so in the absence ofcalcium. 'H-NMR spectra of the fragments indicate that the structures of the heterodimcrs are changed little from that of the intact protein. However, the Ca'+ binding constants of the individual subdomains arc several orders of magnitude lower lhan for lhc corresponding sites in the unclcaved protein.NMR; Site-directed mutagenesis,

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“…Furthermore, Ackers et al (1983) have shown that it is generally possible to extract the value of AAG, from individual-site binding isotherms; and estimates of AAG,. for calbindin Dgk are becoming available from spectroscopic and mutagenesis studies (Linse et al, 1991). Finally, it will be shown that the main results of this paper would be the same, even if the macroscopic definition were used.…”

Section: K Kllmentioning

confidence: 88%

“…However, in the more general case of nonidentical binding sites, the 7-dependent term is negative so that AAG, < -RT ln(4K2/K1); in other words, the macroscopic definition provides an upper limit on the value of the microscopic AAG,. (Linse et al, 1988). It is therefore possible that, in systems with nonidentical sites that bind ligands independently (AAG,.…”

Section: K Kllmentioning

confidence: 99%

Electrostatic coupling to pH‐titrating sites as a source of cooperativity in protein‐ligand binding

Spassov

Bashford

1998

Protein Science

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This paper describes an alternative mechanism for the cooperative binding of charged ligands to proteins. The ligandbinding sites are electrostatically coupled to protein side chains that can undergo protonation and deprotonation. The binding of one ligand alters the protein's protonation equilibrium in a manner that makes the the binding of the second ligand more favorable. This mechanism requires no conformational change to produce a cooperative effect, although it is not exclusive of conformational change. We present a theoretical description of the mechanism, and calculations on three kinds of systems: A model system containing one protonation site and two ligand-binding sites; a model system containing two protonation sites and two ligand-binding sites; and calbindin Dgk, which contains two Ca2+-binding sites and 30 protonation sites. For the one-protonation-site model, it is shown that the influence of the protonation site can only be cooperative. The competition of this effect with the anticooperative effect of ligand-ligand repulsion is studied in detail. For the two-protonation site model, the effect can be either cooperative or, in special cases, anticooperative. For calbindin Dgkr the calculations predict that six protonation sites in or near the ligand-binding sites make a cooperative contribution that approximately cancels the anticooperative effect of Ca2+-Ca2+ repulsion, accounting for more than half of the total cooperative effect that is needed to overcome repulsion and produce the net cooperativity observed experimentally. We argue that cooperative mechanisms of the kind described here are likely when there is more than one ligand-binding site in a protein domain.Keywords: allostery, calbindin Dgk cooperativity, electrostatics, linked equilibrium, multisite titration, pH titration of proteins, pK, values in proteins This paper demonstrates that cooperativity in the binding of charged ligands by proteins can arise from electrostatic couplings of the ligand-binding sites to sites of protonation/deprotonation, such as the ionizable side chains of the protein. We provide a theoretical framework for cooperative effects of this kind, and we present calculations on some simple model systems and on the Ca2+-binding protein, calbindin Dgk. In contrast to traditional models of cooperativity (Monod et al., 1965;Koshland et al., 1966), conformational changes in the protein are not required in our model, and therefore are not included in the present calculations. We study the conditions that cause the effects of electrostatic coupling to protonation sites to be cooperative or anticooperative, and of greater or lesser magnitude. The conditions under which this mechanism can produce a cooperative effect strong enough to overcome the anticooperative effect of direct ligand-ligand repulsion are given particular attention.We believe that effects of the kind described here are present in many cases of cooperative binding of charged ligands to proteins, Reprint requests to: Donald Bashford, Department of M...

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The role of protein surface charges in ion binding

Cited by 140 publications

References 10 publications

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding

Dissection of Calbindin D_9k into two Ca²⁺‐binding subdomains by a combination of mutagenesis and chemical cleavage

Electrostatic coupling to pH‐titrating sites as a source of cooperativity in protein‐ligand binding

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The role of protein surface charges in ion binding

Cited by 140 publications

References 10 publications

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding

Dissection of Calbindin D9k into two Ca2+‐binding subdomains by a combination of mutagenesis and chemical cleavage

Electrostatic coupling to pH‐titrating sites as a source of cooperativity in protein‐ligand binding

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Dissection of Calbindin D_9k into two Ca²⁺‐binding subdomains by a combination of mutagenesis and chemical cleavage