Parvalbumin (PV) is a globular calcium (Ca(2+))-selective protein expressed in a variety of biological tissues. Our computational studies of the rat β-parvalbumin (β-PV) isoform seek to elucidate the molecular thermodynamics of Ca(2+) versus magnesium (Mg(2+)) binding at the protein's two EF-hand motifs. Specifically, we have utilized molecular dynamics (MD) simulations and a mean-field electrolyte model (mean spherical approximation (MSA) theory) to delineate how the EF-hand scaffold controls the "local" thermodynamics of Ca(2+) binding selectivity over Mg(2+). Our MD simulations provide the probability density of metal-chelating oxygens within the EF-hand scaffolds for both Ca(2+) and Mg(2+), as well the conformational strain induced by Mg(2+) relative to Ca(2+) binding. MSA theory utilizes the binding domain oxygen and charge distributions to predict the chemical potential of ion binding, as well as their corresponding concentrations within the binding domain. We find that the electrostatic and steric contributions toward ion binding were similar for Mg(2+) and Ca(2+), yet the latter was 5.5 kcal/mol lower in enthalpy when internal strain within the EF hand was considered. We therefore speculate that beyond differences in dehydration energies for the Ca(2+) versus Mg(2+), strain induced in the β-PV EF hand by cation binding significantly contributes to the nearly 10,000-fold difference in binding affinity reported in the literature. We further complemented our analyses of local factors governing cation binding selectivity with whole-protein (global) contributions, such as interhelical residue-residue contacts and solvent exposure of hydrophobic surface. These contributions were found to be comparable for both Ca(2+)- and Mg(2+)-bound β-PV, which may implicate local factors, EF-hand strain, and dehydration, in providing the primary means of selectivity. We anticipate these methods could be used to estimate metal binding thermodynamics across a broad range of PV sequence homologues and EF-hand-containing, Ca(2+) binding proteins.

Sarcoendoplasmic reticulum Ca 2+-ATPase (SERCA) is a transmembrane pump that plays an important role in transporting calcium into the sarcoplasmic reticulum (SR). While calcium (Ca 2+) binds SERCA with micromolar affinity, magnesium (Mg 2+) and potassium (K +) also compete with calcium (Ca 2+) binding. However, the molecular bases for these competing ions' influence on SERCA function and the selectivity of the pump for Ca 2+ are not well-established. We therefore used in silico methods to resolve molecular determinants of cation binding in the canonical site I and II Ca 2+ binding sites: 1) triplicate molecular dynamics (MD) simulations of Mg 2+ , Ca 2+ and K +-bound SERCA. 2) mean spherical approximation (MSA) theory to determine the affinity and selectivity of cation binding to the MD-resolved structures and 3) state models of SERCA turnover informed from MSA-derived affinity data. Our key findings are that a) coordination at sites I and II are optimized for Ca 2+ and to a lesser extent for Mg 2+ and K + , as determined by MD-derived cation-amino acid oxygen and bound water configurations, b) the impaired coordination and high desolvation cost for Mg 2+ precludes favorable Mg 2+ binding relative to Ca 2+ , while K + has limited capacity to bind site I, c) Mg 2+ most likely acts as inhibitor and K + as intermediate in SERCA's reaction cycle, based on a best-fit state model of SERCA turnover. These findings provide a quantitative basis for SERCA function that leverages molecular-scale thermodynamic data and rationalize enzyme activity across broad ranges of K + , Ca 2+ and Mg 2+ concentrations.

C-terminal domains. Interestingly, a calcium-modulated contractile fabric can be reconstituted with purified full-length Tcb2. The full-length protein and its N-terminal domain are not amenable to high-resolution structure determination, as they tend to aggregate in the presence of calcium and/or upon concentration. However, the C-terminal domain (Tcb2-C) is highly soluble at both low and high calcium concentrations. Solution NMR HSQC spectra of 15N-labeled Tcb2-C indicate that the protein is well folded in the presence and absence of calcium and undergoes a dramatic conformational change upon calcium addition. We expressed and purified 15N, 13C-labeled Tcb2-C, and obtained nearly complete chemical shift assignments for the protein in both the presence and absence of calcium. The solution structure of calcium-free Tcb2-C was determined using NMR-derived experimental distance and torsion angle restraints. The current structural models reveal an architecture exhibited by other calcium-binding proteins, with paired EF-hand motifs connected by a flexible loop. NMR structure determination of calcium-bound Tcb2-C is currently underway. We are also using NMR spectroscopy to quantify the calcium-binding properties of the domain and investigate its conformational dynamics. These studies will establish a structural basis for elucidating the function and unique contractile properties of Tcb2. Calcium-binding proteins play an important role in cellular functions and regulation. Two such proteins are the S100A1 protein and the beta-parvalbumin (PV) protein, both of which contain metal-binding sites called EF-hands that consist of helix-loop-helix regions. Both of these proteins selectively bind calcium ions despite significantly higher (millimolar) intracellular concentrations of other cat-

Sarcoendoplasmic reticulum Ca 2+ -ATPase (SERCA) is a transmembrane pump that plays an important role in transporting calcium into the sarcoplasmic reticulum (SR). While calcium (Ca 2+ ) binds SERCA with micromolar affinity, magnesium (Mg 2+ ) and potassium (K + ) also compete with calcium (Ca 2+ ) binding. However, the molecular bases for these competing ions' influence on SERCA function and the selectivity of the pump for Ca 2+ are not well-established. We therefore used in silico methods to resolve molecular determinants of cation binding in the canonical site I and II Ca 2+ binding sites: 1) triplicate molecular dynamics (MD) simulations of Mg 2+ , Ca 2+ and K + -bound SERCA. 2) mean spherical approximation (MSA) theory to determine the affinity and selectivity of cation binding to the MDresolved structures and 3) state models of SERCA turnover informed from MSA-derived affinity data. Our key findings are that a) coordination at sites I and II are optimized for Ca 2+ and to a lesser extent for Mg 2+ and K + , as determined by MD-derived cation-amino acid oxygen and bound water configurations, b) the impaired coordination and high desolvation cost for Mg 2+ precludes favorable Mg 2+ binding relative to Ca 2+ , while K + has limited capacity to bind site I, c) Mg 2+ most likely acts as inhibitor and K + as intermediate in SERCA's reaction cycle, based on a best-fit state model of SERCA turnover. These findings provide a quantitative basis for SERCA function that leverages molecular-scale thermodynamic data and rationalize enzyme activity across broad ranges of K + , Ca 2+ and Mg 2+ concentrations.

Background The development and optimization of therapies for rheumatoid arthritis (RA) is currently hindered by a lack of methods for early non-invasive monitoring of treatment response. Annexin A2, an inflammation-associated protein whose presence and phosphorylation levels are upregulated in RA, represents a potential molecular target for tracking RA treatment response. Methods LS301, a near-infrared dye-peptide conjugate that selectively targets tyrosine 23-phosphorylated annexin A2 (pANXA2), was evaluated for its utility in monitoring disease progression, remission, and early response to drug treatment in mouse models of RA by fluorescence imaging. The intraarticular distribution and localization of LS301 relative to pANXA2 was determined by histological and immunohistochemical methods. Results In mouse models of spontaneous and serum transfer-induced inflammatory arthritis, intravenously administered LS301 showed selective accumulation in regions of joint pathology including paws, ankles, and knees with positive correlation between fluorescent signal and disease severity by clinical scoring. Whole-body near-infrared imaging with LS301 allowed tracking of spontaneous disease remission and the therapeutic response after dexamethasone treatment. Histological analysis showed preferential accumulation of LS301 within the chondrocytes and articular cartilage in arthritic mice, and colocalization was observed between LS301 and pANXA2 in the joint tissue. Conclusions We demonstrate that fluorescence imaging with LS301 can be used to monitor the progression, remission, and early response to drug treatment in mouse models of RA. Given the ease of detecting LS301 with portable optical imaging devices, the agent may become a useful early treatment response reporter for arthritis diagnosis and drug evaluation.