In vitro and in vivo studies of creatine monohydrate supplementation to Duroc and Landrace pigs.

Duroc and Landrace pigs as well as primary myotubes from these breeds were used to investigate mechanisms behind differences in their response to creatine monohydrate (CMH). Pigs were supplemented with 0, 12.5, 25 or 50g CMH/d for 5 days (n=10 per treatment and breed). Plasma levels of creatine increased dose-dependently in both breeds, while muscle-creatine phosphate content increased only in the Duroc pigs. (1)H NMR metabolic profiling showed a tendency towards clustering according to CMH supplementation only among Duroc pigs, revealing a stronger response compared to Landrace pigs. The abundance of insulin-like growth factor I and myostatin mRNA was decreased by CMH supplementation while that of type 1 IGF-receptor and creatine transporter was unaffected. Protein synthesis, increased in the myotubes from both breeds, indicating protein accretion, but no effect was observed on the mRNA abundance of IGF-I, type 1 IGF-receptor, myostatin or the creatine transporter in myotubes.

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.

Structural dynamics in the C-terminal domain of calmodulin at low calcium levels.

Calmodulin undergoes Ca2+-induced structural rearrangements that are intimately coupled to the regulation of numerous cellular processes. The C-terminal domain of calmodulin has previously been observed to exhibit conformational exchange in the absence of Ca2+. Here, we characterize further the conformational dynamics in the presence of low concentrations of Ca2+ using 15N spin relaxation experiments. The analysis included 1H-15N dipolar/15N chemical shift anisotropy interference cross-correlation relaxation rates to improve the description of the exchange processes, as well as the picosecond to nanosecond dynamics. Conformational transitions on microsecond to millisecond time scales were revealed by exchange contributions to the transverse auto-relaxation rates. In order to separate the effects of Ca2+ exchange from intramolecular conformational exchange processes in the apo state, transverse auto-relaxation rates were measured at different concentrations of free Ca2+. The results reveal a Ca2+-dependent contribution due mainly to exchange between the apo and (Ca2+)1 states with an apparent Ca2+ off-rate of approximately 5115 s(-1), as well as Ca2+-independent contributions due to conformational exchange within the apo state. 15N chemical shift differences estimated from the exchange data suggest that the first Ca2+ binds preferentially to loop IV. Thus, characterization of chemical exchange as a function of Ca2+ concentration has enabled the extraction of unique information on the rapidly exchanging and weakly populated (<10 %) (Ca2+)1 state that is otherwise inaccessible to direct study due to strongly cooperative Ca2+ binding. The conformational exchange within the apo state appears to involve transitions between a predominantly populated closed conformation and a smaller population of more open conformations. The picosecond to nanosecond dynamics of the apo state are typical of a well-folded protein, with reduced amplitudes of motions in the helical segments, but with significant flexibility in the Ca2+-binding loops. Comparisons with order parameters for skeletal troponin C and calbindin D9k reveal key structural and dynamical differences that correlate with the different Ca2+-binding properties of these proteins.

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.

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.

When size is important. Accommodation of magnesium in a calcium binding regulatory domain.

The accommodation of Mg2+ in the N-terminal domain of calmodulin was followed through amide 1H and 15N chemical shifts and line widths in heteronuclear single-quantum coherence spectroscopy NMR spectra. Mg2+ binds sequentially to the two Ca2+-binding loops in this domain, with affinities such that nearly half of the loops would be occupied by Mg2+ in resting eukaryotic cells. Mg2+ binding seems to occur without ligation to the residue in the 12th loop position, previously proven largely responsible for the major rearrangements induced by binding of the larger Ca2+. Consequently, smaller Mg2+-induced structural changes are indicated throughout the protein. The two Ca2+-binding loops have different Mg2+ binding characteristics. Ligands in the N-terminal loop I are better positioned for cation binding, resulting in higher affinity and slower binding kinetics compared with the C-terminal loop II (koff = 380 +/- 40 s-1 compared with approximately 10,000 s-1 at 25 degreesC). The Mg2+-saturated loop II undergoes conformational exchange on the 100-microseconds time scale. Available data suggest that this exchange occurs between a conformation providing a ligand geometry optimized for Mg2+ binding and a conformation more similar to that of the empty loop.

Ca2+ binding and conformational changes in a calmodulin domain.

Calcium activation of the C-terminal domain of calmodulin was studied using 1H and 15N NMR spectroscopy. The important role played by the conserved bidentate glutamate Ca2+ ligand in the binding loops is emphasized by the striking effects resulting from a mutation of this glutamic acid to a glutamine, i.e. E104Q in loop III and E140Q in loop IV. The study involves determination of Ca2+ binding constants, assignments, and structural characterizations of the apo, (Ca2+)1, and (Ca2+)2 states of the E104Q mutant and comparisons to the wild-type protein and the E140Q mutant [Evenäs et al. (1997) Biochemistry 36, 3448-3457]. NMR titration data show sequential Ca2+ binding in the E104Q mutant. The first Ca2+ binds to loop IV and the second to loop III, which is the order reverse to that observed for the E140Q mutant. In both mutants, the major structural changes occur upon Ca2+ binding to loop IV, which implies a different response to Ca2+ binding in the N- and C-terminal EF-hands. Spectral characteristics show that the (Ca2+)1 and (Ca2+)2 states of the E104Q mutant undergo global exchange on a 10-100 micros time scale between conformations seemingly similar to the closed and open structures of this domain in wild-type calmodulin, paralleling earlier observations for the (Ca2+)2 state of the E140Q mutant, indicating that both glutamic acid residues, E104 and E140, are required for stabilization of the open conformation in the (Ca2+)2 state. To verify that the NOE constraints cannot be fulfilled in a single structure, solution structures of the (Ca2+)2 state of the E104Q mutant are calculated. Within the ensemble of structures the precision is good. However, the clearly dynamic nature of the state, a large number of violated distance restraints, ill-defined secondary structural elements, and comparisons to the structures of calmodulin indicate that the ensemble does not provide a good picture of the (Ca2+)2 state of the E104Q mutant but rather represents the distance-averaged structure of at least two distinct different conformations.

Calcium.

Ca2+ is involved in an intriguing variety of different biological events. The rapid development of techniques such as region- or organelle-directed fluorescent probes and laser scanning confocal microscopy for studying cellular biological events at a molecular level provides us with a rich daily intake of new results. While detailed three-dimensional structures of many intracellular and extracellular Ca2+-binding proteins have become available, structural information on key membrane proteins is still lacking. An integrated picture of the molecular events behind the multifunctional roles of Ca2+ in biological systems is still pending.

Sequence and context dependence of EF-hand loop dynamics. An 15N relaxation study of a calcium-binding site mutant of calbindin D9k.

The influence of amino acid sequence and structural context on the backbone dynamics of EF-hand calcium-binding loops was investigated using 15N spin relaxation measurements on the calcium-free state of the calbindin D9k mutant (A14D+A15Delta+P20Delta+N21G+P43M), in which the N-terminal pseudo-EF-hand loop, characteristic of S100 proteins, was engineered so as to conform with the C-terminal consensus EF-hand loop. The results were compared to a previous study of the apo state of the wild-type-like P43G calbindin D9k mutant. In the helical regions, the agreement with the P43G data is excellent, indicating that the structure and dynamics of the protein core are unaffected by the substitutions in the N-terminal loop. In the calcium-binding loops, the flexibility is drastically decreased compared to P43G, with the modified N-terminal loop showing a motional restriction comparable to that of the surrounding helixes. As in P43G, the motions in the C-terminal loop are less restricted than in the N-terminal loop. Differences in key hydrogen-bonding interactions correlate well with differences in dynamics and offer insights into the relationship between structure and dynamics of these EF-hand loops. It appears that the entire N-terminal EF-hand is built to form a rigid structure that allows calcium binding with only minor rearrangements and that the structural and dynamical properties of the entire EF-hand--rather than the loop sequence per se--is the major determinant of loop flexibility in this system.

Solution structure of the paramagnetic complex of the N-terminal domain of calmodulin with two Ce3+ ions by 1H NMR.

The solution structure of the dicerium(III) complex of the N-terminal domain of calmodulin (Ce2-TR1C hereafter) has been solved employing paramagnetic T1 relaxation enhancements and pseudocontact shifts introduced by the Ce3+ ions, together with conventional NOE constraints. The use of pseudocontact shift constraints constitutes the first attempt to locate metal ions within a protein structure by NMR. Like calcium(II), paramagnetic cerium(III) has been found to bind to the two metal binding sites of the TR1C fragment of calmodulin in a cooperative manner. Due to the presence of pseudocontact interactions between the Ce3+ ions and protons of the 76-residue protein, the 1H NMR spectra of the complex show resonances shifted between +22 and -9 ppm. Eighty percent of its proton resonances could be assigned through a standard approach using TOCSY/COSY and NOESY spectra and through 1D NOE difference spectra for the broad resonances of protons close to the paramagnetic ions. A family of structures was calculated by means of the torsion angle dynamics program DYANA [Güntert, P., Mumenthaler, C., & Wüthrich, K. (1996) XVIIthInternational Conference on Magnetic Resonance inBiological Systems (Abstract)] using 1012 NOEs. Longitudinal proton relaxation times helped to roughly define the position of the metal ions within the protein. A total of 381 pseudocontact shift constraints, whose evaluation and use are critically discussed, have then been added to further refine the metal coordinates within the protein frame and to improve the structure resolution. A dramatic resolution improvement of the metal coordinates together with a sizable resolution improvement in the regions close to the paramagnetic centers, where the number of NOEs is low, is observed. The good quality of the solution structure permitted a meaningful comparison with the solid-state structure of calcium-loaded calmodulin at 1.7 A resolution [Chattopadhyaya, R., Meador, W. E., Means, A. R., & Quiocho, F. A. (1992) J. Mol. Biol. 228, 1177]. The Ce2-TR1C complex is overall more compact than the Ca form.

Structural basis for the negative allostery between Ca(2+)- and Mg(2+)-binding in the intracellular Ca(2+)-receptor calbindin D9k.

The three-dimensional structures of the magnesium- and manganese-bound forms of calbindin D9k were determined to 1.6 A and 1.9 A resolution, respectively, using X-ray crystallography. These two structures are nearly identical but deviate significantly from both the calcium bound form and the metal ion-free (apo) form. The largest structural differences are seen in the C-terminal EF-hand, and involve changes in both metal ion coordination and helix packing. The N-terminal calcium binding site is not occupied by any metal ion in the magnesium and manganese structures, and shows little structural deviation from the apo and calcium bound forms. 1H-NMR and UV spectroscopic studies at physiological ion concentrations show that the C-terminal site of the protein is significantly populated by magnesium at resting cell calcium levels, and that there is a negative allosteric interaction between magnesium and calcium binding. Calcium binding was found to occur with positive cooperativity at physiological magnesium concentration.

NMR studies of the E140Q mutant of the carboxy-terminal domain of calmodulin reveal global conformational exchange in the Ca2+-saturated state.

In the present investigation, the Ca2+ activation of the C-terminal domain of bovine calmodulin and the effects of replacing the bidentate Ca2+-coordinating glutamic acid residue in the 12th and last position of loop IV with a glutamine are studied by NMR spectroscopy. The mutation E140Q results in sequential Ca2+ binding in this domain and has far-reaching effects on the structure of (Ca2+)2 TR2C, thereby providing further evidence for the critical role of this glutamic acid residue for the Ca2+-induced conformational change of regulatory EF-hand proteins. Analyses of the NOESY spectra of the mutant under Ca2+-saturated conditions, such that 97% of the protein is in the (Ca2+)2 form, revealed two sets of mutually exclusive NOEs. One set of NOEs is found to be consistent with the closed structure observed in the apo state of the C-terminal domain of the wild-type protein, while the other set supports the open structure observed in the Ca2+-saturated state. In addition, several residues in the hydrophobic core exhibit broadened resonances. We conclude that the (Ca2+)2 form of the mutant experiences a global conformational exchange between states similar to the closed and open conformations of the C-terminal domain of wild-type calmodulin. A population of 65 +/- 15% of the open conformation and an exchange rate of (1-7) x 10(4) s(-1) were estimated from the NMR data and the chemical shifts of the wild-type protein. From a Ca2+ titration of the 15N-labeled mutant, the macroscopic binding constants [log(K1) = 4.9 +/- 0.3 and log(K2) = 3.15 +/- 0.10] and the inherent chemical shifts of the intermediate (Ca2+)1 form of the mutant were determined using NMR. Valuable information was also provided on the mechanism of the Ca2+ activation and the roles of the structural elements in the two Ca2+-binding events. Comparison with the wild-type protein indicates that the (Ca2+)1 conformation of the mutant is essentially closed but that some rearrangement of the empty loop IV toward the Ca2+-bound form has occurred.