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PLoS ONEplosplosonePLoS ONE1932-6203 Public Library of Science San Francisco, USA 10-PONE-RA-21673R110.1371/journal.pone.0014718 Research Article Biochemistry Biophysics Chemical Biology Computational Biology Biochemistry/Biomacromolecule-Ligand Interactions Biochemistry/Chemical Biology of the Cell Biophysics/Biomacromolecule-Ligand Interactions Biophysics/Protein Chemistry and Proteomics Biophysics/Theory and Simulation Chemical Biology/Macromolecular Chemistry Computational Biology/Molecular Dynamics The Dynamics of Ca2+ Ions within the Solvation Shell of Calbindin D9kCa2+ Motion on Protein Surface Project Elad Nachliel Esther Gutman Menachem * Laser Laboratory for Fast Reactions, Biochemistry, Tel Aviv University, Tel Aviv, Israel Langowski Jörg Editor German Cancer Research Center, Germany * E-mail:

Conceived and designed the experiments: EP EN MG. Performed the experiments: EP. Analyzed the data: EP EN MG. Wrote the paper: EP EN MG.

The authors have declared that no competing interests exist.

2011 22 2 2011 62e14718 29 7 2010 28 1 2011 2011Project et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The encounter of a Ca2+ ion with a protein and its subsequent binding to specific binding sites is an intricate process that cannot be fully elucidated from experimental observations. We have applied Molecular Dynamics to study this process with atomistic details, using Calbindin D9k (CaB) as a model protein. The simulations show that in most of the time the Ca2+ ion spends within the Debye radius of CaB, it is being detained at the 1st and 2nd solvation shells. While being detained near the protein, the diffusion coefficient of the ion is significantly reduced. However, due to the relatively long period of detainment, the ion can scan an appreciable surface of the protein. The enhanced propagation of the ion on the surface has a functional role: significantly increasing the ability of the ion to scan the protein's surface before being dispersed to the bulk. The contribution of this mechanism to Ca2+ binding becomes significant at low ion concentrations, where the intervals between successive encounters with the protein are getting longer. The efficiency of the surface diffusion is affected by the distribution of charges on the protein's surface. Comparison of the Ca2+ binding dynamics in CaB and its E60D mutant reveals that in the wild type (WT) protein the carboxylate of E60 function as a preferred landing-site for the Ca2+ arriving from the bulk, followed by delivering it to the final binding site. Replacement of the glutamate by aspartate significantly reduced the ability to transfer Ca2+ ions from D60 to the final binding site, explaining the observed decrement in the affinity of the mutated protein to Ca2+.

The authors have no support or funding to report.

Intracellular calcium plays an essential role in the transduction of most hormonal, neuronal and muscular stimuli. Cells have a multi-components calcium signaling toolkits that can be assembled to create a wide range of spatial and temporal signals. This versatility is exploited to control processes as diverse as fertilization, proliferation, development, learning and memory, contraction and secretion [1]. In general, a Ca2+ signal begins with the activation of a cell surface receptor or channel protein by an extracellular stimulus: virtually all tyrosine-kinase-linked receptors, G-protein-linked seven-helix transmembrane receptors, and Ca2+ channels trigger one or more Ca2+ responses [2]. The signal from the cell surface, which arrives at the intracellular Ca2+ store via a second messenger or via direct electrical contact, opens Ca2+ channels and thereby releases Ca2+ into the cytoplasm. The elevated Ca2+ concentration modulates Ca2+ regulatory proteins at key control points in essential physiological pathways, until the Ca2+ is pumped out of the cytoplasm by a Ca2+ ATPase.

Being of crucial importance to all living organisms, Ca2+ binding proteins have been the subject of many studies. One of the most prevalent Ca2+ binding structures is a conserved motif termed the EF-hand [3]. The EF-hand motif is widely distributed in Ca2+ signaling pathways and acts both in modulation, buffering and protein stabilization. The heart of the EF-hand is its Ca2+-binding ‘loop’, consisting of twelve consecutive residues, six of which provide direct or indirect Ca2+ coordination. Bordering the loop are two α-helices which serve to anchor the loop and to communicate the information of Ca2+ binding to distant regions of the molecule. Usually, two EF-hand sites are associated in the same protein domain to yield a highly cooperative Ca2+ binding system.

Various studies on EF-hand containing proteins have yielded different parameters such as specificity of the EF-hand to Ca2+ vs. Mg2+ and the rate constants of the process [2]. The association rate constants (kon) values of the binding process vary over a wide range for different EF-hand proteins and chelators and are considerably slower than the diffusion-controlled limit. This observation is in apparent contrast to the rapid rate of inner sphere water substitution of Ca2+, ranging between 108 and 109 sec−1 [4], suggesting that the binding mechanism conceals a complex sequence of events in addition to the arrival of the Ca2+ ion to the binding site. These events can not be elucidated by measurements of the association rate constants (kon), as this value is calculated indirectly from the equilibrium constants (Keq) and the dissociation constants (koff) of the process (kon = koff * Keq) [5] [6]. Thus, the derived kon value incorporates the overall binding process and does not give insights on the detailed mechanism, which for Ca2+ ions, may be quite complex, as evident by the wide range of association rates found in the organic and inorganic world.

Several aspects of the binding mechanism of Ca2+ to EF-hand proteins can be derived from studies on the properties of the Ca2+ ion and its binding with chelators. The dehydration free energy of Ca2+ is as high as +362 kcal/mol [7], necessitating that any effective chelating site must replace the solvating water with strong coordinating interactions that more than offset this energetic cost. The ionic radius of Ca2+ is such that 7 or 8 coordinating oxygen atoms can be comfortably accommodated around the ion. Thus, these coordination numbers are most frequently observed in inorganic complexes. The kinetics of spherical metal ion binding reveals the key molecular details of the process by which a solvated ion is engulfed by chelators [8], [9]. The association kinetics are considerably faster than would be expected for a transition state involving the bare metal ion; thus the reaction mechanism is actually a stepwise replacement of solvent by ligands of the chelators, so that the ion retains approximately its normal coordination number throughout the chelating reaction.

The modeling of the complex formation should account for the flexibility of the residues in the binding site. High flexibility of the residues may slow the rate of bond formation between the ion and the protein, since the residue may have a lot of conformational space to search for the ion. On the other hand, a rigid binding site may also hinder the binding process as the ion has to perfectly align itself. What is more, it is logical to assume that the interaction of the ion with protein causes structural rearrangements in the protein. This has been witnessed in several experimental and computational studies [10], [11], [12], [13], [14], [15], [16], where removal of Ca2+ from the binding sites of Calmodulin (CaM) significantly altered the stability of the protein. Such conformational changes may expedite the binding process.

Beside the stepwise bond formation that seems to be the rate limiting step of the binding process, the encounter of the Ca2+ ion with the binding site, which is the first step in the binding process, raises interesting questions regarding the mechanism by which the ion approaches the binding site. This event may occur by a simple Brownian diffusion from the bulk. Yet it is very likely that the ion first encounters a negatively charged group on the surface of the protein and is detained in the group's Coulomb cage. In the detained state the ion may shuttle to nearby negative groups, progressing from one carboxyl to another, until it reaches the correct binding site, a concept known as the antenna effect [17]. Ca2+ ions, having a greater charge density and a more intricate coordination system than a proton, Na+ or Cl ions, poses new challenges to the suggested mechanism since the solvent-ion interactions are more complex.

Site specific mutations of Calbindin D9k (CaB) demonstrate that its surface charges play a significant role in the binding of Ca2+. In these experiments, charged surface residues of CaB, which do not serve as direct ligands of the Ca2+, were neutralized and the overall rate constant for Ca2+ binding (kon = koff * Keq) was shown to reduce significantly in a low ionic strength environment [5]. In physiological ionic strength, the reduction of the rate constant was much less dramatic. This observation demonstrates the crucial role of electrostatics in the binding process. Since the charges on the surface of the protein are unevenly dispersed, the detailed electrostatic potential around the protein must be considered [18]. The detailed treatment of the local electrostatic potential is of special importance at high ionic strength solution (I≥0.1 M), where the Coulomb cage radius is in the order of 2–3 water molecules. Under conditions where the electrostatic potential is screened at a short range, a negative lobe of the Coulomb cage can be broken into a set of local potential traps that may delay the ion, reducing its ability to rapidly scan the protein's surface. Conversely, one can argue that when an ion is in close proximity to the binding site, attractive surface charges not located at the binding site may deflect it from reaching the binding site, causing a decrease in the kon values as compared to the same protein where these surface charges are neutralized. It is therefore clear that a more detailed evaluation of the reaction mechanism is needed.

The present research investigates the mechanism of Ca2+ binding to EF-hand proteins. While it is problematic to devise an experimental system suitable to monitor the dynamics of Ca2+ on a protein, one can use molecular dynamics to simulate the process and gain insights on the mechanism of the process. By using the simulations, the underlying processes which make up the overall binding process can be elucidated, breaking up the experimentally measured kon to its constituent components. The ion detainment is a general feature reflecting the effect of local fields on the diffusion of ions. In the case of S6, where the protein does not utilize any ion for its activity, the ion detainment appears as a simple consequence of the ions interacting with the protein's electrostatic field [17]. In case of Calbindin D9k, the detainment may have a functional role.

The structure of the CaB protein [19] is of a single domain that resembles the Calmodulin's Ca2+ binding domain [20]. It is used as a model for many Ca2+ binding studies in EF-hand proteins [21] and its small size (78 residues) makes it ideal for molecular dynamics simulations. What is more, the experimental measurements on CaB mutants can be used as references for simulations on similar mutants. In the present study, we performed 100 ns long simulation of the WT protein and the E60D mutant, which is known experimentally to have a reduced affinity to Ca2+ [22]. The simulations show that in most of the time the Ca2+ ion spends within the Debye radius of CaB, it is detained at the 2nd and 1st solvation shells. While being detained near the protein, the diffusion coefficient of the ion is significantly reduced. However, due to the relative long period of detainment, the ion can scan an appreciable surface of the protein. This surface scanning may play a functional role, significantly increasing the surface area of the protein available for the Ca2+ ion, especially at low Ca2+ concentrations. This mechanism was demonstrated by comparison of the Ca2+ binding dynamics in CaB and its E60D mutant. In the WT CaB, the side chain carboxylate of E60 was witnessed to be an intermediate station for the Ca2+ ion arriving from the bulk, on their way to the binding residues. As witnessed from the simulations, the E60D mutant has a significantly reduced ability to transfer Ca2+ ions from D60 to important binding residues, thereby decreasing the overall binding affinity, explaining the experimental observations.

Results and Discussion The ion binding process

The mechanism leading to the binding of an ion (or any other small molecule) to its specific binding site consists of three processes: (1) The encounter of the ion with the perimeter of the protein (usually considered as the Debye radius of the protein) (2) Wandering of the ion in the immediate proximity of the protein and (3) The final encounter with the specific binding site and the formation of the stable complex. The velocity of the first process is of a diffusion controlled reaction and varies with the concentrations of the reactants. The second process is concentration independent and the last one may reflect some rate limiting steps like preparatory conformational changes or those that follow the complex formation and stabilize its structure.

In this study we focused our attention on the 2nd and 3rd aspects of the binding process, using molecular dynamics simulations. The system consisted of a single protein molecule, stripped from the Ca2+ ion and fully relaxed, embedded in a box stretching a little more than 1.2 nm from the protein in the presence of sufficient Na+ and Cl ions to attain electroneutrality and maintain ionic strength of ∼0.1 M. The system contained also two Ca2+ ions, sufficient to saturate both binding site. The observed frequency of encounter between the Ca2+ ions with the protein was 1–2 per ns, in accord with the rate estimated by the Debye-Smoluchowski equation. It should be stressed that the nominal concentration of the Ca2+ was ∼25 µM, which is ∼3 orders of magnitude above the physiologic concentration of the ion. However, any attempt to adhere to the physiologic Ca2+ concentration will waste most of the simulation time waiting for primary encounters of the ion with the protein.

The electrostatic potential surrounding the protein

The encounters of the ions with the protein are sensitive not only to the total charge of the protein but also to its precise distribution over the protein's surface [23]. The electrostatic field around CaB, accounting for the individual location of the charges and for the high ionic strength of the solution (150 mM) was calculated by the APBS program and is presented in Figure 1. Due to nature of the calculation, which is performed on a static structure and is dependent on the selected dielectric constants, some variations are expected. Thus, the results of the calculation demonstrate the trends rather than supply accurate quantitative values.

10.1371/journal.pone.0014718.g001 The electrostatic iso-potential surfaces of Apo-CaB.

The electrostatic potential as calculated for the protein in ionic strength of 150 mM. The dielectric constant of the protein was set as εp = 2 and εw = 78. (A) 1 kBT surface, (B) 4 kBT surface and (C) 10 kBT surface.

Figure 1 depicts the surface of the electrostatic potential at 1, 4 and 10 kBT levels at panels A, B and C, respectively. The 1 kBT potential (Figure 1, A) surface appears to be smooth and homogenous, located at distance of ∼10 Å from the surface of the protein (which we define as the distance to the nearest protein atom). The potential surface exhibits a major negative lobe (red) and a smaller positive one (blue). This presentation can be fairly approximated by the homogenous spherical Coulomb cage. As the Ca2+ ions penetrate the 1 kBT surface, the electrostatic potential increase steeply and in parallel looses its smooth appearance. At the level of 4 kBT (Figure 1, B), the surface is already bumpy and positive (repulsive) domains appear to be located below the smooth −1 kBT outer envelop, at a distance of up to 5 Å from the protein surface. Thus, as the Ca2+ ion is propagating towards the protein, its trajectory is affected by the local non homogenous field and specific routes are preferred. The 10kBT electrostatic potential (Figure 1, C) stretches to at most 3 Å from the protein surface (the first hydration shell) so while bound to the protein, the ion is trapped within fairly deep energetic wells, severely limiting its motional liberation.

Diffusion of the ion from the bulk to the vicinity of the protein

To evaluate the effect of the increasing electrostatic potential on the translational motion of the Ca2+ ion, we calculated the diffusion coefficient of the ion, D(Ca2+), as a function of its distance from the protein (defined as the distance between the Ca2+ ion and its nearest protein atom). The results, presented in Figure 2, demonstrate the steep variation of D(Ca2+) as the distance from the protein's surface changes. Ions located within the first shell (the definitions of the shells are detailed in Table 1) exhibit a diffusion coefficient of 0.166×10−5 cm2s−1 ±0.049×10−5, which only slightly exceeds the diffusion coefficient of the protein (0.110×10−5 cm2s−1 ±0.022×10−5). Ions in the second solvation shell are almost as immobile as those in the first shell. The intensity of the electrostatic potential in the second shell is in the order of 4 kBT, explaining the restriction on the translational motion of the ions at that distance from the protein. Ions located out of the 1 kBT boundary, about 10 Å from the surface of the protein, exhibit diffusion coefficient of 1.303×10−5 cm2s−1 ±0.140×10−5, that is close to the one computed for a free ion, indicating that there are no restrictions on their ability to propagate in the diffusion space.

10.1371/journal.pone.0014718.g002 The variation of the Ca2+ ion's diffusion coefficient in the successive shells of Apo-CaB.

The bulk columns (either from simulations or measured experimentally) refer to Ca2+ in protein-free solutions. * The calculated value is comparable to that of a globular protein of the same size [24], [25]. ** The calculated value is comparable with the experimental diffusion coefficient of free Ca2+ ions [26].

10.1371/journal.pone.0014718.t001 Residence time of Ca2+ ions near the CaB protein.
Shell distance range from the surface of the protein (nm) τ1 (ps) τ2 (ps) Relative RMS error of fit
d<0.35 1.389 2670.862 0.742
0.35<d<0.6 3.611 212.193 0.663
0.6<d<0.85 1.388 22.131 0.164
0.85<d<1.1 1.072 14.341 0.057
1.1<d<1.35 0.969 13.516 0.269
1.35<d<1.6 1.145 13.774 0.183
1.6<d<1.85 1.631 15.502 0.123
1.85<d<2.1 1.876 14.695 0.223

The accuracy of the calculation is supported by the comparison of the diffusion coefficient of CaB, derived from the simulations (0.110×10−5 cm2s−1 ±0.022×10−5), with that measured for Lysozyme (0.111×10−5 cm2s−1) [24],[25], a protein with comparable shape and mass. Similarly, the diffusion coefficient calculated from the simulation for the Ca2+ in water is only slightly smaller than the experimental of 1.584×10−5 cm2s−1 [26], which can be attributed to the non-vanishing concentration of the ion in the simulation system [27].

The residence time of Ca2+ at the vicinity of the protein

The reduction in the translational diffusivity of the Ca2+ ions near the protein implies that the residence time of the ions should also vary as a function of the ion's distance from the protein. During the simulation time, the Ca2+ ions were scanning the whole aqueous phase around the protein, but not at homogenous distribution. Figure 3 (black curve) relates the percentage of time a Ca2+ ion spends at a given distance from the protein, out of the total time it spends within a distance of 1.2 nm from the protein. The trace clearly indicates that the highest probability to find the ion is within the first shell. A secondary (significantly smaller) peak that corresponds with the second shell is also clearly visible. The accumulation of the calculated curve (red) indicates that at ∼65% of the time, the ion was located within the first shell of the protein, and at another 20% of the time, it was in the second shell. Close inspection of the histogram reveals hints for more remote shells, however these are hardly ordered and we have thus defined the remaining shells consecutively with a fixed width of 2.5 Å (detailed in Table 1). It should be noticed that the second peak has its maximum at ∼4.5 Å, compatible with the sum of a water molecule diameter and the ionic radius of Ca2+. Thus, it appears that the Ca2+ ion can either be directly interacting with the polar residues of CaB or through a single water molecule that bridge between them.

10.1371/journal.pone.0014718.g003 Ca2+ distribution around Apo-CaB.

A histogram of the percentage of time a Ca2+ ion spends at a specific distance from the Apo-CaB, out of the total time it is within a distance of 1.2 nm from the protein (black curve). The accumulation of the histogram is presented in the red curve.

The preferential dwelling of the ions within the first two discrete shells is reflected by the Ca2+ residence time. Figure 4 depicts the residence decay curves of the Ca2+ ions in the 2nd (top panel) and 3rd (bottom panel) shells. The residence decay of the Ca2+ near CaB, as computed from the simulations, is denoted in the black curves. As the residence time is an exponentially decaying process, an exponential fit was applied and is given in the green curve. As can be seen, in both shells the single exponential fit does not accurately describe the decay curve. This can be explained by recalling that the protein exhibits positive and negative electrostatic lobes, in which the residence time of the Ca2+ ion is expected to be completely different. Thus, we have performed bi-exponential fits (blue curves) and summarize the results for all shells in Table 1. Indeed, for the 3rd shell and up, the bi-exponential function describes well the residence dynamics (as can be seen from the RMS errors in Table 1). Yet, for the 1st and 2nd solvation shells, it can be seen (according to the RMS error and visual inspection of the graphs) that a two exponential function does not accurately describe the residence curve.

10.1371/journal.pone.0014718.g004 Ca2+ residence times.

The main frame kinetics of Ca2+ ions residence in the 2nd (top panel) and 3rd (bottom panel) shell of Apo-CaB. The inset depicts the dynamics on a semi log scale. The residence time decay curves are in black. One exponential fits are in green and a bi-exponential fits are in blue.

All dynamics exhibit a very fast component with time constant in the order of 1–3 ps. This fast phase is attributed to Ca2+ molecules located in the positive lobe of the electrostatic potential. The slower phase, which relates to long residence times, is attributed to ions located within the attractive negative lobe. The ions located in the first shell exhibit an extremely long residence, in the order of 2.5 ns, while ions in the second shell dwelled in the layer with time constant of ∼200 ps. Further on along the radius vector the dwell time is reduced to ∼15 ps. On the 1st and 2nd shells, the residence process is more complex, due to the great inhomogeneity of the proteins surface (as can be seen in Figure 1, panels B and C). A Ca2+ ion in these shells is affected by the individual charges of the polarized moieties and thus the residence process becomes a complex process, which cannot be readily described by just two independent events. This local field effect is significantly reduced starting from the 3rd shell and outwards. Ca2+ ions in these shells are affected by the charges on the protein as a whole rather than the individual moieties.

It is of interest to compare the residence time of ions leaving towards the bulk to those leaving towards the protein. The flux from the first shell is, naturally, only to the second shell. The residence time of ions in the second shell flowing inwards is 2.5 times larger than the residence time of ions flowing outwards. This reflects the high gradient of the electrostatic potential at the ∼0.3 nm range from the surface of the protein. Beyond the 2nd shell, the inward and outward ion fluxes are essentially the same with a value comparable with the residence time.

Propagation of the Ca2+ within the protein's Coulomb cage i. The lateral translation of the ions in the respective solvation shells

The approach of Ca2+ ions towards the protein is accompanied by decrement of its diffusion coefficient and longer residence time. The lower diffusivity tends to reduce the translational motion of the ion while the longer residence time will allow it more time to propagate within the shell. The combined effect of the two terms on the ability of ion to scan its immediate vicinity can be evaluated by the function presented in Figure 5. The figure is a histogram of the maximum mean square deviation (MSD) of the Ca2+ ions while residing in the 1st (solid) and 2nd (dashed) solvation shells. For this calculation we compiled the data of all CaB simulations (totaling 2 µs) screening for the events where the Ca2+ ions resided in a specific shell. The curves in the figure represent the percentage of events (within a specific shell) in which the Ca2+ ion propagated (measured as MSD) more than the value in the abscissa, relative to the surface of the protein.

10.1371/journal.pone.0014718.g005 The diffusion mobility of Ca2+ ions on the surface of the protein.

The figure relates the translational motion of Ca+2 ions expressed by the MSD of a Ca2+ ion as a function of the time the ion remained with the first (solid line) or the second (dashed line) shell of Apo-CaB. The translational motion of the ion was corrected for the rotation and translation of the protein. Straight lines indicate events where MSD > = 1 nm2.

The results of this analysis clearly imply that the fraction of Ca2+ ions that propagated more than 1 nm (MSD ≥1 nm2) while in the 1st solvation shell (∼13%) is larger than that of the ions in the 2nd shell (∼4%). Considering that the length of a glutamate side chain is ∼0.4 nm, a ∼1 nm displacement cannot be achieved through a “piggy back” riding over the rotameric space of the residues. There must be a net motion on the surface of the protein. In contrast, when the ion is detained in the 2nd solvation shell, it moves more than 1 nm only 4% of the time (dashed line). Thus, despite the restricted diffusion coefficient of the ion in the first solvation shell, the long dwell time allows the ion to translate over the protein's surface.

Based on these observations we conclude that ions located in the first solvation shell are mobile enough to skim over the surface of the protein and search for alternative sites. Thus the Ca2+ ion may end up in quite a different site than the one it first encountered.

ii. The encounter of Ca2+ ions with Apo-CaB

Figure 6, frame A, shows the overall structure of Calbindin D9k with the two bound Ca2+ ions, as taken from the crystallographic structure by Szebenyi and Moffat [28]. A detailed view of the binding sites, shown in frame B, demonstrates that residue E60 takes part in the coordination of the Ca2+ ions on both sites (either directly or via a water molecule). During the molecular dynamics simulations, the protein was ‘stripped’ from the Ca2+ ions and allowed to relax into a solution structure of the Apo state, both for the WT and the E60D mutation. This observation is in accord with our previous report that the Amber 94 force field indeed retains the structure of CaB intact, both in the Apo and Holo states [29]. On accumulation of 1 µs for each of the structures, the protein exercised structural fluctuations, but basically retained its shape without any deviations that should be accounted for (data not shown). Accordingly, we can discuss the motion of the Ca2+ ions without the need to evaluate the changes experienced by the protein.

10.1371/journal.pone.0014718.g006 The structure of Holo Calbindin D9k.

The CaB (backbone cartoon) with two bound Ca2+ ions (blue VDW) as taken from the crystal structure 3ICB. The binding residues are denoted as lines. Protein atoms closer than 0.3 nm are denoted as CPK (oxygens in red, carbons in cyan). Frame A depicts the whole protein. Frame B depicts the binding sites with the names of the binding residues. The E60 hydrogen bonded water oxygen that coordinate site I Ca2+ is also shown (in CPK) along with the relevant distances.

The mechanism by which a Ca2+ ion propagates on the surface of the protein was investigated by examining the encounters of the Ca2+ ions with the individual residues on the surface of the CaB protein. The formal concentration of Ca2+ in the simulations was 25 mM, leading to an average frequency of one encounter every 1.3 ns. These encounters, where the ion came to contact with a residue on the surface after diffusion in the bulk, are defined as primary encounters. We define secondary encounters as events where the Ca2+ ion came to contact with a residue due to transfer from another surface residue, without leaving the first solvation shell. The compilations of all these events are given in Tables 2, S1, and S2.

10.1371/journal.pone.0014718.t002 Total binding times, primary hit count and secondary hit count of Ca2+ ion to Apo-CaB residues, calculated from ten 100 ns WT and ten 100 ns E60D simulations.
Residue Binding time (ps) Primary encounters Secondary encounters Binding time (ps) Primary encounters Secondary encounters
E4 4681 5 0 3282 4 0
E5 1 1 0 0 0 0
E11 3769 6 1 1331 1 1
Y13 0 0 0 1 0 1
A14 0 0
A15 0 0 0 1080 5 1
K16 2966 4 2 3296 5 2
E17 139336 51 10 269478 57 8
G18 1434 5 1 2878 4 0
D19 15339 10 7 1520 8 2
A21 4491 4 2 45288 6 3
Q22 67512 52 16 176169 37 18
L23 55 19 1 0 0 0
S24 1463 3 8 21335 15 11
E26 18961 6 5 18842 8 6
E27 24807 4 5 29762 6 5
K41 657 0 3 722 3 1
G42 841 9 2 1935 3 1
P43 89505 16 11 31363 8 2
S44 3254 4 10 1469 2 2
T45 1874 0 2 1 0 1
D47 62462 37 11 40492 25 4
E48 176802 55 7 92731 10 7
E51 159179 62 14 130802 42 8
E52 12894 15 3 3911 9 1
L53 0 0 0 2 0 1
D54 96269 17 22 94526 19 4
K55 1427 7 3 8392 16 2
A56 36670 28 6 85218 17 11
G57 73675 28 11 7737 11 2
D58 191748 58 19 385846 88 12
F59 20194 9 10 486 2 7
E60 269285 71 35 *527753 *165 *27
S62 1033 14 16 3118 175 14
E64 31634 26 11 17635 28 2
E65 157857 26 12 21236 24 7
Q67 0 0 0 1 0 1
K72 0 0 0 56253 15 2
I73 22122 6 3 7630 15 2
S74 2531 16 2 11640 33 6
Q75 80 1 0 1238 2 1

Table 2 lists the compilation of the total time of contact between the ion and the residues (expressed as number of snapshots) and the number of primary and secondary encounters made with the residues. The data summed in the table corresponds with 1 µs of total simulation time (10 independent runs lasting 100 ns) for each of the WT and E60D mutant. During this period the Ca2+ ions were free in solution for 44% of the time and, when bound to the protein, interacted simultaneously with (average) ∼1.6 nearby residues. Inspection of Table 2 reveals that not all residues were attractive to Ca2+ ions by the same extent. Few residues experienced many encounters and accumulated extended contact time (for example: D58, D60, D17, E19) while others, of equal charge, hardly encountered with the ion (E4, E5, E11). Inspection of the table reveals that the high frequency of encounters, and a long binding time is not limited to the binding sites (marked by bold letters). It should be mentioned that A14, which in the crystalline state of the protein is a ligand to the Ca2+ in site I, did not make a single contact with the Ca2+ during the whole simulation.

Comparison between the abundance of primary encounters to the secondary ones reveals that the ratio between the total secondary encounters to the total primary encounters per residue is ∼0.3, meaning that of all contacts made by the ion with the protein about one quarter are mediated through a previous encounter of the ion a with some other site on the protein rather than coming directly from the bulk. The average dwell time of the ions on the binding residues were widely spread; some residues (like E65, P43) retained contact for few ns, while other residues (like S62) release the Ca2+ ion within few tens of ps.

Examination of the accumulative time of contact of the residues associated with binding sites I and II reveals that from all residues making binding site I, only E17, and to a lesser extant Q22, were sufficiently attractive to retain the ion for 14% and 7% of the total simulation time, respectively. The residues of binding site II appeared to be more reactive both in accumulated contact time (up to ∼29% for D58 and ∼40% for E60/D60) and in the number of primary and secondary encounters. These findings are correlated with experimental indications that site II is occupied first during the binding process [22]. Finally, there are residues that are not associated directly with the specific binding sites (P43, D47, E48, E51) that are attractive both for primary and secondary encounters, suggesting that they may function in passing the ion between nearby residues.

A further breakdown to the specific binding atoms is given in Tables S1 and S2. The first lists the encounters of Ca2+ with the oxygen atoms of backbone carbonyls while the latter reports encounters with the more polarized side chain oxygen atoms. The overall ratio between secondary and primary encounters for all the protein oxygens is 0.38, which is somewhat higher than 0.3 ratio observed at the per-residue resolution. This implies that intra-residue Ca2+ transfer occur quite frequently. The overall ratio between secondary and primary hits for carboxylates and carbonyls is 0.33 and 0.55, respectively.

The approximated rate of encounter between the Ca2+ ions with calbindin, derived by the Debye-Smoluchowski equation is ∼3×1010 M−1s−1 and the apparent rate constant at 25 mM Ca2+ will be ∼0.8×109 s−1 or encounters at average intervals of 1.3 ns. The value derived from the number of primary hits that occurred during the 2 µs trajectories is quite comparable, ∼1.3 ns. Thus, the encounter of the ion with the surface of the protein is compatible with that of a diffusion controlled reaction and depends on the concentration of the Ca2+ ions in the solution. Once a primary encounter occurs, the following secondary encounters are independent of the free Ca2+ concentration and occur at average intervals of ∼4 ns. Under physiologic conditions, were the free Ca2+ concentration is few µM, the time interval between primary encounters will be stretched to the µs time frame. However, many of the Ca2+ ions that encounter the protein's surface will be transferred to a nearby site (secondary encounter) within the ns time frame. Thus, as at physiologic Ca2+ concentration, the primary encounters will serve as efficient donors to the nearby sites, rendering the protein's surface as a collecting antenna, and increasing the surface of the protein which is scanned by the ions.

The Binding of Ca2+ to the E60D mutant of Calbindin D9k

The measured rate constant for the binding of the Ca2+ ion to the specific binding sites (kon = 2×107 M−1s−1) [30] implies that within the present simulation time, we are still far from the formation of the final stable complex as determined by the X ray diffraction of the crystal. Consequently our simulation can reflect only on the initial events associated with the binding of Ca2+ to the specific sites. Still these simulations are sufficient to reveal how a single mutation can alter the reaction pathway.

The functional role of the Ca2+ shuttling mechanism can be evaluated in the CaB itself by comparing the WT simulations with the simulations of the E60D mutant. This conservative mutation has a highly similar structure to the WT CaB, yet at physiologic ionic strength its affinity to Ca2+ is reduced by ∼8 fold [22]. This effect was partly attributed [22] to a more labile water molecule that binds site I Ca2+ (the left binding site, as seen in Figures 6). As shown in Figure 6B, the side chain of E60, as determined for the crystalline protein, indirectly coordinates the Ca2+ ion in site I via a water molecule. This water molecule is bound to the site I Ca2+ and hydrogen bonded to the side chain carboxylate of E60. The shortening of the E60 side chain by a carbon in the E60D mutant increased the length of the hydrogen bond from ∼2.65 Å to ∼2.95 Å, making the water molecule more labile. Since the experimental evidence indicates that both sites show similar reduction in affinity and that site II is occupied first (and that the binding is a cooperative process), we wished to investigate the mechanism leading to the lower affinity of site II.

The interactions between the Ca2+ ion with its surrounding ligands are detailed in Figure 6 frame B. The ion in site I is bound to the carboxylate of E27 and the backbone carbonyls of A14, E17, D19 and Q22. These coordinating species are in good agreement with the NMR structure 1B1G [31], with the exception that in the NMR structures the carbonyl of G18 and one of the D19 carboxylate oxygens can also serve as binding atoms. Site II Ca2+ is bound in the crystal structure to the carbonyl of E60, a single carboxylate oxygen of D54, a single carboxylate oxygen of D58, the amide oxygen of N56 and the carboxylate of E65. In the NMR structures, only D54 and E65, with one or two carboxylate oxygen coordination appear in all structures. D58 carboxylate and E60 carbonyl appear in the majority of the structures and N56 is missing altogether, replaced by the carbonyl of G58 or E59.

In order to resolve these apparent discrepancies, we have to consider that both crystallographic and NMR structure determination involve inherent biases. In crystallography, the crystal structure is created in non-physiological conditions. The NMR structures reflect some sort of average, and not necessarily a structure that existed in the solution. Both methods carry a bias caused by the FF used to refine them. In a recent study by Paquin et. al., NMR was used to measure the S2 order parameter of amide carbonyl and carboxyl groups in the side chains of aspartic acid, asparagine, glutamic acid, and glutamine of Ca2+-loaded calbindin D9k P43G [32]. Since the S2 order parameter is model independent, and measured in solution, some of the biases described above are eliminated. The results of Paquin et. al. show that the Ca2+ binding residues (as witnessed from crystallographic and NMR structures) are associated with high S2 values in the range of 0.68<S2<0.94. It can also be witnessed that three of the binding carboxylates, E27, D54 and E65, show exceptionally high S2 values of 0.836±0.041, 0.938±0.047 and 0.913±0.056, respectively, indicating an extremely low motional liberation. These values are significantly higher than those of the other binding residues, ASN56 and D58, which exhibit values of 0.685±0.037 and 0.681±0.075, respectively. For E27, there was a consensus between the crystal and NMR structures that it binds Ca2+ in a bi-dentate manner, using both of its carboxylate oxygens. The high S2 values of D54 and E65 suggests that D54 and E65 both bind the site II Ca2+ ion using both their carboxylate oxygens. This will form tight binding with very little motional liberation of both residues.

This conclusion also conforms well to the fact that these two residues appear as binding residues in all crystallographic and NMR structures. The lower S2 values of the ASN56 and D58 residues indicate that they bind the Ca2+ less tightly. For the ASN56 this is probably due to the weaker electrostatic attraction of the amide carbonyl oxygen. For the D58 this is probably due to being bound by only one of the carboxylate oxygens (as is also seen in the crystal structures and some of the NMR structures). To conclude, the experimental evidence suggests that the key residues involved in a stable Ca2+ binding in site II are D54 and E65.

As can be seen in Table 2, residue 60 (either aspartate or glutamate) is the main Ca2+ attractor in CaB. This is mostly due to its side chain carboxylate (as can be seen in Tables S1 and S2), even that the side chain is not involved in the final coordination of the bound site II Ca2+. In the case of the WT protein, the carboxylate moiety of E60 retains the Ca2+ for ∼25% of the total accumulative time, with an average dwell time of ∼2.4 ns.

Figure 7 summarizes the total binding time (panel A), primary encounters (panel B) and secondary encounters (panel C) of the side chain carboxylates of D54, D58, E60/D60 and E65 in the WT and E60D mutant (as summarized in Table S2). As can be seen in the top panel, the replacement of E60 by aspartate enhances the binding time of the D60 carboxylate with the ion. As can be deduced from Figure 7 (middle and bottom panels), the increased binding time was due to increased primary encounters, with no change in the average dwell time (∼2.4 ns). For D58, the increase in total binding time was even more dramatic, which was also due to increase in primary hits. As can be calculated from table S2, the average dwell time increased from ∼1.3 ns to 3.6 ns.

10.1371/journal.pone.0014718.g007 The Ca2+ binding characteristics of specific anionic residues Associated with the Ca+2 binding sites.

(A) Total binding time, (B) number of primary hits and (C) number of secondary hits. Data is presented for the WT (blue) and E60D (red).

In contrast to the increased affinity shown by D58 and D60, the two key residues for Ca2+ binding in site II, D54 and E65, showed decreased affinity for Ca2+, possibly explaining the experimentally measured reduced affinity of the E60D mutant to Ca2+. In the WT protein, the carboxylate side chain of D54 shows a relatively moderate overall binding time to the ion (∼7%). The E65, on the other hand, shows a relatively high binding time (∼16%). It is interesting that in the mutant both these residues show substantial decrease in the overall binding time. As demonstrated in Figure 7, panel A, for D54 there is a ∼4 fold decrease in the binding time and in the E65 there is a ∼7 fold decrease in the total binding time. A more in-depth look at the mode of encounter for these residues shows that for both D54 and E65 the main cause for the reduction in total binding time is a significant reduction in secondary hits. For E65, there was also a decrease in the dwell time.

Figure 8 top panel, shows the amount of Ca2+ mediated interactions between specific pairs of residues: D54 and D58, D54 and D/E60, E65 and D58, E65 and D/E60. It can be seen that there is a significant reduction in Ca2+ transfers between these residues, which can quantitatively explain the reduction in binding time observed for D54 and E65. However, when looking at the Ca2+ transfers between the carboxylates of these residues, it can be seen that the Ca2+ transfer between the carboxylates of residues D60 and E65 was only mildly reduced. This discrepancy can be explained by the major reduction observed between the E65 carboxylate and the D60 carbonyl. It is also worth noting that a 2-fold increase in Ca2+ transfer is witnessed between D60 and D58 carboxylates (data not shown).

10.1371/journal.pone.0014718.g008 Inter-residue Ca2+ transfers.

Number of Ca2+ transfers between residues 58 and 60 with residues 54 and 65. The top panel counts the Ca2+ transfer between the whole residues where the bottom panel counts the Ca2+ transfer between the carboxylates. All events, in any direction were counted for both the WT (blue) and E60D (red) simulations.

All the above data leads to a mechanism by which in the WT CaB, Ca2+ arrives from the bulk mainly to the carboxylate side chain of E60. E60 can than transfer the Ca2+ ions to the key binding residues D54 and E65. Shortening the length of its side chain significantly reduces the efficiency of these Ca2+ transfer reactions and alternatively increases the Ca2+ transfer of D60 with D58. Effectively, in the E60D mutant, both D54 and E65 become “starved” for Ca2+ and the overall binding affinity in site II diminishes significantly.

For D54, the reduction in Ca2+ binding is due to a reduction of Ca2+ transfers between its carboxylate and the carboxylates of D60, D58 and E65 (the latter is a consequence of its own decreased affinity).

For E65, the reduction is due to a reduction in both inter-carboxylate interactions with the carboxylates of D60 and D58, and also from a reduction in its interaction with the backbone carbonyl of D60. The latter case can be explained by the increased level of Ca2+ transfers observed between these three species (the carboxylates of D60 and D58 and the carbonyl of D60), reducing their availability to transfer Ca2+ to E65.

Concluding remarks

The interaction of the Ca2+ ion with the protein starts when the ion encounters the Coulomb cage of the protein. Out of the Coulomb cage the ion is unaware of the protein, having a diffusion coefficient of a free ion. Once it is within the cage, the motion is restricted by the local electrostatic fields and its diffusion coefficient decreases as it gets closer to the protein. Although the mobility of the ion is hindered by the decreased diffusion coefficient, as the ion gets closer to the protein's surface, its residence time increases, enabling it to skim over longer distances in the 1st shell than in the 2nd one. Thus, the sites first encountered by the Ca2+ ion need not necessarily be the final binding site. The Ca2+ can propagate on the surface by hopping from residue to residue.

For carbonyls, which are weaker binding species, the Ca2+ binding mostly occurs by hopping from a nearby residue. For carboxylates, the encounter occurs mainly from the bulk, with 33% from nearby residues. In low Ca2+ concentration, when encounters are rare, the secondary encounters may significantly increase the protein surface available to the Ca2+ ion, thus making the region around the binding site a collecting antenna.

On average, the ion on the surface is interacting with more than single residue, keeping it bound for a long time, reducing the probability of getting lost to the bulk.

The Ca2+ shuttling mechanism was observed in simulations of Calbindin D9k where the main Ca2+ attractor is the side chain carboxylate of E60, which is not a part of the binding site, according to experimental structures. Thus, this site only serves as a midway station for Ca2+ arriving from the bulk on their way to the proper binding site. In the calbindin E60D mutant, the ability of D60 to transfer the Ca2+ ion to key binding residues in site II (D54 and E65) is significantly reduced, which we suggest as a possible cause for the reduced affinity observed in experiments.

The number of simulations that were carried out, their lengths and compositions are given in Table 3.

10.1371/journal.pone.0014718.t003 Simulations' details and composition.
Name Duration of simulation (ns) Number of simulations Number of water molecules Ionic composition
Ca2+ in water 2 20 2174 1Ca2+, 2Cl
WT Apo-CaB 100 10 4472 2Ca2+, 8Cl, 11Na+
E60D Apo-CaB 100 10 4317 2Ca2+, 7Cl, 10Na+

The force field selected for the simulation was Amber94 [33] ported by Sorin and Pande [34]. To optimize the interaction of the Ca2+ ion with carboxylate we modulated the Lennard–Jones parameters as described in [35], using the values: σ = 0.300616 nm; ε = 1.87 KJ·mol−1. The TIP3P [36] water model was used in all simulations. The simulations were performed using the GROMACS 4 package of programs [37], [38], [39], [40]. The WT Calbindin protein structure (PDB code 3ICB) given in the Protein Data Bank(PDB) [41] was determined by X-ray crystallography at 0.23 nm [28]. In order to simulate a Ca2+ depleted CaB (Apo-CaB), the two Ca2+ ions were removed from their binding sites and added at random locations. The water box extended to at least 1.2 nm from the molecule, for CaB simulations, and at least 2.0 nm from the Ca2+ ion for the free Ca2+ simulations. D, E and LYS residues were charged to reflect their state at physiological pH. Electroneutrality and physiological ionic strength of ∼0.150 M for the CaB simulations and 0.1 M for the free Ca2+ simulations were reached by adding appropriate amounts of Na+ and Cl ions to the simulation boxes (as detailed in Table 3). Prior to the dynamics simulation, internal constraints were relaxed by energy minimization. Following the minimization, an MD equilibration run was performed for 20 ps under position restraints. Finally, before all the production runs, further unconstrained equilibrations were performed for 200 ps. After the equilibration, an MD production run was performed for extended time frame, as described in Table 3. The time step for the simulation was 2 fs. The simulations were run under NPT conditions, using Berendsen's coupling algorithm for keeping the temperature and the pressure constant (P = 1 bar; τP = 0.5 ps; T = 300 K; τT = 0.1 ps) [42]. VDW and short range electrostatic forces were treated using a cutoff of 1.2 nm. All simulations were carried out with periodic boundary conditions, using the particle mesh Ewald (PME) method [43] for long range electrostatic forces. During the MD runs, the LINCS algorithm [44] was used in order to constrain the lengths of all bonds; the water molecules were constrained using the SETTLE algorithm [45]. Coordinates were saved every 1 ps. Protein images in Figure 6 were generated using VMD [46].

The diffusion coefficients were calculated from the mean square displacement (MSD) curves between specific time points selected to give the most reliable results. For Ca2+ at specific shells from the protein, the MSD statistics used for calculation of the diffusion constant was such that it contained at least 8 different simulations which contributed to the MSD average and the MSD graph was near linear. The diffusion coefficient errors stated in the manuscript are estimates based on multiple calculations using different sets of simulations.

Residence times were calculated by fitting single and double exponential functions to the residence time decay curves. The residence time decay curves denote the number of times a Ca2+ ion was found at the desired distance from the protein up to a given time span marked by the he abscissa. In these calculations an event where the Ca2+ ion changed its distance from the protein's surface for a period of 2 ps or less was not considered as termination of its residence period. All Apo-CaB simulations were used to generate these curves. For the exponential fitting procedure, only sections of the graph with a count higher than 10 were considered.

Electrostatic potential calculation were performed using the APBS software 1.2.1 [47], with a grid spacing of 1.096×1.096×1.096 Å. The calculations were carried out for a solution having an ionic strength of 150 mM. The dielectric constant of the protein was set as 2 and solvent dielectric of 78.54.

Supporting Information

Total binding times, primary hit count and secondary hit count of Ca2+ ion to carbonyl oxygen atoms, calculated from ten 100 ns WT and ten 100 ns E60D simulations.

(0.06 MB DOC)

Total binding times, primary hit count and secondary hit count of Ca2+ ion to side chain oxygens of charged and polar residues (carboxylate oxygens treated as one), calculated from ten 100 ns WT and ten 100 ns E60D simulations.

(0.07 MB DOC)

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Biochemistry 24 6740 6743 Komeiji Y Ueno Y Uebayasi M 2002 Molecular dynamics simulations revealed Ca(2+)-dependent conformational change of Calmodulin. FEBS Lett 521 133 139 Project E Friedman R Nachliel E Gutman M 2006 A molecular dynamics study of the effect of Ca2+ removal on calmodulin structure. Biophysical Journal 90 3842 3850 Project E Friedman R Nachliel E Gutman M 2006 A molecular dynamics study of the effect of Ca2+ removal on calmodulin structure. Biophys J 90 3842 3850 Friedman R Nachliel E Gutman M 2005 Molecular dynamics of a protein surface: Ion-residues interactions. Biophysical Journal 89 768 781 Patargias GN Harris SA Harding JH A demonstration of the inhomogeneity of the local dielectric response of proteins by molecular dynamics simulations. The Journal of Chemical Physics 132 235103 235108 Szebenyi DM Moffat K 1986 The refined structure of vitamin D-dependent calcium-binding protein from bovine intestine. 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Anfinsen CB Edsall JT Richards FM Advances in Protein Chemistry Academic Press 239 345 David RL 2010 CRC Handbook of Chemistry and Physics 90th edition. 90th ed Robinson RA Stokes RH 1959 Electrolyte Solutions. 2nd ed London, U.K Butterworths, Boston Szebenyi DME Moffat K 1986 The Refined Structure of Vitamin-D-Dependent Calcium-Binding Protein from Bovine Intestine - Molecular Details, Ion Binding, and Implications for the Structure of Other Calcium-Binding Proteins. Journal of Biological Chemistry 261 8761 8777 Project E Nachliel E Gutman M 2010 Force Field-Dependant Structural Divergence Revealed During Long Time Simulations of Calbindin d9k. Journal of Computational Chemistry 31 1864 1872 Forsen S Kordel J Grundstrom T Chazin WJ 1993 The Molecular Anatomy of a Calcium-Binding Protein. Accounts of Chemical Research 26 7 14 Kordel J Pearlman DA Chazin WJ 1997 Protein solution structure calculations in solution: Solvated molecular dynamics refinement of calbindin D-9k. Journal of Biomolecular Nmr 10 231 243 Paquin R Ferrage F Mulder FAA Akke M Bodenhausen G 2008 Multiple-Timescale Dynamics of Side-Chain Carboxyl and Carbonyl Groups in Proteins by C-13 Nuclear Spin Relaxation. Journal of the American Chemical Society 130 15805 15807 Cornell WD Cieplak P Bayly CI Gould IR Merz KM 1995 A 2Nd Generation Force-Field for the Simulation of Proteins, Nucleic-Acids, and Organic-Molecules. Journal of the American Chemical Society 117 5179 5197 Sorin EJ Pande VS 2005 Exploring the helix-coil transition via all-atom equilibrium ensemble simulations. Biophysical Journal 88 2472 2493 Project E Nachliel E Gutman M 2008 Parameterization of Ca+2-protein interactions for molecular dynamics simulations. J Comput Chem 29 1163 1169 Jorgensen WL Chandrasekhar J Madura JD Impey RW Klein ML 1983 Comparison of Simple Potential Functions for Simulating Liquid Water. Journal of Chemical Physics 79 926 935 Berendsen HJC van der Spoel D van Drunen R 1995 GROMACS: A message-passing parallel molecular dynamics implementation. Comp Phys Comm 91 43 56 Lindahl E Hess B Van der Spoel E 2001 GROMACS 3.0:a package for molecular simulation and trajectory analysis. J Mol Model 7 306 317 Van der Spoel D Lindahl E Hess B Groenhof G Mark AE 2005 GROMACS: Fast, flexible, and free. Journal of Computational Chemistry 26 1701 1718 Hess B Kutzner C van der Spoel D Lindahl E 2008 GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular simulation. Journal of Chemical Theory and Computation 4 435 447 Berman HM Westbrook J Feng Z Gilliland G Bhat TN 2000 The Protein Data Bank. Nucleic Acids Res 28 235 242 Berendsen HJC Postma JPM DiNola A Haak JR 1984 Molecular dynamics with coupling to an external bath. J Chem Phys 81 3684 3690 Essman U Perela L Berkowitz ML Darden T Lee H 1995 A smooth particle mesh Ewald method. J Chem Phys 103 8577 8592 Hess B Bekker H Berendsen HJC JGEM. F 1997 LINCS: A linear constraint solver for molecular simulations. J Comp Chem 18 1463 1472 Miyamoto S Kollman PA 1992 SETTLE: An Analytical Version of the SHAKE and RATTLE Algorithms for Rigid water models. J Comp Chem 13 952 962 Humphrey W Dalke A Schulten K 1996 VMD: Visual molecular dynamics. Journal of Molecular Graphics 14 33 38 Baker NA Sept D Joseph S Holst MJ McCammon JA 2001 Electrostatics of nanosystems: application to microtubules and the ribosome. Proc Natl Acad Sci U S A 98 10037 10041


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Cooking Over My Head

Home  /  Cooking Over My Head

coverJoyce’s inspirational memoir, full of photos, recipes, and images of her paintings.

“Ignoring the molecular explanations of the phenomena, seeing only this beautiful tarantella of the water, I gently immerse one strand of angel-hair into the fray and watch as minute bubbles embrace all its surfaces. I cook for the sheer joy of observation, grateful beyond words. My first time watching butter foam was momentous, seeing a pan deglazed with a cup of wine was magic. True, these processes had been taking place before me for years, but I hadn’t been able to see them. All the stoves had been over my head.”

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Friday, October 31, 2008

So Few Words

Back during my first stint in graduate school, right before I dropped out and wandered into my "lost" years, I used to visit a friend down in Galveston, Texas. It was a messy time for both of us, probably best forgotten, but there was something about her insistence that we go to to the beach to swim in the Gulf that sticks with me. The sky was dark, the winds were cool, the beach was dirty, and we were totally and utterly lost. I still hear the weird sounds of the beach: the odd percussive 'whoosh' of waves, the reverse echo of seagull voices, the silence between us. So anyway, many years later, thinking about all these chasms, I wrote a song and recorded it immediately. Although I say "Sheraton Bay" in the song, I actually mean "Galveston Bay." I recorded the electric guitar in my bathroom to get a natural echo. The song is called "So Few Words."

Joy - So Few Words [mp3]

Another song I recorded, more recently entitled "1989 Or so." I was trying to do a power pop song, and play around with bars and meters. This is one of the few songs I'd had in my mind as a complete work and just tried to record it exactly the way it was in my mind. I was trying to get that feeling I used to get while flying a kite in a Dallas suburb on a spring day.

Joy - 1989 Or So [mp3]

Thursday, October 30, 2008

Man Of The World

I discovered the late '60s version of Fleetwood Mac from the better known late '70s version of Fleetwood Mac. One of my favorite albums as a kid (well, I guess 10th grade or so) was Fleetwood Mac's Live, which, a gajillion years later, still stands up as a pretty good classic rock live album. I have a soft spot for the Lindsey Buckingham-Stevie Nicks era Fleetwood Mac that is not rational at all, and best left explained in another posting, but the point is that on Live, they did a riproaring song called "Oh Well" which I later discovered was actually originally done by the late '60s version of the band, the one with Peter Green, variously described as insane and a genius and both. Anyway, all of this led me back over the years to rediscover the blues-based music of Peter Green's old Fleetwood Mac, particularly their stellar album Then Play On, a title apparently a paraphrase from Shakespeare's "if music be the food of love, play on." The thing that really stands about this album (and the others from that period) is the utter sophistication of the guitar playing. These guys had a triple guitar playing field (Peter Green, Jeremy Spencer, and Danny Kirwan) but boy, they knew when to pull back and when to hit the notes. Basically, these guys were ten times better than that other absurdly overrated and mediocre guitarist Eric Clapton (who also played recycled but amped up blues). Unfortunately, as far as I can tell, all three of them actually went insane. Then in 1975 or so, the rhythm section took on Buckingham and Nicks, and the rest, is history.

Anyway, all this for a coupla songs which I wanted to post. Neither of these songs were originally featured in any original Fleetwood Mac and seem to be notoriously hard to track down as they were released on CD on only a few places. Both are soft mellow songs, and actually not at all blues-based like most of the band's other stuff from the period. Both, written by Peter Green, are deceptively simple but incredibly beautiful pieces of music. The first is Fleetwood Mac's first single, released in 1967, "Albatross."

The second is even better, "Man of the World," a single released in 1969. The lyrics are so ridiculously simple but these sentiments could not have been better put in another way. I totally 100% understand what he's talking about. And check out the totally understated triple guitar play during the bridge. Lovely.

And just to show that they could indeed rock out. Here's the original version of "Oh Well."

I'm posting mp3s of both songs for a short while. They are supposedly remastered versions from the rare (and expensive) The Chain 4-CD set put out by Fleetwood Mac in 1992.

Fleetwood Mac -- Albatross [mp3]
Fleetwood Mac -- Man Of The World [mp3]

Thursday, October 09, 2008

Young Man Blues / TV On The Radio

I would pretty much give up my real job to play in a band. I say "pretty much" because in actual real life, I haven't and I won't. But you know that's kinda what I really want to do. Everything else is just bullshit that I do to get by. I have a job at a university and it's OK, I suppose. I get health insurance and I have an office with a computer. I work with more or less nice people. Although some of them are rather weird in the socially-awkward-kind-of way. But nothing that would drive me crazy or anything. The university runs like it's the movie Brazil in that things work very slowly and you have to fill out everything in triplicate. One of the drawbacks of my job is that I have to deal with a lot of people all the time. For someone who has determinedly tried to avoid people his entire life, this is obviously rather ironic that I ended up in a job where I'm constantly bombarded by people who have some need to speak to me. (Just to be clear, they need to talk to me not because they enjoy conversing with me but because they actually really need to do so to do their work).

I've been thinking that I'd really like to go to the TV On The Radio show next week in Brooklyn (actually, they are playing three nights in a row) but as usual, because this is a show in New York, all the shows are sold out. I enjoyed watching the clip of them performing on David Letterman. See / watch / look:

The other day, I was walking by a bar in Cambridge, Massachusetts, and I randomly ran into a person I had not seen in at least ten years. She used to be this cool chick who (along with her husband) were the landlords of the house where I lived in Amherst. I'm not sure what she used to do, but it had something to do with the film industry. She would always go to the Sundance film festival or Cannes or something. We had some crazy ass party times in Amherst, Massachusetts. It was at a completely insane phase of my life and our little house, which we used to call the "White House," singlehandedly kept afloat the alcohol industry in America. (Thanks Dave!) Anyway, so I run into this chick and she was smoking outside a bar and we exchanged pleasantries and I got her number. I had no intention of calling her back but accidentally a couple of days ago hit her number on my cell phone. Fuck! So I had to talk to her. But strangely it turned out to be a good conversation. She has like two kids now. That is some weird shit. I cannot imagine her being a mom and everything. We is getting old old old.

Anyway, one of my favorite songs from that period in Amherst was the Who's "Young Man Blues." This was some badass rock and roll shit. It's worth it to watch this video (from 1970), especially because this is the Who, firing on all cylinders. Check out the one minute stretch from 3:45 to 4:45 in the video. That's exactly how I feel sometimes.

Everytime I played this song at the "White House," I would turn up my stereo and wanna smash the whole fucking house down. I'm sure she didn't appreciate the volume. But I needed volume. Did I mention that I would rather play in an ass-kicking rock'n'roll band than do anything else?

Tuesday, October 07, 2008

shows 2008 so far

- the verve / april 29, 2008 / new york / good
- swell season / may 19, 2008 / new york / good
- m.i.a. / june 6, 2008 / brooklyn / good
- mogwai / september 18, 2008 / new york / awesome
- my bloody valentine / september 22, 2008 / new york / fucking awesome

tuesdays = old

listening to the new M83 album

it's very eighties
but it's really good
so different from the last album

happy but sad
old but young
eighties but now
songs but music

my life sucks
have no friends / want no friends / have no friends / want no friends
the new m83 album is called "saturdays = youth


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Perfect Loaf Sourdough Bread

Perfect Loaf Sourdough Bread

Finally! I’ve found a recipe for Sourdough Bread that satisfies my soul….I’m not kidding. Well, so far as bread can satisfy ones soul…Maybe it’s just my idea of what a good loaf of sourdough bread ought to be.

perfect sourdough bread loaf2

After trying 7 or 8 recipes, this one does everything I’d expect of my sourdough bread. It has a slight crispness on the outside crust with a little bit of chewiness, but not tough, and it’s light and fragrantly soft on the inside.

perfect sourdough bread sliced

Who knew a simple loaf of bread could be so satisfying….

perfect sourdough loaf5

There are several things that make this bread so satisfying to me…other than its wonderful taste.

First off, I let my Kitchen Aid Mixer do the kneading.  This recipe doesn’t need too much kneading, so it’s perfect that the little bit of kneading it needs can be done by my KitchenAid mixer’s dough hook. A few minutes is all you need…to knead. I like saying that….need and knead. 🙂 Can you tell?

perfect sourdough bread loaf2

Another thing I love about this recipe is that it only takes a few minutes to prepare. 10-15 minutes, tops.

All you need to decide is when you want to bake.  If you want to bake at night,  then you begin this recipe the night before. If you want to bake in the morning, then begin it the morning before.

A third thing to love about this recipe is that because it only uses 1/3 cup of starter, it takes lots of time to rise.  What’s so important about that?  It’s this long rising period that breaks down the glutens in the flour and the sugars, making for a healthy loaf.

This is especially important if you or someone in your family has a problem with gluten intolerance or blood sugar issues. THIS is the LOAF you want to bake. It has 2 long rise periods, which makes a perfect loaf of sourdough bread.

And even if you don’t have gluten issues, you may have problems with triglycerides or cholesterol. The culprit is refined white flour (not fatty diets) and this flour breaks down into something healthy without using some funky ‘gluten free flours’ etc. I don’t even like saying this is ‘healthy’ because that in itself can be a turn off….please don’t turn me off.

This is simply the way bread ought to be baked. This is the way our grandmothers baked bread before the advent of commercial breads. This is Real Bread.

And one more thing….I also love that because it comes together so quickly, I can mix one batch and then mix a couple more in no time. So when I do bake, I have 3 loaves, giving me a loaf or two to share.

I’m in love…I hope you will be, too…..Here’s to our good health! Enjoy!

Perfect Loaf Sourdough Bread

  • 1/3 cup Sourdough Starter...that has been fed a couple of times in the past 12 hours
  • 1 cup + 1 TBLS water
  • 3 cups unbleached bread flour
  • 2 tsp Salt
  • 2 TBLS oil

  1. In your mixer combine the sourdough starter and water until blended. Add 1 cup of flour at a time and before the last cup add your salt. Allow mixer to knead 4-5 minutes or by hand for 10 minutes.
  2. Place oil in bowl. Place dough into bowl and flip it over so that top and bottom are oiled. Cover with plastic wrap and allow to rise over night or 8 hours...or longer according to your schedule....
  3. Grease a loaf pan. After dough rises 8-12 hours in bowl, remove from bowl and shape into loaf, kneading very little. Cover with plastic and allow to rise 6-8 hours.
  4. Preheat oven 425*
  5. Immediately, before placing in oven, slice several slits with a bakers lame or very sharp knife. This allows the gasses to escape during baking. And can be a nice decorative touch. 😉
  6. Bake loaf 10 minutes and then reduce heat to 400* and bake for 20-25 minutes longer or until lightly browned.
  7. Turn out onto cutting board and cool a few minutes before slicing.
  8. Turn bread loaf on its side to slice with a serrated knife.

  1. Linda Maples says:

    Love your site!! Just finished checking out your mosaic birdbaths – so beautiful and instructions are great.

    The sourdough breads look scrumptious. However, I do not have a recipe for sourdough starter. I made this type of bread years ago but have since misplaced the starter recipe.

    Would you mind emailing me the starter recipe?

    Many thanks,

    Linda in Alabama

  2. Pam aka Nana in Arkansas says:

    Made your recipe for this sourdough bread it was first time I made sourdough bread and it is so easy and great tasting. Thank you
    Pam aka Nana in Arkansas

  3. What size of loaf pan do you use? I am going to try your recipe. I am new to sourdough and so far have not found a recipe I like for bread. While rising I put a towel over it and end up with a hard, dry, thick layer on top. I’ve seen recipes for bread saying to cover with towel. Is that maybe the problem. I have made your biscuits and they are so good. Another quick question. If I put plastic wrap over it, do I have to oil it so it doesn’t stick to the loaf?

    • Bobbi Jo….I have a large plastic bag that I slip my loaf pan into and allow it to rise. I don’t oil the bag or anything. I do try to fluff the bag so that it doesn’t really touch the loaf pan/dough and leaves room to rise. I have no idea where I found this bag but it works perfectly. It’s even large enough for me to place 2 loaf pans in at a time….My loaf pans are 5×9. The only thing I do is to pour a little oil in the bowl where the dough is to rise and then add the dough, coat on one side and then flip it over to the oiled side is up. I hope this helps.

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Saturday, May 24, 2014

God has prepared me over the past years to work in His mission field. He gave me the opportunity to go to a third world country last year to minister to orphans and families in complete poverty.

Now, I've been asked to work for a rescue mission using my public relation and photojournalism back ground. I am so excited to work for God and God's peoples. The truth is I'm the one who is blessed. God has been speaking to me and preparing my heart for my new job.

After watching a webinar to assist donor relations for rescue missions, one of the speakers shared this verse:

I am not commanding you, but I want to test the sincerity of your love by comparing it with the earnestness of others. For you know the grace of our Lord Jesus Christ, that though he was rich, yet for your sake he became poor, so that you through his poverty might become rich. (2 Corinthians 8:8, 9 NIV)

This verse really touched me because I had randomly read Romans 12 this same day.  While the whole chapter spoke to me, Romans 12:9-13 reflected the same idea as 2 Corinthians 8:8-9.

Love must be sincere. Hate what is evil; cling to what is good. Be devoted to one another in love. Honor one another above yourselves. Never be lacking in zeal, but keep your spiritual fervor, serving the Lord. Be joyful in hope, patient in affliction, faithful in prayer. Share with the Lord’s people who are in need. Practice hospitality. (Romans 12:9-13 NIV)

My God continue to work and prepare me for this new journey.


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Choose amazing.

Yes, I have been a little MIA. It’s just that when I think about sitting down and having to deal with the daunting task of writing a blog. I don’t know why but it always takes me hours to fully get my thoughts down. So, long story short, I’m not dead. Long story a bit longer, I’ve been going to school and coming home and eating, and not really doing much of anything.

Well, okay, that’s a bit of a lie. I’ve gone to plenty of restaurants, and been trying out new things. So, I will try and try to get this shit posted. Kick my ass, tie me down, I’m going to do this. Getting my ass in gear. I’ve been too slacky lately.

Alors, what have I been doing these past couple of months in Toronto with few new friends? Homework, food, parties, and ordering shit online. I love buying things online. Sometimes I’ll go on binges and buy so many things. It’s the part where I receive them in the mail that really gets me off.

So here, a more or less a list of things I’ve bought recently.

1. USB Hub

So, my laptop only has two USB ports and lately, I’ve found myself having to use more than that. It’s very inconvenient. So I bought this little guy, because he’s cute, and he was inexpensive, but mostly because he’s cute. His little heart even lights up when you plug shit into him. 😀

2. Doctor Who : Season Four Specials

They were on sale on Amazon for only 20$! It was a steal, so how could I not buy it. It’s all I’m missing (of the series revival) until the fifth season comes out on DVD (November!). Plus it has shiny David Tennant on the cover. Win!

3. Robots in Disguise

I love this shirt. It was a one-day-only design on Teefury.com and it was only 9$! 12$ with shipping. It is so incredibly nerdy but so incredibly amazing. It fits me real well, and I can wear my Doctor Who pride out in public. The material its made out of feels real nice too. 3-level of nerdiness, I’m not lame, no.

4. iPhone Cases

From the Apple Case program. I love free things. And then some other cases. Why I need more than one case, I don’t know.

5. iPhone case stickers + screen guard

Found for real cheap on the interwebs. Okay, so it isn’t mine, but it still looks pretty rad. This one was for my brother.

  1. The R2D2/Dalek shirt is actually 3 levels of win 🙂

    But you are a big loser for real

  2. Also the USB port guy is actually pretty awesome. Anything cute gets the thumbs up.

  3. MOMMA?!!

    Hahahha. Yeah, as I’ve told you time and time again, I’m pretty rad. 🙂

    Now I reallu=y feel like I need to watch doctor who.

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