Hydrophobic Core Substitutions in Calbindin D9k: Effects on Ca2+ Binding and Dissociation

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• 作者：Birthe B. Kragelund ; Malin Jö ; nsson ; Giuseppe Bifulco ; Walter J. Chazin ; Hanna Nilsson ; Bryan E. Finn ; and Sara Linse
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：June 23, 1998
• 年：1998
• 卷：37
• 期：25
• 页码：8926 - 8937
• 全文大小：280K
• 年卷期：v.37,no.25(June 23, 1998)
• ISSN：1520-4995

文摘

Hydrophobic core residues have a marked influence onthe Ca²⁺-binding properties of calbindinD_9k, even though there are no direct contacts between theseresidues and the bound Ca²⁺ ions.Elevendifferent mutants with substitutions in the hydrophobic core wereproduced, and their equilibrium Ca²⁺-binding constants measured from Ca²⁺ titrations in thepresence of chromophoric chelators. TheCa²⁺-dissociation rate constants were estimated from Ca²⁺titrations followed by ¹H NMR¹ and weremeasuredmore accurately using stopped-flow fluorescence. The parameterswere measured at four KClconcentrations to assess the salt dependence of the perturbations.The high similarity between the NMRspectra of mutants and wild-type calbindin D_9k suggeststhat the structure is largely unperturbed by thesubstitutions. More detailed NMR investigations of the mutant inwhich Val61 is substituted by Alashowed that the mutation causes only very minimal perturbations in theimmediate vicinity of residue 61.Substitutions of alanines or glycines for bulky residues in thecenter of the core were found to havesignificant effects on both Ca²⁺ affinity anddissociation rates. These substitutions caused a reductioninaffinity and an increase in off-rate. Small effects, bothincreases and decreases, were observed forsubstitutions involving residues far from the Ca²⁺ sitesand toward the outer part of the hydrophobiccore. The mutant with the substitution Phe66 Trp behaveddifferently from all other mutants, anddisplayed a 25-fold increase in overall affinity of binding twoCa²⁺ ions and a 6-fold reduction in calciumdissociation rate. A strong correlation (R = 0.94)was found between the observedCa²⁺-dissociationrates and affinities, as well as between the salt dependence of theoff-rate and the distance to the nearestCa²⁺-coordinating atom. There was also a strongcorrelation (R = 0.95) between the Ca²⁺affinity andstability of the Ca²⁺ state and a correlation(R = 0.69) between the Ca²⁺ affinity andstability of the apostate, as calculated from the results in the present and precedingpaper in this issue [Julenius, K., Thulin,E., Linse, S., and Finn, B. E. (1998) Biochemistry 37,8915-8925]. The change in salt dependenciesof k_off and cooperativity were most pronouncedfor residues completely buried in the core of the protein(solvent accessible surface area p.gif"> 0). Altogether, the resultssuggest that the hydrophobic core residuespromote Ca²⁺ binding both by contributing to thepreformation of the Ca²⁺ sites in the apo state andbypreferentially stabilizing the Ca²⁺-boundstate.