Backbone dynamics and energetics of a calmodulin domain mutant exchanging between closed and open conformations.

Previous studies have suggested that the Ca2+-saturated E140Q mutant of the C-terminal domain of calmodulin exhibits equilibrium exchange between "open" and "closed" conformations similar to those of the Ca2+-free and Ca2+-saturated states of wild-type calmodulin. The backbone dynamics of this mutant were studied using15N spin relaxation experiments at three different temperatures. Measurements at each temperature of the15N rate constants for longitudinal and transverse auto-relaxation, longitudinal and transverse cross-correlation relaxation, and the1H-15N cross-relaxation afforded unequivocal identification of conformational exchange processes on microsecond to millisecond time-scales, and characterization of fast fluctuations on picosecond to nanosecond time-scales using model-free approaches. The results show that essentially all residues of the protein are involved in conformational exchange. Generalized order parameters of the fast internal motions indicate that the conformational substates are well folded, and exclude the possibility that the exchange involves a significant population of unfolded or disordered species. The temperature dependence of the order parameters offers qualitative estimates of the contribution to the heat capacity from fast fluctuations of the protein backbone, revealing significant variation between the well-ordered secondary structure elements and the more flexible regions. The temperature dependence of the conformational exchange contributions to the transverse auto-relaxation rate constants directly demonstrates that the microscopic exchange rate constants are greater than 2.7x10(3)s-1at 291 K. The conformational exchange contributions correlate with the chemical shift differences between the Ca2+-free and Ca2+-saturated states of the wild-type protein, thereby substantiating that the conformational substates are similar to the open and closed states of wild-type calmodulin. Taking the wild-type chemical shifts to represent the conformational substates of the mutant and populations estimated previously, the microscopic exchange rate constants could be estimated as 2x10(4)to 3x10(4)s-1at 291 K for a subset of residues. The temperature depen dence of the exchange allows the characterization of apparent energy barriers of the conformational transition, with results suggesting a complex process that does not correspond to a single global transition between substates.     

Dynamics of the transition between open and closed conformations in a calmodulin C-terminal domain mutant

BACKGROUND: Calmodulin is a ubiquitous Ca(2+)-activated regulator of cellular processes in eukaryotes. The structures of the Ca(2+)-free (apo) and Ca(2+)-loaded states of calmodulin have revealed that Ca(2+) binding is associated with a transition in each of the two domains from a closed to an open conformation that is central to target recognition. However, little is known about the dynamics of this conformational switch. RESULTS: The dynamics of the transition between closed and open conformations in the Ca(2+)-loaded state of the E140Q mutant of the calmodulin C-terminal domain were characterized under equilibrium conditions. The exchange time constants (tau(ex)) measured for 42 residues range from 13 to 46 micros, with a mean of 21 +/- 3 micros. The results suggest that tau(ex) varies significantly between different groups of residues and that residues with similar values exhibit spatial proximity in the structures of apo and/or Ca(2+)-saturated wild-type calmodulin. Using data for one of these groups, we obtained an open population of p(o) = 0.50 +/- 0.17 and a closed --> open rate constant of k(o) = x 10(4) s(-1). CONCLUSIONS: The conformational exchange dynamics appear to involve locally collective processes that depend on the structural topology. Comparisons with previous results indicate that similar processes occur in the wild-type protein. The measured rates match the estimated Ca(2+) off rate, suggesting that Ca(2+) release may be gated by the conformational dynamics. Structural interpretation of estimated chemical shifts suggests a mechanism for ion release.     

15N NMR assignments and chemical shift analysis of uniformly labeled 15N calbindin D9k in the apo, (Cd2+)1 and (Ca2+)2 states.

15N has been uniformly incorporated into the EF-hand Ca(2+)-binding protein calbindin D9k so that heteronuclear experiments can be used to further characterize the structure and dynamics of the apo, (Cd2+)1 and (Ca2+)2 states of the protein. The 15N NMR resonances were assigned by 2D 15N-resolved 1H experiments, which also allowed the identification of a number of sequential and medium-range 1H-1H contacts that are obscured by chemical shift degeneracy in homonuclear experiments. The 15N chemical shifts are analyzed with respect to correlations with protein secondary structure. In addition, the changes in 15N chemical shift found for the apo----(Cd2+)1----(Ca2+)2 binding sequence confirm that the effects on the protein are mainly associated with chelation of the first ion.     

Comparison of the structural and dynamical properties of holo and apo bovine alpha-lactalbumin by NMR spectroscopy

In the presence of 0.5 M NaCl at pH 7.1, the Ca(2+)-free apo form of recombinant bovine alpha-lactalbumin (BLA) is sufficiently stabilised in its native state to give well-resolved NMR spectra at 20 degrees C. The (1)H and (15)N NMR resonances of native apo-BLA have been assigned, and the chemical-shifts compared with those of the native holo protein. Large changes observed between the two forms of BLA are mainly limited to the Ca(2+)-binding region of the protein. These data suggest that Na(+) stabilises the native apo state through the screening of repulsive negative charges, at the Ca(2+)-binding site or elsewhere, rather than by a specific interaction at the vacant Ca(2+)-binding site. The hydrogen exchange protection of residues in the Ca(2+)-binding loop and the C-helix is reduced in the apo form compared to that in the holo form. This indicates that the dynamic behaviour of this region of the protein is substantially increased in the absence of the bound Ca(2+). Real-time NMR experiments show that the rearrangements of the structure associated with the conversion of the holo to apo form of the protein do not involve the detectable population of partially unfolded intermediates. Rather, the conversion appears to involve local reorganisations of the structure in the vicinity of the Ca(2+)-binding site that are coupled to the intrinsic fluctuations in the protein structure.     

Solution structure and internal dynamics of NSCP, a compact calcium-binding protein

The solution structure of Nereis diversicolor sarcoplasmic calcium-binding protein (NSCP) in the calcium-bound form was determined by NMR spectroscopy, distance geometry and simulated annealing. Based on 1859 NOE restraints and 262 angular restraints, 17 structures were generated with a rmsd of 0.87 A from the mean structure. The solution structure, which is highly similar to the structure obtained by X-ray crystallography, includes two open EF-hand domains, which are in close contact through their hydrophobic surfaces. The internal dynamics of the protein backbone were determined by studying amide hydrogen/deuterium exchange rates and 15N nuclear relaxation. The two methods revealed a highly compact and rigid structure, with greatly restricted mobility at the two termini. For most of the amide protons, the free energy of exchange-compatible structural opening is similar to the free energy of structural stability, suggesting that isotope exchange of these protons takes place through global unfolding of the protein. Enhanced conformational flexibility was noted in the unoccupied Ca2+-binding site II, as well as the neighbouring helices. Analysis of the experimental nuclear relaxation and the molecular dynamics simulations give very similar profiles for the backbone generalized order parameter (S2), a parameter related to the amplitude of fast (picosecond to nanosecond) movements of N(H)-H vectors. We also noted a significant correlation between this parameter, the exchange rate, and the crystallographic B factor along the sequence.     

Nuclear magnetic resonance studies of the internal dynamics in Apo, (Cd2+)1 and (Ca2+)2 calbindin D9k. The rates of amide proton exchange with solvent.

The backbone dynamics of the EF-hand Ca(2+)-binding protein, calbindin D9k, has been investigated in the apo, (Cd2+)1 and (Ca2+)2 states by measuring the rate constants for amide proton exchange with solvent. 15N-1H correlation spectroscopy was utilized to follow direct 1H-->2H exchange of the slowly exchanging amide protons and to follow indirect proton exchange via saturation transfer from water to the rapidly exchanging amide protons. Plots of experimental rate constants versus intrinsic rate constants have been analyzed to give qualitative insight into the opening modes of the protein that lead to exchange. These results have been interpreted within the context of a progressive unfolding model, wherein hydrophobic interactions and metal chelation serve to anchor portions of the protein, thereby damping fluctuations and retarding amide proton exchange. The addition of Ca2+ or Cd2+ was found to retard the exchange of many amide protons observed to be in hydrogen-bonding environments in the crystal structure of the (Ca2+)2 state, but not of those amide protons that were not involved in hydrogen bonds. The largest changes in rate constant occur for residues in the ion-binding loops, with substantial effects also found for the adjacent residues in helices I, II and III, but not helix IV. The results are consistent with a reorganization of the hydrogen-bonding networks in the metal ion-binding loops, accompanied by a change in the conformation of helix IV, as metal ions are chelated. Further analysis of the results obtained for the three states of metal occupancy provides insight into the nature of the changes in conformational fluctuations induced by ion binding.     

Conformational dynamics of yeast calmodulin in the Ca(2+)-bound state probed using NMR relaxation dispersion

Most calmodulin (CaM) in apo and Ca(2+)-bound states show a dumb-bell-like structure, involving the N- and C-terminal domains, connected with a flexible linker. However, Ca(2+)-bound yeast calmodulin (yCaM) takes on a unique globular structure; the target-binding site of this protein is autoinhibited. We applied NMR relaxation dispersion experiments to yCaM in the Ca(2+)-bound state. The amide (15)N and (1)H(N) relaxation dispersion profiles indicated the presence of conformational dynamics for specific residues at the interface between the N- and C-terminal domains. We conclude that these conformational dynamics were derived from the mobility of the C-terminal domain.     

Quantification of the calcium-induced secondary structural changes in the regulatory domain of troponin-C.

The backbone resonance assignments have been completed for the apo (1H and 15N) and calcium-loaded (1H, 15N, and 13C) regulatory N-domain of chicken skeletal troponin-C (1-90), using multidimensional homonuclear and heteronuclear NMR spectroscopy. The chemical-shift information, along with detailed NOE analysis and 3JHNH alpha coupling constants, permitted the determination and quantification of the Ca(2+)-induced secondary structural change in the N-domain of TnC. For both structures, 5 helices and 2 short beta-strands were found, as was observed in the apo N-domain of the crystal structure of whole TnC (Herzberg O, James MNG, 1988, J Mol Biol 203:761-779). The NMR solution structure of the apo form is indistinguishable from the crystal structure, whereas some structural differences are evident when comparing the 2Ca2+ state solution structure with the apo one. The major conformational change observed is the straightening of helix-B upon Ca2+ binding. The possible importance and role of this conformational change is explored. Previous CD studies on the regulatory domain of TnC showed a significant Ca(2+)-induced increase in negative ellipticity, suggesting a significant increase in helical content upon Ca2+ binding. The present study shows that there is virtually no change in alpha-helical content associated with the transition from apo to the 2Ca2+ state of the N-domain of TnC. Therefore, the Ca(2+)-induced increase in ellipticity observed by CD does not relate to a change in helical content, but more likely to changes in spatial orientation of helices.     

Magnesium promotes structural integrity and conformational switching action of a calcium sensor protein

Calcium binding proteins carry out various signal transduction processes upon binding to Ca2+. In general, these proteins perform their functions in a high background of Mg2+. Here, we report the role of Mg2+ on a calcium sensor protein from Entamoeba histolytica (EhCaBP), containing four Ca2+-binding sites. Mg2+-bound EhCaBP exists as a monomer with a conformation different from that of the holo- and apo-EhCaBP. NMR and biophysical data on EhCaBP demonstrate that Mg2+ stabilizes the closed conformation of the apo form. In the presence of Mg2+, the partially collapsed apo-EhCaBP gains stability and structural integrity. Mg2+ binds to only 3 out of 4 calcium binding sites in EhCaBP. The Ca2+ binding affinity and cooperativity of the conformational switching from the "closed" to the "open" state is significantly modulated by the presence of Mg2+. This fine-tuning of the Ca2+ concentration to switch its conformation is essential for CaBPs to carry out the signal transduction process efficiently.     

Intrinsically disordered PEP-19 confers unique dynamic properties to apo and calcium calmodulin

PEP-19 (Purkinje cell protein 4) is an intrinsically disordered protein with an IQ calmodulin (CaM) binding motif. Expression of PEP-19 was recently shown to protect cells from apoptosis and cell death due to Ca(2+) overload. Our initial studies showed that PEP-19 causes novel and dramatic increases in the rates of association of Ca(2+) with and dissociation of Ca(2+) from the C-domain of CaM. The goal of this work was to study interactions between the C-domain of CaM (C-CaM) and PEP-19 by solution nuclear magnetic resonance (NMR) to identify mechanisms by which PEP-19 regulates binding of Ca(2+) to CaM. Our results show that PEP-19 causes a greater structural change in apo C-CaM than in Ca(2+)-C-CaM, and that the first Ca(2+) binds preferentially to site IV in the presence of PEP-19 with exchange characteristics that are consistent with a decrease in Ca(2+) binding cooperativity. Relatively weak binding of PEP-19 has distinct effects on chemical and conformational exchange on the microsecond to millisecond time scale. In apo C-CaM, PEP-19 binding causes a redistribution of residues that experience conformational exchange, leading to an increase in the number of residues around Ca(2+) binding site IV that undergo conformational exchange on the microsecond to millisecond time scale. This appears to be caused by an allosteric effect because these residues are not localized to the PEP-19 binding site. In contrast, PEP-19 increases the number of residues that exhibit conformational exchange in Ca(2+)-C-CaM. These residues are primarily localized to the PEP-19 binding site but also include Asp93 in site III. These results provide working models for the role of protein dynamics in the regulation of binding of Ca(2+) to CaM by PEP-19.     

Tissue inhibitor of metalloproteinases-1 undergoes microsecond to millisecond motions at sites of matrix metalloproteinase-induced fit

The N-terminal, matrix metalloproteinase (MMP)-inhibitory fragment of recombinant, human tissue inhibitor of metalloproteinases (TIMP-1) exhibits varied backbone dynamics and rigidity. Most striking is the presence of chemical exchange in the MMP-binding ridge reported to undergo conformational change upon MMP binding. Conformational exchange fluctuations in microseconds to milliseconds map to the sites of MMP-induced fit at residues Val29 through Leu34 of the AB loop and to the Ala65 and Cys70 "hinges" of the CD loop of TIMP-1. Slow chemical exchange is also present at the type I turn of the EF loop at the base of the MMP-binding ridge. These functional slow motions and other fast internal motions are evident from backbone (15)N spin relaxation at 500 and 750 MHz, whether interpreted by the model-free formalism with axial diffusion anisotropy or by the reduced spectral density approach. The conformational exchange is confirmed by its deviation from the trend between R(2) and the cross-correlation rate eta. The magnetic field-dependence indicates that the chemical exchange broadening in the AB and CD loops is fast on the time-scale of chemical shift differences. The conformational exchange rates for most of these exchanging residues, which can closely approach MMP, appear to be a few thousand to several thousand per second. The slow dynamics of the TIMP-1 AB loop contrast the picosecond to nanosecond dynamics reported in the longer TIMP-2 AB loop.     

Interactions of calcineurin A, calcineurin B, and Ca2+.

Calcineurin B (CN-B) is the Ca(2+)-binding, regulatory subunit of the phosphatase calcineurin. Point mutations to Ca(2+)-binding sites in CN-B were generated to disable individual Ca(2+)-binding sites and evaluate contributions from each site to calcineurin heterodimer formation. Ca(2+)-binding properties of four CN-B mutants and wild-type CN-B were analyzed by flow dialysis confirming that each CN-B mutant binds three Ca2+ and that wild-type CN-B binds four Ca2+. Macroscopic dissociation constants indicate that N-terminal Ca(2+)-binding sites have lower affinity for Ca2+ than the C-terminal sites. Each CN-B mutant was coexpressed with the catalytic subunit of calcineurin, CN-A, to produce heterodimers with specific disruption of one Ca(2+)-binding site. Enzymes containing CN-B with a mutation in Ca(2+)-binding sites 1 or 2 have a lower ratio of CN-B to CN-A and a lower phosphatase activity than those containing wild-type CN-B or mutants in sites 3 or 4. Effects of heterodimer formation on Ca2+ binding were assessed by monitoring (45)Ca2+ exchange by flow dialysis. Enzymes containing wild-type CN-B and mutants in sites 1 and 2 exchange (45)Ca2+ slowly from two sites whereas mutants in sites 3 and 4 exchange (45)Ca2+ slowly from a single site. These data indicate that the Ca2+ bound to sites 1 and 2 is likely to vary with Ca2+ concentration and may act in dynamic modulation of enzyme function, whereas Ca(2+)-binding sites 3 and 4 are saturated at all times and that Ca2+ bound to these sites is structural.     

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.     

Thermal stability of calmodulin and mutants studied by (1)H-(15)N HSQC NMR measurements of selectively labeled [(15)N]Ile proteins

Calmodulin, the Ca(2+)-dependent activator of many cellular processes, contains two well-defined structural domains, each of which binds two Ca(2+) ions. In its Ca(2+)-free (apo) form, it provides an attractive model for studying mechanisms of protein unfolding, exhibiting two separable, reversible processes, indicating two structurally autonomous folding units. (1)H-(15)N HSQC NMR in principle offers a detailed picture of the behavior of individual residues during protein unfolding transitions, but is limited by the lack of dispersion of resonances in the unfolded state. In this work, we have used selective [(15)N]Ile labeling of four distinctive positions in each calmodulin domain to monitor the relative thermal stability of the folding units in wild-type apocalmodulin and in mutants in which either the N- or C-domain is destabilized. These mutations lead to a characteristic perturbation of the stability (T(m)) of the nonmutated domain relative to that of wild-type apocalmodulin. The ability to monitor specific (15)N-labeled residues, well-distributed throughout the domain, provides strong evidence for the autonomy of a given folding unit, as well as providing accurate measurements of the unfolding parameters T(m) and DeltaH(m). The thermodynamic parameters are interpreted in terms of interactions between one folded and one unfolded domain of apocalmodulin, where stabilization on the order of a few kilocalories per mole is sufficient to cause significant changes in the observed unfolding behavior of a given folding unit. The selective (15)N labeling approach is thus a general method that can provide detailed information about structural intermediates populated in complex protein unfolding processes.     

Ca2+ binding to calbindin D9k strongly affects backbone dynamics: measurements of exchange rates of individual amide protons using 1H NMR

One- and two-dimensional 1H NMR have been used to study the backbone dynamics in Ca2(+)-free (apo) and Ca2(+)-loaded (Ca2) calbindin D9k at pH 7.5 and 25 degrees C. Hydrogen exchange rates of all 71 backbone amide protons (NH's) have been measured for the Ca2 form by both a direct exchange-out experiment and another experiment that measures the transfer of saturation from water protons to amide protons. A large number of NH's are found to be highly protected against exchange with solvent protons. The results for the Ca2 form are related to solvent accessibility and hydrogen bonding obtained in molecular dynamics simulations of calcium-loaded calbindin. The correlation with these parameters is strong within the N-terminal half of calbindin, which is found to be more stable than the C-terminal half. The amide proton exchange in the apo form is much faster than in the Ca2 form and was studied in a series of experiments in which the exchange was quenched after different times by Ca2+ addition. This experiment is applicable to all amide hydrogens that exchange slowly in the Ca2 form. For these NH's the effects of Ca2+ removal span from a 10(2)-fold decrease to a 10(5)-fold increase of the exchange rate, and the average is a 220-fold increase. The effects on individual NH exchange rates show that the four alpha-helices are almost intact after calcium removal and that the changes in dynamics involve not only the Ca2(+)-binding region. Hydrogen bonds involving backbone NH's in the Ca2+ loops appear to be broken or weakened when calbindin releases Ca2+, whereas the beta-sheet between the Ca2+ loops is found to be present in both the Ca2 and apo forms. Large Ca2(+)-induced effects on NH exchange rates were measured for a few residues at alpha-helix ends far from the two Ca2(+)-binding sites. This may be the result of a change in interhelix angles (or the rate of interhelix angle fluctuations) on calcium binding.     

Calcium-induced changes in calmodulin structural dynamics and thermodynamics

The thermodynamics of the interaction between Ca(2+) and calmodulin (CaM) was examined using isothermal titration calorimetry (ITC). The chemical denaturation of calmodulin was monitored spectroscopically to determine the stability of Ca(2+)-free (apo) and Ca(2+)-loaded (holo) CaMs. We explored the conformational and structural dynamics of CaM using amide hydrogen-deuterium (H-D) exchange coupled with Fourier transform infrared (FT-IR) spectroscopy. The results of H-D exchange and FT-IR suggest that CaM activation by Ca(2+) binding involves significant conformational changes. The results have also revealed that while the overall conformation of holo-CaM is more stable than that of the apo-CaM, some part of its α-helix structures, most likely the EF-hand domain region, has more solvent exposure, thus, has a faster H-D exchange rate than that of the apo-CaM. The ITC method provides a new strategy for obtaining site-specific Ca(2+) binding properties and a better estimation of the cooperativity and conformational change contributions of coupled EF-hand proteins.     

Conformational change upon ligand binding and dynamics of the PDZ domain from leukemia-associated Rho guanine nucleotide exchange factor

Leukemia-associated Rho guanine nucleotide exchange factor (LARG) is a RhoA-specific guanine nucleotide exchange factor (GEF) that can activate RhoA. The PDZ (PSD-95/Disc-large/ZO-1 homology) domain of LARG interacts with membrane receptors, which can relay extracellular signals to RhoA signal transduction pathways. Until now there is no structural and dynamic information about these interactions. Here we report the NMR structures of the LARG PDZ in the apo form and in complex with the plexin-B1 C-terminal octapeptide. Unobservable resonances of the residues in betaB/betaC and betaE/alphaB loops in apo state were observed in the complex state. A distinct region of the binding groove in the LARG PDZ was found to undergo conformational change compared with other PDZs. Analysis of the (15)N relaxation data using reduced spectral density mapping shows that the apo LARG PDZ (especially its ligand-binding groove) is flexible and exhibits internal motions on both picosecond to nanosecond and microsecond to millisecond timescales. Mutagenesis and thermodynamic studies indicate that the conformation of the betaB/betaC and betaE/alphaB loops affects the PDZ-peptide interaction. It is suggested that the conformational flexibility could facilitate the change of structures upon ligand binding.     

The second Ca(2+)-binding domain of NCX1 binds Mg2+ with high affinity

We report the effects of binding of Mg(2+) to the second Ca(2+)-binding domain (CBD2) of the sodium-calcium exchanger. CBD2 is known to bind two Ca(2+) ions using its Ca(2+)-binding sites I and II. Here, we show by nuclear magnetic resonance (NMR), circular dichroism, isothermal titration calorimetry, and mutagenesis that CBD2 also binds Mg(2+) at both sites, but with significantly different affinities. The results from Mg(2+)-Ca(2+) competition experiments show that Ca(2+) can replace Mg(2+) from site I, but not site II, and that Mg(2+) binding affects the affinity for Ca(2+). Furthermore, thermal unfolding circular dichroism data demonstrate that Mg(2+) binding stabilizes the domain. NMR chemical shift perturbations and (15)N relaxation data reveal that Mg(2+)-bound CBD2 adopts a state intermediate between the apo and fully Ca(2+)-loaded forms. Together, the data show that at physiological Mg(2+) concentrations CBD2 is loaded with Mg(2+) preferentially at site II, thereby stabilizing and structuring the domain and altering its affinity for Ca(2+).     

Battle for the EF-hands: magnesium-calcium interference in calmodulin.

The ubiquitous Ca(2+)-regulatory protein calmodulin activates target enzymes as a response to submicromolar Ca(2+) increases in a background of millimolar Mg(2+). The potential influence of Mg(2+)/Ca(2+) competition is especially intriguing for the N-terminal domain of the protein which possesses the sites with the lowest Ca(2+) specificity. The interdependence of Ca(2+) and Mg(2+) binding in the N-terminal domain of calmodulin was therefore studied using (43)Ca NMR, (1)H-(15)N NMR, and fluorescent Ca(2+) chelator techniques. The apparent affinity for Ca(2+) was found to be significantly decreased at physiological Mg(2+) levels. At Ca(2+) concentrations of an activated cell the (Ca(2+))(2) state of the N-terminal domain is therefore only weakly populated, indicating that for this domain Ca(2+) binding is intimately associated with binding of target molecules. The data are in good agreement with a two-site model in which each site can bind either Ca(2+) or Mg(2+). The Mg(2+)-Ca(2+) binding interaction is slightly positively allosteric, resulting in a significantly populated (Mg(2+))(1)(Ca(2+))(1) state. The Ca(2+) off-rate from this state is determined to be at least one order of magnitude faster than from the (Ca(2+))(2) state. These two findings indicate that the (Mg(2+))(1)(Ca(2+))(1) state is structurally and/or dynamically different from the (Ca(2+))(2) state. The (43)Ca quadrupolar coupling constant and the (1)H and (15)N chemical shifts of the (Mg(2+))(1)(Ca(2+))(1) state were calculated from titration data. The values of both parameters suggest that the (Mg(2+))(1)(Ca(2+))(1) state has a conformation more similar to the "closed" apo and (Mg(2+))(2) states than to the "open" (Ca(2+))(2) state.     

Effect of metal ion binding on the secondary structure of bovine alpha-lactalbumin as examined by infrared spectroscopy

We have examined the influence of monovalent and divalent cations on the secondary structure of bovine alpha-lactalbumin at neutral pH using Fourier-transform infrared spectroscopy. Our present studies are based on previously reported amide I' component band assignments for this protein [Prestrelski, S. J., Byler, D. M., & Thompson, M. P. (1991) Int. J. Pept. Protein Res. 37, 508-512]. The results indicate that upon dissolution, alpha-lactalbumin undergoes a small, but significant, time-dependent conformational change, regardless of the ions present. Additionally, these studies provide the first quantitative measure of the well-known secondary structural change which accompanies calcium binding. Results indicate that removal of Ca2+ from holo alpha-lactalbumin results in local unfolding of the Ca(2+)-binding loop; the spectra indicate that approximately 16% of the backbone chain changes from a rigid coordination complex to an unordered loop. We have also examined the effects of binding of several other metal ions. Our studies have revealed that binding of Mn2+ to apo alpha-lactalbumin (Ca(2+)-free), while inducing a small, but significant, conformational change, does not cause the alpha-lactalbumin backbone conformation to change to that of the holo (Ca(2+)-bound) form as characterized by infrared spectroscopy. Similar changes to those induced by Mn2+ are observed upon binding of Na+ to apo alpha-lactalbumin, and furthermore, even at very high concentrations (0.2 M), Na+ does not stabilize a structure similar to the holo form. Binding of Zn2+ to the apo form of alpha-lactalbumin does not result in significant backbone conformational changes, suggesting a rigid Zn(2+)-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)