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The intracellular [Ca.sup.2+]-activated targeting of the isolated protein kinase C[alpha] (PKC[alpha])- and cPL[A.sub.2][alpha]-C2 domains in a macrophage model system were compared. The results show that phosphatidylinositol-4,5-biphosphate (PI[P.sub.2]) is essential for efficient membrane docking at physiological [Ca.sup.2+] concentrations.

Molecular dynamics simulations were conducted for adipocyte lipid-binding protein by utilizing the apo and holo forms bound with stearic and oleic acid. The charged holo forms showed more electrostatic binding energy and more stable hydrogen bonds compared to the neutral forms. However, fatty acid behavior was barely affected by electrostatics. Instead, fatty acid reactions were related to the absence or presence of an unsaturated bond within the acyl chain.

[Super 1]H NMR was used to study the degree of H-bonding of mammalian heme oxygenase due to the coupling of the ligated water molecule to the H-bond network. The results provide insight into much larger effect of pH on the H-bonding network in human HO which is transmitted much further from the iron than in a pathogenic bacterial HO and their implications for the HO mechanism of the H-bond of Tyr58 to Asp140.

A bioinformatics study is conducted to analyze the polyelectrolyte domains, as well as the intrinsic disorder observed within the intrinsically disordered proteins (IDP) family, Asprich. The functions of the domains of these proteins are shown to be highly controlled by the structural and surface charge density features.

The two-dimensional NMR spectroscopy studies are conducted to explain the electronic structure of the orbital ground state of the azide-substrate complex of human heme oxygenase 1 (hHO). The azide, as well as the cyanide hHO complexes are shown to exhibit a low-spin ground state.

The bioinformatics studies related to the identification of the molecular traits that classify 12 amelogenin proteins as members of the intrinsically disordered or unstructured protein family (IDPs) which are capable of transformation to a folded state as part of their overall function is described. The studies confirmed labile structure of one of the amelogenins, recombinant porcine rP172, that facilitated interactions of amelogenin with other macromolecules or with minerals to yield stable internal protein assembly.

A novel research on AP7, a member of the intrinsically disordered protein class (IDP) and also an extracellular aragonite-associated protein, and the new aspects of AP7 mineralization activity is presented. The ability of the partially disordered C-RING protein to perform inorganic phase stabilization and aggregate formation has provided insight into the protein-mediated nacre formation process.

The structure-function properties of an unusual 36 AA C-terminal sequence of an extracellular matrix mollusk shell protein, AP7 that exhibits partial homology to the really interesting new gene (RING) family are reported. The results suggest that cationic pseudo-RING region is utilized by the AP7 protein for the tasks such as protein-protein interaction within the mollusk shell matrix.

The [super 1]H NMR hyperfine shift pattern for the substrate and axial His [C.sub.[beta]]H's and the substrate-protein contacts of the cyanide-inhibited protohemin and 2,4-dimethyldeuterohemin complexes of the triple mutant are analyzed and compared to data for the WT complex. The results have shown that the pattern of heme methyl hyperfine shifts is not used alone to determine the in-plane orientation of the substrates as it is related to the stereospecificity of heme cleavage.

The role of residue histidine ligand ([His.sup.172]) in the mechanism of peptidylglycine [alpha]-hydroxylating monooxygenase (PHM) is analyzed. The disruption of a hydrogen-bonded network through the water-filled cavity connecting the two copper sites might interfere with the preorganization and subsequent reorganization that promotes effective hydrogen wave function overlap and transfer of the hydrogen from the [alpha]-carbon of substrate to the activated electron transfer copper oxygen species.

The amino acids Agr183, Lys18 and Tyr134 are mutated to completely remove the interactions between the protein and the propionate groups that anchor the heme and Asp140 and Tyr58 are mutated to the network to evaluate the contribution of the distal hydrogen bonding network to control the regiospecificity. The analysis of the Heme seating in a triple mutant with almost completely altered regiochemistry has clarified the determinants of the regiospecificity of human heme oxygenase.

Betaine-homocysteine S-methyltransferase(BHMT) is a zinc dependent enzyme that catalyzes the transfer of a methyl group from glycine betaine to homocysteine to form dimethylglycine and methionine. the crystal structure of BHMT, the amino acids Trp44 and Tyr160 are involved in binding Bet, and Glu 159 in binding Hcy.

The triple mutant of the solubilized, 265-residue construct of human heme oxygenase, K I8E/E29K/R183E-hHO, has been shown to redirect the exclusive α-regioselectivity of wild-type hHO to primarily β,δ-selectivity in the cleavage of heme (Wang, J., Evans, J. P., Ogura, H., La Mar, G. N., and Ortiz de Montellano, P. R. (2006) Biochemistry 45, 61-73). The 1H NMR hyperfine shift pattern for the substrate and axial His CβH's .and the substrate-protein contacts of the cyanide-inhibited protohemin and 2,4-dimethyldeuterohemin complexes of the triple mutant have been analyzed in detail and compared to data for the WT complex. It is shown that protein contacts for the majo/solution isomers for both substrates in the mutant dictate ∼90° in-plane clockwise rotation relative to that in the WT. The conventional interpretation of the pattern of substrate methyl hyperfine shifts, however, indicates substrate rotations of only ∼50°. This paradox is resolved by demonstrating that the axial His25 imidazole ring also rotates counterclockwise with respect to the protein matrix in the mutant relative to that in the WT. The axial His25 CβH hyperfine shifts are shown to serve as independent probes of the imidazole plane orientation relative to the protein matrix. The analysis indicates that the pattern of heme methyl hyperfine shifts cannot be used alone to determine the in-plane orientation of the substrate as it relates to the stereospecificity of heme cleavage, without explicit consideration of the orientation of the axial His imidazole plane relative to the protein matrix. [ABSTRACT FROM AUTHOR]

The C2 domain is a ubiquitous, conserved protein signaling motif widely found in eukaryotic signaling proteins. Although considerable functional diversity exists, most C2 domains are activated by Ca2+ binding and then dock to a specific cellular membrane. The C2 domains of protein kinase Ca (PKCα) and cytosolic phospholipase A2α (cPLA2α), for example, are known to dock to different membrane surfaces during an intracellular Ca2+ signal. Ca2+ activation targets the PKCα C2 domain to the plasma membrane and the cPLA2α C2 domain to the internal membranes, with no detectable spatial overlap. It is crucial to determine how such targeting specificity is achieved at physiological bulk Ca2+ concentrations that during a typical signaling event rarely exceed 1 µM. For the isolated PKCα C2 domain in the presence of physiological Ca2+ levels, the target lipids phosphatidylserine (PS) and phosphatidylinositol-4,5- bisphosphate (PIP2) are together sufficient to recruit the PKCα C2 domain to a lipid mixture mimicking the plasma membrane inner leaflet. For the cPLA2α C2 domain, the target lipid phosphatidyicholine (PC) appears to be sufficient to drive membrane targeting to an internal membrane mimic at physiological Ca2+ levels, although the results do not rule out a second, unknown target molecule. Stopped-flow kinetic studies provide additional information about the fundamental molecular events that occur during Ca2+-activated membrane docking. In principle, C2 domain-directed intracellular targeting, which requires coincidence detection of multiple signals (Ca2+ and one or more target lipids), can exhibit two different mechanisms: messenger-activated target affinity (MATA) and target-activated messenger affinity (TAMA). The C2 domains studied here both utilize the TAMA mechanism, in which the C2 domain Ca2+ affinity is too low to be activated by physiological Ca2+ signals in most regions of the cell. Only when the C2 domain nears its target membrane, which provides a high local concentration of target lipid, is the effective Ca2+ affinity increased by the coupled binding equilibrium to a level that enables substantial Ca2+ activation and target docking. Overall, the findings emphasize the importance of using physiological ligand concentrations in targeting studies because super-physiological concentrations can drive docking interactions even when an important targeting molecule is missing. [ABSTRACT FROM AUTHOR]