A set of accurate experimental data is provided for Ca2+ ion binding to calbindin D9k, a protein in the calmodulin superfamily of intracellular regulatory proteins. The study comprises both the role of protein surface charges and the effects of added electrolyte. The two macroscopic Ca2(+)-binding constants K1 and K2 are determined for the wild-type and eight mutant calbindins in 0, 0.05, 0.10, and 0.15 M KCl from titrations in the presence of Quin 2 or 5,5'-Br2BAPTA. The mutations involve replacement of surface carboxylates (of Glu17, Asp19, Glu26, and Glu60) with the corresponding amides. It is found that K1K2 may decrease by a factor of up to 2.5 x 10(5) (triple mutant in 0.15 M KCl as compared to the wild-type protein in 0 M KCl). Ca2(+)-binding constants of the individual Ca2+ sites (microscopic binding constants) have also been determined. The positive cooperativity of Ca2+ binding, previously observed at low salt concentration [Linse et al. (1987) Biochemistry 26, 6723-6735], is also present at physiological ionic strength and amounts to 5 kJ.mol-1 at 0.15 M KCl. The electrolyte concentration and some of the mutations are found to affect the cooperativity. 39K NMR studies show that K+ binds weakly to calbindin. Two-dimensional 1H NMR studies show, however, that potassium binding does not change the protein conformation, and the large effect of KCl on the Ca2+ affinity is thus of unspecific nature. Two-dimensional 1H NMR has also been used to assess the structural consequences of the mutations through assignments of the backbone NH and C alpha H resonances of six mutants.(ABSTRACT TRUNCATED AT 250 WORDS)
Genes encoding the minor A component of bovine calbindins D9k--the smallest protein known with a pair of EF-hand calcium-binding sites--with amino acid substitutions and/or deletions have been synthesized and expressed in Escherichia coli and characterized with different biophysical techniques. The mutations are confined to the N-terminal Ca2+-binding site and constitute Pro-20----Gly (M1), Pro-20----Gly and Asn-21 deleted (M2), Pro-20 deleted (M3), and Tyr-13----Phe (M4). 1H, 43Ca, and 113Cd NMR studies show that the structural changes induced are primarily localized in the modified region, with hardly any effects on the C-terminal Ca2+-binding site. The Ca2+ exchange rate for the N-terminal site changes from 3 s-1 in the wild-type protein (M0) and M4 to 5000 s-1 in M2 and M3, whereas there is no detectable variation in the Ca2+ exchange from the C-terminal site. The macroscopic Ca2+-binding constants have been obtained from equilibration in the presence of the fluorescent chelator 2-[[2-[bis(carboxymethyl)-amino]- 5-methylphenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline or by using a Ca2+-selective electrode. The Ca2+ affinity of M4 was similar to that of M0, whereas the largest differences were found for the second stoichiometric step in M2 and M3. Microcalorimetric data show that the enthalpy of Ca2+ binding is negative (-8 to -13 kJ.mol-1) for all sites except the N-terminal site in M2 and M3 (+5 kJ.mol-1). The binding entropy is strongly positive in all cases. Cooperative Ca2+ binding in M0 and M4 was established through the values of the macroscopic Ca2+-binding constants. Through the observed changes in the 1H NMR spectra during Ca2+ titrations we could obtain ratios between site binding constants in M0 and M4. These ratios in combination with the macroscopic binding constants yielded the interaction free energy between the sites delta delta G as -5.1 +/- 0.4 kJ.mol-1 (M0) and less than -3.9 kJ.mol-1 (M4). There is evidence (from 113Cd NMR) for site-site interactions also in M1, M2, and M3, but the magnitude of delta delta G could not be determined because of sequential Ca2+ binding.
Protein engineering is a means of probing the role of electrostatic interactions in protein functions; this elegant technique has been applied to the elucidation of electrostatic effects in enzyme catalysis. Here we show how the use of mutant proteins allows the determination of the contributions of individual charges to the free energy of ion binding to proteins. We have investigated the importance of three negatively charged side chains in the binding of Ca2+ to bovine calbindin D9K (ref.2): these are clustered around the calcium sites but are not directly involved as ligands. Each of these charges is found to contribute approximately 7 kJ mol-1 to the free energy of binding of two Ca2+ ions and to affect the cooperativity of Ca2+ binding. The influence of surface charges on ion binding to proteins may be more common than generally supposed and could have important consequences for protein function.
7,10) spectrum of r-calbindin D9k (9) acquired at 300 K from a 4 mM self-buffered solution at pH 6.0. Acquisition and data-processing parameters are described in ref. 11. Cross-peaks are labeled with the sequence-specific assignment (one-letter amino acid symbol and residue number). The major form is boxed and the minor form, where distinguishable, is circled. terization (including gel electrophoresis, isoelectric focusing, amino acid composition, and amino acid sequence) of each preparation shows that the protein is purified to homogeneity. In contrast, approximately half ofthe protons in 1H NMR spectra exhibit resolved major and minor resonances in a ratio of 3:1. This is illustrated in Fig. 1, which shows The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
The importance of the aspartate ligand in the +Y Ca2+ coordinating position of two EF-hands of calmodulin has been investigated. Synthetic calmodulin genes were used to produce engineered proteins with the wild-type bovine sequence as well as with aspartate 58 in Ca(2+)-binding site II and/or aspartate 95 in site III changed to asparagine. The macroscopic Ca(2+)-binding constants of the intact calmodulins and of tryptic fragments comprising the N- and C-terminal domains were determined from titrations with Ca2+ in the presence of 5,5'-Br2BAPTA. Substitution of aspartate by asparagine in Ca(2+)-binding site II led to a slight increase in the total free energy change on Ca2+ binding, and the cooperativity of Ca2+ binding to the N-terminal sites was substantially increased. The change from aspartate to asparagine in site III decreased the Ca2+ affinity and also appeared to decrease the positive cooperativity between the sites in the C-terminal domain. Thus, identical mutations in sites II and III were found to result in opposite effects. The data imply that involvement of liganding side chains in interactions other than direct calcium attraction and calcium coordination is of considerable importance for the Ca(2+)-binding process, particularly for the cooperativity.
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