Cerebellum Purkinje cell vulnerability in aged rats with memory impairment.

The cerebellum is involved in higher order cognitive function and is susceptible to age-related atrophy. However, limited evidence has directly examined the cerebellum's role in cognitive aging. To interrogate potential substrates of the relationship between cerebellar structure and memory in aging, here we target the Purkinje cells (PCs). The sole output neurons of the cerebellum, PC loss and/or degeneration underlie a variety of behavioral abnormalities. Using a rat model of normal cognitive aging, we immunostained sections through the cerebellum for the PC-specific protein, calbindin-D28k. Although morphometric quantification revealed no significant difference in total PC number as a function of age or cognitive status, regional cell number was a more robust correlate of memory performance in the young cerebellum than in aged animals. Parallel biochemical analysis of PC-specific protein levels in whole cerebellum additionally revealed that calbindin-D28k and Purkinje cell protein-2 (pcp-2) levels were lower selectively in aged rats with spatial memory impairment compared to both young animals and aged rats with intact memory. These results suggest that cognitive aging is associated with cerebellum vulnerability, potentially reflecting disruption of the cerebellum-medial temporal lobe network.

Engineering a calcium-dependent conformational change in Calbindin D9k by secondary elements replacement.

EF-hand is a common motif in Ca2+-binding proteins, some of which present a conformational change upon Ca2+-binding, a relevant property for signal transduction. In the present work, we investigated the behavior of Calbindin D9k, a modulator protein with a high affinity for Ca2+ but structurally insensitive to its presence. Its non-canoncal N-terminal EF-hand was replaced by chimeric motifs, containing increasing structural elements from the sensor troponin C SCIII motif. We demonstrated that the loop and helix II were the necessary elements for a conformational change promoted by calcium in chimeric Calbindin D9k. Fusion of the isolated chimeric motifs to an activity reporter gene showed the loop as the minimal element to promote a conformational change. The discrepancy between these results is discussed in the light of inter-motif interactions and helix I participation in modulating the Ca2+ affinity and restricting motif conformation.

Mouse S100G protein exhibits properties characteristic of a calcium sensor.

Bovine S100 G (calbindin D9k, small Ca2+-binding protein of the EF-hand superfamily) is considered as a calcium buffer protein; i.e., the binding of Ca2+ practically does not change its general conformation. A set of experimental approaches has been used to study structural properties of apo- and Ca2+-loaded forms of mouse S100 G (81.4% identity in amino acid sequence with bovine S100 G). This analysis revealed that, in contrast to bovine S100 G, the removal of calcium ions increases α-helices content of mouse S100 G protein and enhances its accessibility to digestion by α-chymotrypsin. Furthermore, mouse apo-S100 G is characterized by a decreased surface hydrophobicity and reduced tendency for oligomerization. Such behavior is typical of calcium sensor proteins. Apo-state of mouse S100 G still has rather compact structure, which can be cooperatively unfolded by temperature and GdnHCl. Computational analysis of amino acid sequences of S100 G proteins shows that these proteins could be in a disordered state upon a removal of the bound calcium ions. The experimental data show that, although mouse apo-S100 G is flexible compared to the Ca2+-loaded state, the apo-form is not completely disordered and preserves some cooperatively meting structure. The origin of the unexpectedly high stability of mouse S100 G can be rationalized by an exceptionally strong association of its N- and C-terminal parts containing the EF-hands I and II, respectively.

Nuclear overhauser spectroscopy of chiral CHD methylene groups.

Nuclear magnetic resonance spectroscopy (NMR) can provide a great deal of information about structure and dynamics of biomolecules. The quality of an NMR structure strongly depends on the number of experimental observables and on their accurate conversion into geometric restraints. When distance restraints are derived from nuclear Overhauser effect spectroscopy (NOESY), stereo-specific assignments of prochiral atoms can contribute significantly to the accuracy of NMR structures of proteins and nucleic acids. Here we introduce a series of NOESY-based pulse sequences that can assist in the assignment of chiral CHD methylene protons in random fractionally deuterated proteins. Partial deuteration suppresses spin-diffusion between the two protons of CH2 groups that normally impedes the distinction of cross-relaxation networks for these two protons in NOESY spectra. Three and four-dimensional spectra allow one to distinguish cross-relaxation pathways involving either of the two methylene protons so that one can obtain stereospecific assignments. In addition, the analysis provides a large number of stereospecific distance restraints. Non-uniform sampling was used to ensure optimal signal resolution in 4D spectra and reduce ambiguities of the assignments. Automatic assignment procedures were modified for efficient and accurate stereospecific assignments during automated structure calculations based on 3D spectra. The protocol was applied to calcium-loaded calbindin D9k. A large number of stereospecific assignments lead to a significant improvement of the accuracy of the structure.

A microfluidic platform for quantitative measurements of effective protein charges and single ion binding in solution.

The charge state of proteins in solution is a key biophysical parameter that modulates both long and short range macromolecular interactions. However, unlike in the case of many small molecules, the effective charges of complex biomolecules in solution cannot in general be predicted reliably from their chemical structures alone. Here we present an approach for quantifying the effective charges of solvated biomolecules from independent measurements of their electrophoretic mobilities and diffusion coefficients in free solution within a microfluidic device. We illustrate the potential of this approach by determining the effective charges of a charge-ladder family of mutants of the calcium binding protein calbindin D9k in solution under native conditions. Furthermore, we explore ion-binding under native conditions, and demonstrate the ability to detect the chelation of a single calcium ion through the change that ion binding imparts on the effective charge of calbindin D9k. Our findings highlight the difference between the dry sequence charge and the effective charge of proteins in solution, and open up a route towards rapid and quantitative charge measurements in small volumes in the condensed phase.

Molecular recognition study on the binding of calcium to calbindin D9k based on 3D reference interaction site model theory.

Ca(2+)-binding proteins are widely distributed throughout cells and play various important roles. Calbindin D9k is a member of the EF-hand Ca(2+)-binding protein family. In this study, we examined the binding of Ca(2+) to calbindin D9k in terms of the free energy of solvation, as obtained by 3D reference interaction site model theory, which describes the statistical mechanics of liquids. We also investigated the main structural biological factor using spatial decomposition analysis in which the solvation free energy values are decomposed into the residue. We found some characteristic residues that contribute to stabilization of the holo-structure (Ca(2+)-binding structure). These results indicated that, in the holo-structure, these residues are newly exposed to solvent. Subsequently, the gain in solvation free energy, involving a conformational change and exposure to solvent, forms the driving force for binding of the Ca(2+) ion to the EF-hand.

Rapid calculation of protein pKa values using Rosetta.

We developed a Rosetta-based Monte Carlo method to calculate the pK(a) values of protein residues that commonly exhibit variable protonation states (Asp, Glu, Lys, His, and Tyr). We tested the technique by calculating pK(a) values for 264 residues from 34 proteins. The standard Rosetta score function, which is independent of any environmental conditions, failed to capture pK(a) shifts. After incorporating a Coulomb electrostatic potential and optimizing the solvation reference energies for pK(a) calculations, we employed a method that allowed side-chain flexibility and achieved a root mean-square deviation (RMSD) of 0.83 from experimental values (0.68 after discounting 11 predictions with an error over 2 pH units). Additional degrees of side-chain conformational freedom for the proximal residues facilitated the capture of charge-charge interactions in a few cases, resulting in an overall RMSD of 0.85 pH units. The addition of backbone flexibility increased the overall RMSD to 0.93 pH units but improved relative pK(a) predictions for proximal catalytic residues. The method also captures large pK(a) shifts of lysine and some glutamate point mutations in staphylococcal nuclease. Thus, a simple and fast method based on the Rosetta score function and limited conformational sampling produces pK(a) values that will be useful when rapid estimation is essential, such as in docking, design, and folding.

Structural insights into the calcium-dependent interaction between calbindin-D28K and caspase-3.

The regulation of apoptosis involves a complicated cascade requiring numerous protein interactions including the pro-apoptotic executioner protein caspase-3 and the anti-apoptotic calcium-binding protein calbindin-D28K. Using isothermal titration calorimetry, we show that calbindin-D28K binds caspase-3 in a Ca(2+)-dependent fashion. Molecular docking and conformational sampling studies of the Ca(2+)-loaded capase-3/calbindin-D28K interaction were performed in order to isolate potentially crucial intermolecular contacts. Residues in the active site loops of caspase-3 and EF-hands 1 and 2 of calbindin-D28K were shown to be critical to the interaction. Based on these studies, a model is proposed to help understand how calbindin-D28K may deactivate caspase-3 upon binding.

BC(50): a generalized, unifying affinity descriptor.

Assessing binding affinities is an unavoidable step that we come across any time interactions between binding species are investigated. A quantitative evaluation of binding affinities relies on the determination of binding constants but, whilst the binding constant fully defines the affinity of a reagent for a ligand when only one complex species is formed, the same is not true when the interacting partners form more than one complex of different stoichiometry, because all complexes contribute to the overall binding affinity. Unfortunately, this situation is the rule rather than the exception in chemical systems, but a generally accepted solution for this issue has not yet been settled. In this Personal Account, we describe the evolution, from the initial idea to a fully developed stage, of a binding descriptor that has been developed with the aim of filling this gap, thereby providing scientists in all fields of chemistry with a unifying tool for the assessment of binding affinities based on the knowledge of the binding constants in systems that involve any number of complex species.

Calbindin-D32k is localized to a subpopulation of neurons in the nervous system of the sea cucumber Holothuria glaberrima (Echinodermata).

Members of the calbindin subfamily serve as markers of subpopulations of neurons within the vertebrate nervous system. Although markers of these proteins are widely available and used, their application to invertebrate nervous systems has been very limited. In this study we investigated the presence and distribution of members of the calbindin subfamily in the sea cucumber Holothuria glaberrima (Selenka, 1867). Immunohistological experiments with antibodies made against rat calbindin 1, parvalbumin, and calbindin 2, showed that these antibodies labeled cells and fibers within the nervous system of H. glaberrima. Most of the cells and fibers were co-labeled with the neural-specific marker RN1, showing their neural specificity. These were distributed throughout all of the nervous structures, including the connective tissue plexi of the body wall and podia. Bioinformatics analyses of the possible antigen recognized by these markers showed that a calbindin 2-like protein present in the sea urchin Strongylocentrotus purpuratus, corresponded to the calbindin-D32k previously identified in other invertebrates. Western blots with anti-calbindin 1 and anti-parvalbumin showed that these markers recognized an antigen of approximately 32 kDa in homogenates of radial nerve cords of H. glaberrima and Lytechinus variegatus. Furthermore, immunoreactivity with anti-calbindin 1 and anti-parvalbumin was obtained to a fragment of calbindin-D32k of H. glaberrima. Our findings suggest that calbindin-D32k is present in invertebrates and its sequence is more similar to the vertebrate calbindin 2 than to calbindin 1. Thus, characterization of calbindin-D32k in echinoderms provides an important view of the evolution of this protein family and represents a valuable marker to study the nervous system of invertebrates.

Structural characterization of two alternate conformations in a calbindin D9k-based molecular switch.

We have demonstrated that calbindin D(9k) can be converted into a calcium-sensing switch (calbindin-AFF) by duplicating the C-terminal half of the protein (residues 44-75) and appending it to the N-terminus (creating residues 44'-75'). This re-engineering results in a ligand-driven interconversion between two native folds: the wild-type structure (N) and a circularly permuted form (N'). The switch between N and N' is predicted to involve exchange of the 44-75 and 44'-75' segments, possibly linked to their respective folding and unfolding. Here we present direct structural evidence supporting the existence of N and N'. To isolate the N' and N conformations, we introduced the knockdown Ca(2+) binding mutation Glu → Gln at position 65 (E65Q mutant) or at the analogous position 65' (E65'Q mutant). E65Q and E65'Q are therefore expected to adopt conformations N' and N, respectively, in the presence of calcium. Though the amino acid sequences of E65Q and E65'Q differ at only these two positions, nuclear magnetic resonance resonance assignments, chemical shifts, and paramagnetic relaxation enhancement data reveal that they take on separate structures when bound to calcium. Both proteins are comprised of a well-folded domain and a disordered region. However, the segment that is disordered in E65Q (residues 44-75) is folded in E65'Q, and the region that is disordered in E65'Q (residues 44'-75') is structured in E65Q. The results demonstrate that the N' N' conformational change is mediated by a mutually exclusive folding reaction in which folding of one segment of the protein is coupled to unfolding of another segment, and vice versa.

NMR order parameters calculated in an expanding reference frame: identifying sites of short- and long-range motion.

NMR order parameters are calculated from molecular dynamics computer simulations of ubiquitin and the apo (Ca(2+)-free) state of calbindin D(9k). Calculations are performed in an expanding reference frame so as to discriminate between the effects of short- and long-range motions. This approach reveals that the dominant contributions to the order parameters are short-range. Longer-range contributions are limited to specific sites, many of which have been recognized in previous studies of correlated motions. These sites are identified on the basis of an effective reorientational number, n ( eff ). Not only does this parameter identify sites of short- and long-range motion, it also provides a way of evaluating the separability condition that is key to the Lipari-Szabo model-free method. When analyzed in conjunction with the Prompers-Brüschweiler separability index, the n ( eff ) values indicate that longer-range motions play a more prominent role in apo calbindin than they do in ubiquitin.

The dynamics of Ca2+ ions within the solvation shell of calbindin D9k.

The encounter of a Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) ions from D60 to the final binding site, explaining the observed decrement in the affinity of the mutated protein to Ca(2+).

Calreticulin is a thermostable protein with distinct structural responses to different divalent cation environments.

Calreticulin is a soluble calcium-binding chaperone of the endoplasmic reticulum (ER) that is also detected on the cell surface and in the cytosol. Calreticulin contains a single high affinity calcium-binding site within a globular domain and multiple low affinity sites within a C-terminal acidic region. We show that the secondary structure of calreticulin is remarkably thermostable at a given calcium concentration. Rather than corresponding to complete unfolding events, heat-induced structural transitions observed for calreticulin relate to tertiary structural changes that expose hydrophobic residues and reduce protein rigidity. The thermostability and the overall secondary structure content of calreticulin are impacted by the divalent cation environment, with the ER range of calcium concentrations enhancing stability, and calcium-depleting or high calcium environments reducing stability. Furthermore, magnesium competes with calcium for binding to calreticulin and reduces thermostability. The acidic domain of calreticulin is an important mediator of calcium-dependent changes in secondary structure content and thermostability. Together, these studies indicate interactions between the globular and acidic domains of calreticulin that are impacted by divalent cations. These interactions influence the structure and stability of calreticulin, and are likely to determine the multiple functional activities of calreticulin in different subcellular environments.

100% complete assignment of non-labile (1)H, (13)C, and (15)N signals for calcium-loaded Calbindin D(9k) P43G.

Here we present the 100% complete assignment chemical shift of non-labile (1)H, (15)N and (13)C nuclei of Calbindin D(9k) P43G. The assignment includes all non-exchangeable side chain nuclei, including ones that are rarely reported, such as LysNζ as well as the termini. NMR experiments required to achieve truly complete assignments are discussed. To the best of our knowledge our assignments for Calbindin D(9k) extend beyond previous studies reaching near-completeness (Vis et al. in Biochem 33:14858-14870, 1994; Yamazaki et al. in J Am Chem Soc 116:6464-6465, 1994; Yamazaki et al. in Biochem 32:5656-5669, 1993b).

Structural basis of carbohydrate recognition by calreticulin.

The calnexin cycle is a process by which glycosylated proteins are subjected to folding cycles in the endoplasmic reticulum lumen via binding to the membrane protein calnexin (CNX) or to its soluble homolog calreticulin (CRT). CNX and CRT specifically recognize monoglucosylated Glc(1)Man(9)GlcNAc(2) glycans, but the structural determinants underlying this specificity are unknown. Here, we report a 1.95-Å crystal structure of the CRT lectin domain in complex with the tetrasaccharide α-Glc-(1→3)-α-Man-(1→2)-α-Man-(1→2)-Man. The tetrasaccharide binds to a long channel on CRT formed by a concave ß-sheet. All four sugar moieties are engaged in the protein binding via an extensive network of hydrogen bonds and hydrophobic contacts. The structure explains the requirement for glucose at the nonreducing end of the carbohydrate; the oxygen O(2) of glucose perfectly fits to a pocket formed by CRT side chains while forming direct hydrogen bonds with the carbonyl of Gly(124) and the side chain of Lys(111). The structure also explains a requirement for the Cys(105)-Cys(137) disulfide bond in CRT/CNX for efficient carbohydrate binding. The Cys(105)-Cys(137) disulfide bond is involved in intimate contacts with the third and fourth sugar moieties of the Glc(1)Man(3) tetrasaccharide. Finally, the structure rationalizes previous mutagenesis of CRT and lays a structural groundwork for future studies of the role of CNX/CRT in diverse biological pathways.

On the mechanism of protein fold-switching by a molecular sensor.

Alternate frame folding (AFF) is a mechanism by which conformational change can be engineered into a protein. The protein structure switches from the wild-type fold (N) to a circularly-permuted fold (N'), or vice versa, in response to a signaling event such as ligand binding. Despite the fact that the two native states have similar structures, their interconversion involves folding and unfolding of large parts of the molecule. This rearrangement is reported by fluorescent groups whose relative proximities change as a result of the order-disorder transition. The nature of the conformational change is expected to be similar from protein to protein; thus, it may be possible to employ AFF as a general method to create optical biosensors. Toward that goal, we test basic aspects of the AFF mechanism using the AFF variant of calbindin D(9k). A simple three-state model for fold switching holds that N and N' interconvert through the unfolded state. This model predicts that the fundamental properties of the switch--calcium binding affinity, signal response (i.e., fluorescence change upon binding), and switching rate--can be controlled by altering the relative stabilities of N and N'. We find that selectively destabilizing N or N' changes the equilibrium properties of the switch (binding affinity and signal response) in accordance with the model. However, kinetic data indicate that the switching pathway does not require whole-molecule unfolding. The rate is instead limited by unfolding of a portion of the protein, possibly in concert with folding of a corresponding region.

Control of crystal polymorph in microfluidics using molluscan 28 kDa Ca²(+)-binding protein.

Biominerals produced by biological systems in physiologically relevant environments possess extraordinary properties that are often difficult to replicate under laboratory conditions. Understanding the mechanism that underlies the process of biomineralisation can lead to novel strategies in the development of advanced materials. Using microfluidics, we have demonstrated for the first time, that an extrapallial (EP) 28 kDa protein, located in the extrapallial compartment between mantle and shell of Mytilus edulis, can influence, at both micro- and nanoscopic levels, the morphology, structure and polymorph that is laid down in the shell ultrastructure. Crucially, this influence is predominantly dependent on the existence of an EP protein concentration gradient and its consecutive interaction with Ca²(+) ions. Novel lemon-shaped hollow vaterite structures with a clearly defined nanogranular assembly occur only where particular EP protein and Ca²(+) gradients co-exist. Computational fluid dynamics enabled the progress of the reaction to be mapped and the influence of concentration gradients across the device to be calculated. Importantly, these findings could not have been observed using conventional bulk mixing methods. Our findings not only provide direct experimental evidence of the potential influence of EP proteins in crystal formation, but also offer a new biomimetic strategy to develop functional biomaterials for applications such as encapsulation and drug delivery.

Global and local Voronoi analysis of solvation shells of proteins.

This paper presents the structure and dynamics of hydration shells for the three proteins: ubiquitin, calbindin, and phospholipase. The raw data derived from molecular dynamics simulations are analyzed on the basis of fully atomistic Delaunay tesselations. In order to cope with the high numerical effort for the computation of these Voronoi shells, we have implemented and optimized an intrinsically periodic algorithm. Based on this highly efficient Voronoi decomposition, a variety of properties is presented: three dimensional water and ion nuclear densities as well as the geometrical packing of water molecules are discussed. Thereby, we develop Voronoi interface surface area, the Voronoi analog of the well known solvent accessible surface area. The traditional radial distribution functions are resolved into Voronoi shells as a transient device to the new concept of shell-grained orientational order. Thus, we analyze the donor-acceptor property as well as the amount of dielectric screening. Shell dynamics is described in terms of mean residence times. In this way, a retardation factor for different shells can be derived and was compared to experimental values. All these results and properties are presented both at the global protein level as well as at the local residue level.