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PubMed2006

Upregulation of calbindin-D28k expression during regeneration in the adult fish cerebellum.

In contrast to mammals, fish are distinguished by their enormous potential for brain repair after injuries. This phenomenon has been well studied after application of stab-wound lesions to the corpus cerebelli, a cerebellar subdivision, in the teleost fish Apteronotus leptorhynchus. By combining this lesion paradigm with immunohistochemical staining, we examined the potential role of the calcium-binding protein calbindin-D(28k) in the process of regeneration. Calbindin-D(28k)-immunoreactive cell bodies and fibers were evident in the lesion path and the immediate vicinity of the lesion in the period between 16 h and 7 days after the lesion but absent from this region at shorter or longer postlesion survival times and in the intact brain. Both the number of immunolabeled cells and the intensity of the label were most pronounced 1-3 days postlesion. Analysis of the morphology of the immunostained cells by confocal microscopy suggested that most, and perhaps all of them, were granular neurons. Since the transient upregulation of calbindin-D(28k) is paralleled by a decline in the number of cells undergoing apoptotic cell death, we hypothesize that this protein exerts a neuroprotective function, probably by buffering free intracellular Ca(2+), whose concentration is elevated after brain insults.

Animals↗
PubMed2006

Effects of calcium binding on the side-chain methyl dynamics of calbindin D9k: a 2H NMR relaxation study.

The effects of Ca(2+) binding on the side-chain methyl dynamics of calbindin D(9k) have been characterized by (2)H NMR relaxation rate measurements. Longitudinal, transverse in-phase, quadrupolar order, transverse anti-phase and double quantum relaxation rates are reported for both the apo and Ca(2+)-loaded states of the protein at two magnetic field strengths. The relatively large size of the data set allows for a detailed analysis of the underlying conformational dynamics by spectral density mapping and model-free fitting procedures. The results reveal a correlation between a methyl group's distance from the Ca(2+) binding sites and its conformational dynamics. Several methyl groups segregate into two limiting classes, one proximal and the other distal to the binding sites. Methyl groups in these two classes respond differently to Ca(2+) binding, both in terms of the timescale and amplitude of their fluctuations. Ca(2+) binding elicits a partial immobilization among methyl groups in the proximal class, which is consistent with previous studies of calbindin's backbone dynamics. The distal class, however, exhibits a trend that could not be inferred from the backbone data in that its mobility actually increases with Ca(2+) binding. We have introduced the term polar dynamics to describe this type of organization across the molecule. The trend may represent an important mechanism by which calbindin D(9k) achieves high affinity binding while minimizing the corresponding loss of conformational entropy.

Animals↗
PubMed2004

Potential pathways for intercellular communication within the calbindin subnucleus of the hamster suprachiasmatic nucleus.

In mammals, the suprachiasmatic nucleus (SCN) is the master circadian pacemaker. Within the caudal hamster SCN, a cluster of neurons containing the calcium binding protein, calbindin-D28K (CB), has been implicated in circadian locomotion. However, calbindin-immunoreactive (CB+) neurons in the calbindin subnucleus (CBsn) do not display a circadian rhythm in spontaneous firing [Eur J Neurosci 16 (2002) 2469]. Previously, we proposed that intercellular communication might be essential in integrating outputs from rhythmic (CB-) neurons and nonrhythmic (CB+) neurons to produce a circadian output in the intact animal. The primary aim of this study is to provide a neuroanatomical framework to better understand intercellular communication within the CBsn. Using reconstructions of previously recorded neurons, we demonstrate that CB+ neurons have significantly more dendrites than CB- neurons. In addition, CBsn neurons have dorsally oriented dendritic arbors. Using double-label confocal microscopy, we show that GABA colocalizes with CB+ neurons and GABA(A) receptor subunits make intimate contacts with neurons in the CBsn. Transforming growth factor alpha (TGFalpha), a substance shown to inhibit locomotion [Science 294 (2001) 2511], is present within the CBsn. In addition, neurons in this region express the epidermal growth factor receptor, the only receptor for TGFalpha. Lastly, we show that CB+ neurons are coupled to CB+ and CB- neurons by gap junctions. The current study provides a structural framework for synaptic communication, electrical coupling, and signaling via a growth factor within the CBsn of the hamster SCN. Our results reveal connections that have the potential for integrating cellular communication within a subregion of the SCN that is critically involved in circadian locomotion.

Animals↗
PubMed2004

Calbindin D-28k distribution in odontoblasts underneath tertiary dentine in human carious teeth.

OBJECTIVE: The aim of this study was to examine the calbindin D-28k immunoreactivity in carious teeth to know whether this protein may have a function in tertiary dentine formation. METHODS: Human extracted teeth with or without carious lesions were immersion-fixed with Zamboni fixative, demineralized in 4.13% EDTA solution (pH 7.4), frozen-sectioned, and processed for calbindin immunoreactivity and hematoxylin-eosin stain. The intensity of the immunostaining was evaluated by quantitative densitometry. RESULTS: In intact teeth, numerous odontoblasts were aligned underneath the secondary dentine and their cell bodies showed the immunoreactivity. In carious teeth, tertiary dentine had poor- or rich tubular patterns under the carious lesion. Underneath the tubule-poor tertiary dentine, distinct odontoblasts could not be seen at the central site. However, some cells with a flat appearance were located at this site and were immunonegative for calbindin D-28k. On the other hand, columnar odontoblasts were seen at the peripheral site, and their cell bodies and processes showed strong immunoreactivity. Underneath the tubule-rich tertiary dentine, columnar odontoblasts were abundantly distributed, and the strong immunoreactivity was observed in their cell bodies and processes. The immunoreactivity in odontoblasts underneath the tertiary dentine with poor or rich tubular pattern was more intense than that for the secondary dentine in intact teeth (P<0.05). On the other hand, the intensity of the immunoreactivity in odontoblasts was similar underneath the secondary dentine in intact and carious teeth. CONCLUSIONS: The present study demonstrated that calbidin D-28k was actively synthesised by odontoblasts under the carious lesion. These findings may suggest that this protein plays an important role in the tertiary dentine formation.

Adult↗
PubMed2000

Binding kinetics of calbindin-D(28k) determined by flash photolysis of caged Ca(2+)

We have used UV flash photolysis of DM-nitrophen in combination with model-based analysis of Oregon Green 488 BAPTA-5N fluorescence transients to study the kinetics of Ca(2+) binding to calbindin-D(28K). The experiments used saturated DM-nitrophen at a [Ca(2+)] of 1.5 microM. Under these conditions, UV laser flashes produced rapid steplike increases in [Ca(2+)] in the absence of calbindin-D(28K), and in its presence the decay of the flash-induced fluorescence was due solely to the Ca(2+) buffering by the protein. We developed a novel method for kinetic parameter derivation and used the synthetic Ca(2+) buffer EGTA to confirm its validity. We provide evidence that calbindin-D(28K) binds Ca(2+) in at least two distinct kinetic patterns, one arising from high-affinity sites that bind Ca(2+) with a k(on) comparable to that of EGTA (i.e., approximately 1 x 10(7) M(-1) s(-1)) and another with lower affinity and an approximately eightfold faster k(on). In view of the inability of conventional approaches to adequately resolve rapid Ca(2+) binding kinetics of Ca(2+) buffers, this method promises to be highly valuable for studying the Ca(2+) binding properties of other biologically important Ca(2+) binding proteins.

Acetates↗
PubMed2003

The 5-HT1A serotonin receptor is located on calbindin- and parvalbumin-containing neurons in the rat brain.

The 5-HT(1A) receptor is a well-characterized serotonin receptor playing a role in many central nervous functions and known to be involved in depression and other mental disorders. In situ hybridization, immunocytochemical, and binding studies have shown that the 5-HT(1A) receptor is widely distributed in the rat brain, with a particularly high density in the limbic system. The receptor's localization in the different neuronal subtypes, which may be of importance for understanding its role in neuronal circuitries, is, however, unknown. In this study we show by immunocytochemical double-labeling techniques, that the 5-HT(1A) receptor is present on both pyramidal and principal cells, and calbindin- and parvalbumin-containing neurons, which generally define two different subtypes of interneurons. Moreover, semiquantitative analysis showed that the receptor's distribution in the different neuronal types varies between brain areas. In cortex, hippocampus, hypothalamus, and amygdala the receptor was located on both principal cells and calbindin- and parvalbumin-containing neurons. In septum and thalamus, the receptor was mostly present on calbindin- and parvalbumin-containing cells. Especially in the medial septum and thalamic reticular nucleus, the receptor highly colocalized with parvalbumin-positive neurons. These results suggest a diverse function of the 5-HT(1A) receptor in modulating neuronal circuitry in different brain areas, that may depend on the type of neuron the receptor is predominantly located on.

Animals↗
PubMed1996

Differential age-dependent effects of retinal deafferentation upon calbindin- and parvalbumin-immunoreactive neurons in the superficial layers of the rat's superior colliculus.

Several recent studies have reported varied effects of different forms of visual deprivation on the expression of calcium-binding proteins in the CNS. Most of these studies have surveyed only a single protein from this family and have not systematically evaluated the influence of the age of the animal upon the effects observed. The present study combined immunocytochemistry and quantitative morphometry to determine the effects of eye removal in fetal life, at birth, or in adulthood upon the expression of calbindin and parvalbumin by neurons in the retinorecipient laminae (the stratum griseum superficiale (SGS) and stratum opticum (SO)) of the rat's superior colliculus (SC). Both fetal and neonatal enucleation significantly reduced the total number of neurons in the SGS. Eye removal at any age did not significantly affect the number of neurons in the SO or the proportion of SGS or SO cells that expressed calbindin. Adult enucleation produced a significant increase in the percentage of SGS cells expressing parvalbumin. These results suggest that calbindin expression is highly stable in visual neurons while parvalbumin expression is more plastic and appears to be suppressed by retinal input.

Age Factors↗
PubMed1997

Parvalbumin- and calbindin-containing neurons express c-fos protein in primary and secondary (mirror) epileptic foci of the rat neocortex.

The present experiments aimed at the description and further immunocytochemical characterization of activated neocortical neurons expressing the c-fos gene. Focal seizures were induced by the topical application of isotonic, isohydric 4-aminopyridine solution to the frontal neocortex of adult anesthetized Wistar rats. The EEG of both hemispheres was recorded from the surface of the skull. The animals were perfused with fixative, coronal plane vibratome sections were cut and stained with cocktails containing polyclonal c-fos and monoclonal calbindin or parvalbumin antibodies. The polyclonal c-fos antibody was tested with Western blotting and the diffusion of 4-aminopyridine investigated with autoradiography of [3H]4-aminopyridine. The c-fos protein was detected in every layer of the neocortex (primary focus) and in some allocortical areas of the treated hemisphere. Scattered immunostained nuclei were observed in layers II, III, IV and VI of the contralateral neocortex (mirror focus). Several parvalbumin- and calbindin-positive neurons contained the c-fos protein in both foci. The medium-sized non-pyramidal parvalbumin neurons were found in layers II-IV and VI of the neocortex and in stratum multiforme of the prepiriform cortex. The c-fos protein was colocalized with calbindin mainly in layers II and III in small and medium-sized non-pyramidal neurons. The results prove that focal epileptiform activity of the neocortex activates diverse inhibitory neuronal populations. As concluded, the inhibitory control is probably more effective in the contralateral hemisphere (mirror focus) than on the side of 4-APY treatment (primary focus).

Animals↗
PubMed1995

Calbindin immunoreactivity of horizontal cells in the developing rabbit retina.

Horizontal cells are retinal interneurons that establish inhibitory feedback loops within the outer plexiform layer of the primary visual pathway. Most mammalian retinas contain two types of horizontal cells. A-type horizontal cells have neuritic branches that contact cone photoreceptors exclusively, while the B-type horizontal cells have dendritic branches that contact cones, in addition to axons that form synapses with rod photoreceptors. Immunoreactivity for calbindin, a calcium binding protein involved in calcium transport, was used as a marker for horizontal cells during post-natal development of the rabbit retina. On post-natal days 1, 3 and 5, calbindin immunoreactivity is limited to a single population of A-type horizontal cells. They appear as a monolayer of cells with broad tapering processes, establishing the proximal border of the nascent outer plexiform layer and forming a target for ingrowing cone photoreceptor terminals. The size and density of the cell bodies and the length of neuritic processes are essentially unchanged during this period, which corresponds to the time of peak expression of GABAergic markers in horizontal cells. Coincident with a decrease in GABAergic markers and the completion of cone-to-horizontal cell synaptogenesis by day 7, changes within the horizontal cell mosaic are detected morphometrically. A delayed phase of overall cell growth results in a 70% increase in average somal diameter (representing a 3.7-fold increase in spherical volume), a six-fold increase in mean neurite length and a decrease in cell density to one-third of that found in the newborn. We conclude that the process of terminal differentiation of horizontal cells is not complete until some time after the second post-natal week. Furthermore, the expression of GABAergic markers is associated primarily with early maturational events, whereas expression of calbindin is sustained throughout post-natal development, suggesting a prominent role for calcium dependent mechanisms at all development stages.

Animals↗
PubMed2002

Increased inter-spike intervals and fast after-hyperpolarization of action potentials in rat hippocampal pyramidal cells accompanied with altered calbindin immunoreactivity 10-12 months after global forebrain ischemia.

In vivo electrophysiological recordings of CA1/CA2 pyramidal cells were performed 10-12 months after global forebrain ischemia (four-vessel occlusion, 15 mm) and were compared to levels of calbindin expression. Ischemic animals were subdivided in non-sclerotic ischemic (NSI) and sclerotic ischemic (SI) groups depending on the absence or presence of hippocampal sclerosis. A decreased excitability was observed in neurons from both groups, as shown by significant prolongation of inter-spike intervals (ISI) of evoked action potentials and by increased amplitude of fast after-hyperpolarization (fAHP). The ratio of calbindin-positive CA1/CA2 pyramidal cells decreased from 59% in control to 33% and 8% in NSI and SI animals, respectively. These results suggest that decreased excitability of CA1/CA2 pyramidal cells represents a protective mechanism against ischemia-induced neurodegeneration and might be related to decreased calbindin expression.

Animals↗
PubMed2001

Parvalbumin-containing neurons in the rat basolateral amygdala: morphology and co-localization of Calbindin-D(28k).

Parvalbumin is a calcium-binding protein that is contained in certain neuronal populations in the brain. Although the exact function of parvalbumin is not clear, it has been found to be a useful marker for studying the connections of specific cell types in immunohistochemical studies. In the present investigation immunohistochemical techniques were used to study the morphology of parvalbumin-containing neurons in the rat basolateral amygdala. These neurons were found to be a morphologically heterogeneous subpopulation of non-pyramidal interneurons. Parvalbumin-positive axons in the basolateral amygdala were observed to form "pericellular baskets" that enveloped the perikarya of pyramidal neurons. In addition, some parvalbumin-immunoreactive axons formed "cartridges" that appeared to surround non-immunoreactive processes. The morphology of parvalbumin-positive neurons closely resembled that of neurons containing calbindin, a related calcium-binding protein. Analysis of adjacent sections stained for each protein using the mirror technique revealed that approximately 80% of parvalbumin neurons also contained calbindin, and that approximately 60% of calbindin neurons also contained parvalbumin. This study demonstrates that parvalbumin-containing neurons constitute an important subpopulation of non-pyramidal interneurons in the rat basolateral amygdala. The axonal configurations of these cells indicate that they may exert a potent inhibitory influence over pyramidal projection neurons. We suggest that parvalbumin-containing neurons can control emotional responses mediated by the basolateral amygdala by controlling the output from this important brain region.

Amygdala↗
PubMed1997

Colocalization of parvalbumin and calbindin D-28k in neurons including chandelier cells of the human temporal neocortex.

Chandelier cells are cortical GABAergic interneurons with a unique synaptic specificity enabling them to exert a strong inhibitory influence on pyramidal cells. By using immunocytochemistry for the calcium-binding protein calbindin D-28k in the human temporal neocortex, we have found numerous immunoreactive processes that were identified as chandelier cell axon terminals. This was a striking find since in previous immunocytochemical studies of the primate neocortex, chandelier cell axon terminals had been shown to be immunoreactive for another calcium-binding protein, parvalbumin, and colocalization studies indicate that parvalbumin and calbindin are present in almost completely separate neuronal populations. Here, we present double-label immunofluorescence experiments showing that parvalbumin and calbindin immunoreactivities are colocalized in certain neurons that include a subpopulation of chandelier cells whose cell bodies are located mainly in layers V and VI of the human temporal neocortex. The results suggest a selective laminar distribution of neurochemical subtypes of chandelier cells which is a peculiar feature of the organization of the human neocortex.

Adolescent↗
PubMed1993

Effects of ion binding on the backbone dynamics of calbindin D9k determined by 15N NMR relaxation.

The backbone dynamics of apo- and (Cd2+)1-calbindin D9k have been characterized by 15N nuclear magnetic resonance spectroscopy. Spin-lattice and spin-spin relaxation rate constants and steady-state [1H]-15N nuclear Overhauser effects were measured at a magnetic field strength of 11.74 T by two-dimensional, proton-detected heteronuclear NMR experiments using 15N-enriched samples. The relaxation parameters were analyzed using a model-free formalism that characterizes the dynamics of the N-H bond vectors in terms of generalized order parameters and effective correlation times. The data for the apo and (Cd2+)1 states were compared to those for the (Ca2+)2 state [Kördel, J., Skelton, N. J., Akke, M., Palmer, A. G., & Chazin, W. J. (1992) Biochemistry 31, 4856-4866] to ascertain the effects on ion ligation on the backbone dynamics of calbindin D9k. The two binding loops respond differently to ligation by metal ions: high-frequency (10(9)-10(12) s-1) fluctuations of the N-terminal ion-binding loop are not affected by ion binding, whereas residues G57, D58, G59, and E60 in the C-terminal ion-binding loop have significantly lower order parameters in the apo state than in the metal-bound states. The dynamical responses of the four helices to binding of ions are much smaller than that for the C-terminal binding loop, with the strongest effect on helix III, which is located between the linker loop and binding site II. Significant fluctuations on slower time scales also were detected in the unoccupied N-terminal ion-binding loop of the apo and (Cd2+)1 states; the apparent rates were greater for the (Cd2+)1 state. These results on the dynamical response to ion binding in calbindin D9k provide insights into the molecular details of the binding process and qualitative evidence for entropic contributions to the cooperative phenomenon of calcium binding for the pathway in which the ion binds first in the C-terminal site.

Amino Acid Sequence↗
PubMed1992

Backbone dynamics of calcium-loaded calbindin D9k studied by two-dimensional proton-detected 15N NMR spectroscopy.

Backbone dynamics of calcium-loaded calbindin D9k have been investigated by two-dimensional proton-detected heteronuclear nuclear magnetic resonance spectroscopy, using a uniformly 15N enriched protein sample. Spin-lattice relaxation rate constants, spin-spin relaxation rate constants, and steady-state [1H]-15N nuclear Overhauser effects were determined for 71 of the 72 backbone amide 15N nuclei. The relaxation parameters were analyzed using a model-free formalism that incorporates the overall rotational correlation time of the molecule, and a generalized order parameter (S2) and an effective internal correlation time for each amide group. Calbindin D9k contains two helix-loop-helix motifs joined by a linker loop at one end of the protein and a beta-type interaction between the two calcium-binding loops at the other end. The amplitude of motions for the calcium-binding loops and the helices are similar, as judged from the average S2 values of 0.83 +/- 0.05 and 0.85 +/- 0.04, respectively. The linker region joining the two calcium-binding subdomains of the molecule has a significantly higher flexibility, as indicated by a substantially lower average S2 value of 0.59 +/- 0.23. For residues in the linker loop and at the C-terminus, the order parameter is further decomposed into separate order parameters for motional processes on two distinct time scales. The effective correlation times are significantly longer for helices I and IV than for helices II and III or for the calcium-binding loops. Residue by residue comparisons reveal correlations of the order parameters with both the crystallographic B-factors and amide proton exchange rates, despite vast differences in the time scales to which these properties are sensitive. The order parameters are also utilized to distinguish regions of the NMR-derived three-dimensional structure of calbindin D9k that are poorly defined due to inherently high flexibility, from poorly defined regions with average flexibility but a low density of structural constraints.

Amino Acid Sequence↗
PubMed1991

Electrostatic contributions to the binding of Ca2+ in calbindin D9k.

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)

Amino Acid Sequence↗
PubMed1994

Binding of Ca2+ to calbindin D9k: structural stability and function at high salt concentration.

Calcium binding constants of wild-type calbindin D9k and mutant forms with one, two, and three neutralized negative charges in the vicinity of the Ca2+ binding sites are determined at varying KCl concentrations from 2 mM to 1 M. The results indicate that the added salt does not cause significant structural changes in calbindin D9k and, along with site-directed mutagenesis, can be used as a well-controlled means for modulating electrostatic interactions. The lack of structural changes at high salt concentrations is also supported by two-dimensional 1H NMR data. High salt concentrations are observed to substantially reduce the cooperativity of calcium binding to calbindin D9k. This suggests that the cooperativity is strongly dependent on electrostatic interactions. The data have been used to test a dielectric continuum model for protein electrostatics using a macroscopic dielectric constant of water throughout the system. Excellent agreement between experiment and Monte Carlo simulations is observed for the whole set of data covering changes in the binding constant of more than 6 orders of magnitude. A simplified theoretical treatment using the Kirkwood-Tanford formula, based on the Debye-Hückel approximation, yields an almost equally good agreement with the experiment.

Binding Sites↗
PubMed1989

1H NMR studies of porcine calbindin D9k in solution: sequential resonance assignment, secondary structure, and global fold.

The 1H nuclear magnetic resonance (NMR) spectrum of Ca2+-saturated porcine calbindin D9k (78 amino acids, Mr 8800) has been assigned. Greater than 98% of the 1H resonances, including spin systems for each amino acid residue, have been identified by using an approach that integrates data from a wide range of two-dimensional scalar correlated NMR experiments [Chazin, Rance, & Wright (1988) J. Mol. Biol. 202, 603-626]. Due to the limited quantity of sample and conformational heterogeneity of the protein, two-dimensional nuclear Overhauser effect (NOE) experiments also played an essential role in the identification of spin systems. On the basis of the pattern of scalar connectivities, 43 of the 78 spin systems could be directly assigned to the appropriate residue type. This provided an ample basis for obtaining the sequence-specific resonance assignments. The elements of secondary structure are identified from sequential and medium-range NOEs, values of 3JNH alpha, and the location of slowly exchanging backbone amide protons. Four well-defined helices and a mini beta-sheet between the two calcium binding loops are present in solution. These elements of secondary structure and a few key long-range NOEs provided sufficient information to define the global fold of the protein in solution. Generally good agreement is found between the crystal structure of the minor A form of bovine calbindin D9k and the solution structure of intact porcine calbindin D9k. The only significant difference is a short one-turn helix in the loop between helices II and III in the bovine crystal structure, which is clearly absent in the porcine solution structure.

Amino Acid Sequence↗
PubMed2001

Symmetrical stabilization of bound Ca2+ ions in a cooperative pair of EF-hands through hydrogen bonding of coordinating water molecules in calbindin D(9k).

Water molecules are found to complete the Ca2+ coordination sphere when a protein fails to provide enough ligating oxygens. Hydrogen bonding of these water molecules to the protein backbone or side chains may contribute favorably to the Ca2+ affinity, as suggested in an earlier study of two calbindin D(9k) mutants [E60D and E60Q; Linse et al. (1994) Biochemistry 33, 12478-12486]. To investigate the generality of this conclusion, another side chain, Gln 22, which hydrogen bonds to a Ca2+-coordinating water molecule in calbindin D(9k), was mutated. Two calbindin D(9k) mutants, (Q22E+P43M) and (Q22N+P43M), were constructed to examine the interaction between Gln 22 and the water molecule in the C-terminal calcium binding site II. Shortening of the side chain, as in (Q22N+P43M), reduces the affinity of binding two calcium ions by a factor of 18 at low ionic strength, whereas introduction of a negative charge, as in (Q22E+P43M), leads to a 12-fold reduction. In 0.15 M KCl, a 7-fold reduction in affinity was observed for both mutants. The cooperativity of Ca2+ binding increases for (Q22E+P43M), while it decreases for (Q22N+P43M). The rates of Ca2+ dissociation are 5.5-fold higher for the double mutants than for P43M at low ionic strength. For both mutants, reduced strength of hydrogen bonding to calcium-coordinating water molecules is a likely explanation for the observed effects on Ca2+ affinity and dissociation. In the apo forms, the (Q22E+P43M) mutant has lower stability toward urea denaturation than (Q22N+P43M) and P43M. 2D (1)H NMR and crystallographic experiments suggest that the structure of (Q22E+P43M) and (Q22N+P43M) is unchanged relative to P43M, except for local perturbations in the loop regions.

Amino Acids↗