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Calmodulin is a central mediator of calcium-dependent signal transduction pathways and regulates the activity of a large number of diverse targets. Calcium-dependent interactions of calmodulin with regulated proteins are of generally high affinity but of quite variable thermodynamic origins. Here we investigate the influence of the binding of the calmodulin-binding domain of calmodulin kinase I on the fast internal dynamics of calcium-saturated calmodulin. NMR relaxation was used to probe motion on the backbone (viewed through the backbone amide NH group) and the side chains (viewed through methyl groups). The distribution of the amplitudes of side chain dynamics is trimodal. The microscopic details of side chain motion are compared with those of a thermodynamically and structurally similar complex of calmodulin with the calmodulin-binding domain of the smooth muscle myosin light chain kinase. While there are no significant differences in backbone dynamics and no net change in methyl- bearing side chain dynamics, a large redistribution of the amplitude of methyl dynamics is observed between the two complexes. The variation in dynamics was largely localized to the heterogeneously dynamic target-binding interface, suggesting that differential dynamics of the binding surface plays a functional role in the high-affinity binding interactions of calmodulin. These results begin to reveal a fundamental role for residual protein entropy in molecular recognition by calmodulin. [ABSTRACT FROM AUTHOR]

ThermoFluor (a miniaturized high-throughput protein stability assay) was used to analyze the linkage between protein thermal stability and ligand binding. Equilibrium binding ligands increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. Binding constants (Kb) were measured by examining the systematic effect of ligand concentration on protein stability. The precise ligand effects depend on the thermodynamics of protein stability: in particular, the unfolding enthalpy. An extension of current theoretical treatments was developed for tight binding inhibitors, where ligand effect on Tm can also reveal binding stoichiometry. A thermodynamic analysis of carbonic anhydrase by differential scanning calorimetry (DSC) enabled a dissection of the Gibbs free energy of stability into enthalpic and entropic components. Under certain conditions, thermal stability increased by over 30 °C; the heat capacity of protein unfolding was estimated from the dependence of calorimetric enthalpy on Tm. The binding affinity of six sulfonamide inhibitors to two isozymes (human type 1 and bovine type 2) was analyzed by both ThermoFluor and isothermal titration calorimetry (ITC), resulting in a good correlation in the rank ordering of ligand affinity. This combined investigation by ThermoFluor, ITC, and DSC provides a detailed picture of the linkage between ligand binding and protein stability. The systematic effect of ligands on stability is shown to be a general tool to measure affinity. [ABSTRACT FROM AUTHOR]

Investigates the calcium-induced conformational switching of Paramecium calmodulin (CaM) to provide evidence for domain coupling by CaM. Nuclear magnetic resonance of Paramecium CaM; Titration of sites I and II; Changes at the domain boundaries.

Focuses on the dissection of the pathway of molecular recognition by calmodulin. Use of amide hydrogen exchange; Decline of individual protection factors; Disruption of ion pairs between calmodulin and the bound model.

Presents a study which examines the functions of the carboxamide moiety which links the carbohydrate and protein moieties in N-linked glycoproteins. Assessment of methodology used; Findings of the study.

Reports on the preparation of L-lactate dehydrogenase. Catalytic behavior toward the representative gamma-keto acid substrates; Comparison of the level of protein expression; Resistance to thermal denaturation; Support of the validity of ion-pairing approach.

Investigates the role of the exposed loop in transforming growth factor-beta (TGF-beta) centered at residue 71. Synthesis and purification of mutant TGF-beta molecules; Biological properties of the molecules; Growth inhibition assays; Receptor cross-linking; Northern blot assay.

Investigates two possible mechanisms for enzyme-catalyzed N-glycosylation using chemically synthesized peptide and lipid disaccharide substrates. Fate of deuterium in three stereospecifically deuterated peptides.

Folylpoly-γ-glutamate synthetase (FPGS, EC 6.3.2.17) is an ATP-dependent ligase that catalyzes formation of poly-γ-glutamate derivatives of reduced folates and antifolates such as methotrexate and 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDAH4PteGlu1). While the chemical mechanism of the reaction catalyzed by FPGS is known, it is unknown whether single or multiple glutamate residues are added following each folate binding event. A very sensitive high-performance liquid chromatography method has been used to analyze the multiple ligation reactions onto radiolabeled DDAH4PteGlu1 catalyzed by FPGS to distinguish between distributive or processive mechanisms of catalysis. Reaction time courses, substrate trapping, and pulse-chase experiments were used to assess folate release during multiple glutamate additions. Together, the results of these experiments indicate that hFPGS can catalyze the processive addition of approximately four glutamate residues to DDAH4PteGlu1. The degree of processivity was determined to be dependent on the concentration of the folate substrate, thus suggesting a mechanism for the regulation of folate polyglutamate synthesis in cells. [ABSTRACT FROM AUTHOR]

Two transformed murine macrophage cell lines (RAW 264.7 ATCC TIB-71 and CRL-2278) were examined for oxidant production at various times following activation by using a set of fluorescence and ESR-active probes. Stimulation with a soluble agonist or activation with bacterial lipopolysaccharide plus v-interferon caused only very small initial increases in O2 consumption above basal rates; however, at 2–4 h post-activation, respiration increased to 2–3-fold and remained at these elevated levels over the subsequent lifetime of the cell (20–30 h). Oxidation reactions were confined primarily within the cell, as was demonstrated by using phagocytosable dichlorodihydrofluorescein-conjugated latex beads and cyclic hydroxylamines with differing membrane permeabilities. From the intrinsic reactivities of these probes and the time course of their oxidations, one infers the induction of apparent peroxidase activity beginning at ∼2 h post-activation coinciding with the increase in overall respiratory rate; this acquired capability was accompanied by accumulation of a stable horseradish peroxidase-reactive oxidant, presumably H2O2, in the extracellular medium. Nitrite ion rapidly accumulated in the extracellular medium over a period of 5–8 h post-activation in both cell lines, indicating the presence of active nitric oxide synthase (iNOS) during that period. Prostaglandin endoperoxide H synthase (COX-2) activity was detected at 15–20 h post-activation by the use of a sensitive peroxide assay in conjunction with a COX-2 specific inhibitor (DuP-697). Superoxide formation was detected by reaction with hydroethidine within the first hour following activation, but not thereafter. Consistent with the absence of significant respiratory stimulation, the amount of O2̇- formed was very small; comparative reactions of cyclic hydroxylamine probes indicated that virtually none of the O2̇- was discharged into the external medium. Myeloperoxidase (MPO) activity was probed at various times post-activation by using fluorescein-conjugated polyacrylamide beads, which efficiently trap MPO-generated HOCI in neutrophils to give stable chlorofluorescein products. However, chlorination of the dye was not detected under any conditions in RAW cells, virtually precluding MPO involvement in their intracellular reactions. This same probe was used to determine changes in intraphagosomal pH, which increased slowly from ∼6.5 to ∼8.2 over a 20 h post-phagocytosis period. The cumulative data suggest that activation is followed by sequential induction of an endogenous peroxidase, iNOS, and COX-2, with NADPH oxidase-derived O2̇- playing a minimal role in the direct generation of intracellular oxidants. To account for reported observations of intracellular tyrosine nitration late in the life cycles of macrophages, we propose a novel mechanism wherein iNOS-generated NO2- is used by COX-2 to produce NO2̇ as a terminal microbicidal oxidant and nitrating agent. [ABSTRACT FROM AUTHOR]

Cone short-wave (SWS1) visual pigments can be divided into two categories that correlate with spectral sensitivity, violet sensitive above 390 nm and ultraviolet sensitive below that wavelength. The evolution and mechanism of spectral tuning of SWS1 opsins are proving more complex than those of other opsin classes. Violet-sensitive pigments probably evolved from an ancestral ultraviolet-sensitive opsin, although in birds ultraviolet sensitivity has re-evolved from violet-sensitive pigments. In certain mammals, a single substitution involving the gain of a polar residue can switch sensitivity from ultraviolet to violet sensitivity, but where such a change is not involved, several substitutions may be required to effect the switch. The guinea pig, Cavia porcellus, is a hystricognathous rodent, a distinct suborder from the Sciurognathi, such as rats and mice. It has been shown by microspectrophotometry to have two cone visual pigments at 530 and 400 nm. We have ascertained the sequence of the short-wave pigment and confirmed its violet sensitivity by expression and reconstitution of the pigment in vitro. Moreover, we have shown by site-directed mutagenesis that a single residue is responsible for wavelength tuning of spectral sensitivity, a Va186Phe causing a 60 nm short-wave shift into the ultraviolet and a Va186Tyr substitution shifting the pigment 8 nm long wave. The convergent evolution of this mammalian VS pigment provides insight into the mechanism of tuning between the violet and UV. [ABSTRACT FROM AUTHOR]

The temperature dependence of the fast internal dynamics of calcium-saturated calmodulin in complex with a peptide corresponding to the calmodulin-binding domain of the smooth muscle myosin light chain kinase is examined using [sup 15]N and ²H NMR relaxation methods. NMR relaxation studies of the complex were carded out at 13 temperatures that span 288-346 K. The dynamics of the backbone and over four dozen methyl-beating side chains, distributed throughout the calmodulin molecule, were probed. The side chains show a much more variable and often considerably larger response to temperature than the backbone. A significant variation in the temperature dependence of the amplitude of motion of individual side chains is seen. The amplitude of motion of some side chains is essentially temperature-independent while many show a simple roughly linear temperature dependence. In a few cases, angular order increases with temperature, which is interpreted as arising from interactions with neighboring residues. In addition, a number of side chains display a nonlinear temperature dependence. The significance of these and other results is illuminated by several simple interpretative models. Importantly, analysis of these models indicates that changes in generalized order parameters can be robustly related to corresponding changes in residual entropy. A simple cluster model that incorporates features of cooperative or conditional motion reproduces many of the unusual features of the experimentally observed temperature dependence and illustrates that side chain interactions result in a dynamically changing environment that significantly influences the motion of internal side chains. This model also suggests that the intrinsic entropy of interacting clusters of side chains is only modestly reduced from that of independent side chain motion. Finally, estimates of protein heat capacity support the view that the major contribution to the heat capacity of protein solutions... [ABSTRACT FROM AUTHOR]

Studies the permeation of phospholipid membranes by peroxynitrite. Biochemical reactions of peroxynitrous acid and related reactive nitrogen species as representation of oxidative stress; Putative role of radicals in tissue injury; Minimal reaction scheme of the major pathways for intravesicular oxidation by peroxynitrite; Models of lipid oxidation.

To improve our understanding of the effects of ß-alanine (ß) substitution and the number of heterocycles on DNA binding affinity and selectivity, we investigated the interactions of an eight-ring hairpin polyamide (PA) and two ß derivatives as well as a six-heterocycle analogue with their cognate DNA sequence, 5'-TGGCTT-3'. Binding selectivity and the effects of ß have been investigated with the cognate and five mutant DNAs. A set of powerful and complementary methods have been employed for both energetic and structural evaluations: UV melting, biosensor surface plasmon resonance, isothermal titration calorimetry, circular dichroism, and a DNA ligation ladder global structure assay. The reduced number of heterocycles in the six-ring PA weakens the binding affinity; however, the smaller PA aggregates significantly less than the larger PAs and allows us to obtain the binding thermodynamics. The PA-DNA binding enthalpy is large and negative with a large negative ΔCp and is the primary driving component of the Gibbs free energy. The complete SPR binding results clearly show that ß substitutions can substantially weaken the binding affinity of hairpin PAs in a position-dependent manner. More importantly, the changes in the binding of PA to the mutant DNAs further confirm the position-dependent effects on the PA-DNA interaction affinity. Comparison of mutant DNA sequences also shows a different effect in recognition of T·A versus A·T base pairs. The effects of DNA mutations on binding of a single PA as well as the effects of the position of ß substitution on binding tell a clear and very important story about sequence-dependent binding of PAs to DNA. [ABSTRACT FROM AUTHOR]

Development of efficient microbial biofuel cells requires an ability to exploit interfacial electron transfer reactions to external electron acceptors, such as metal oxides; such reactions occur in the facultative anaerobic Gram-negative bacterium Shewanrila oneidensis MR-i through the catalytic activity of the outer membrane decaheme c-type cytodsmme MtrC. Central to the utility of this pathway to synthetic biology is an understanding of cellular mechanisms that maintain optimal MtrC function cellular localization, and renewal by degradation and resynthesis. In order to monitor trafficking to the outer membrane, and the environmental sensitivity of MtrC, we have engineered a tetracysteine tag (ie, CCPGCC) at its C-terminus that permits labeling by the cell impermeable biarsenical fluorophore carboxy-FIAsH (CrAsH) of MtrC at the surface of living Shewandlla aneidensis MR-I cells. In comparison, the cell ermeable reagent FlAsH permits labeling of the entire population of MtrC, induding proteolytic fragments resulting from incorrect maturation. We demonstrate specific labeling by CrAsH of engineered MtrC (MtrC*) which is dependent on the presence of a fimctional type 2 secretionystem (T2S), as evidenced by T2S system gspl) or gspG deletion mutants which are incapable of CrAsH labeling. Under these latter conditions, MtrC* undergoes proteolytic degradation to fr~rm a large 35-38 kDa fragment~ this degradation product is also resolved during normal turnover of the CrAsH-labeled MtrC protein. Ho MtrC protein is released into the medium during turnover, suggesting the presence of cellular turnover systems involving MtrC reuptake and degradation. The mature MtrC localized on the outer membrane is a long-lived protein, with a turnover rate of 0.043 h 'that is insensitive to °z concentration. Maturation of MtrC is relatively inefficient, with substantial rates of turnover of the immature protein prior to export to the outer membrane (i.e., 0.028 h ) that are consistent with the inherent complexity associated with correct heme insertion and acylation of MtrC that occurs in the periplasm prior to its targeting to the outer membrane. These latter results suggest that MtrC protein trafficking to the outer membrane and its subsequent degradation are tightly regulated, which is consistent with cellular processing pathways that target MtrC to extracellular structures and their possible role in promoting electron transfer from Shewanella to extracellular acceptors. [ABSTRACT FROM AUTHOR]

γ-Glutamyl hydrolase, a cysteine peptidase, catalyzes the hydrolysis of poly-γ-glutamate derivatives of folate cofactors and many antifolate drugs. We have used internally quenched fluorogenic derivatives of glutamyl-γ-glutamate and (4,4-difluoro)glutamyl-γ-glutamate to examine the effect of fluorine substitution adjacent to the scissile isopeptide bond. Using a newly developed continuous fluorescence assay, the hydrolysis of both substrates could be described by Michaelis-Menten kinetics. Fluorine substitution resulted in a significant decrease in observed rates of hydrolysis under steady-state conditions due primarily to a 15-fold increase in Km. Using stopped-flow techniques, hydrolysis of the non-fluorinated isopeptide was characterized by a burst phase followed by a steady-state rate, indicating that formation of the acyl enzyme is not rate-limiting for hydrolysis of this isopeptide. This conclusion was confirmed by analysis of the progress curves over a wide range of substrate concentration, which demonstrated that the acylation rate (k2) is 10-fold higher than the deacylation rate (k3). The increased value of Km associated with the difluoro derivative limited the ability to obtain comparable pre-steady-state kinetics data at saturating concentration of substrate due to inner filter effects. However, even under nonsaturating conditions, a modest burst was observed for the difluoro derivative. These data indicate that either deacylation or rearrangement of the enzyme-product complex is rate-limiting in this isopeptide hydrolysis reaction. [ABSTRACT FROM AUTHOR]

In glycosyltransferase-catalyzed reactions a new carbohydrate—carbohydrate bond is formed between a carbohydrate acceptor and the carbohydrate moiety of either a sugar nucleotide donor or lipid-linked saccharide donor. It is currently believed that most glycosyltransferase-catalyzed reactions occur via an electrophilic activation mechanism with the formation of an oxocarbenium ion-like transition state, a hypothesis that makes clear predictions regarding the charge development on the donor (strong positive charge) and acceptor (minimal negative, charge) substrates. To better understand the mechanism of these enzyme-catalyzed reactions, we have introduced a strongly electron-withdrawing group (fluorine) at C-5 of both donor and acceptor substrates in order to explore its effect on catalysis. In particular, we have investigated the effects of the 5-fluoro analogues on the kinetics of two glycosyltransferase-catalyzed reactions mediated by UDP-GlcNAc:GlcNAc-P-P-Dol N-acetylglucosaminyltransferase (chitobiosyl-P-P-lipid synthase, CLS) and β-N-acetylglucosaminyl-β-1,4 galactosyltransferase (GAIT). The 5-fluoro group has a marked effect on catalysis when inserted into the UDP-GlcNAc donor, with the UDP(5-F)-GlcNAc serving as a competitive inhibitor of CLS rather than a substrate. The (5-F)-GlcNAc β-octyl glycoside acceptor, however, is an excellent substrate for GAIT. Both of these results support a weakly associative transition state for glycosyltransferase-catalyzed reactions that proceed with inversion of configuration. [ABSTRACT FROM AUTHOR]

Escherichia coli were transformed by electroporation to introduce a plasmid harboring a GFP gene-containing vector. The fluorescence of the purified GFP isolated from the transformant was quenched by myeloperoxidase (MPO)-generated HOC!, by peroxynitrous acid (ONOOH) and by enzymatic ally or radiolytically generated N02¨ but not by other putative neutrophil-generated oxidants. Fluorescence from the bacterium was effectively quenched by HOC! but not peroxynitrite, oxidizing radicals derived from its 0-0 bond homolysis, or the other oxidants under study. Exposure of serum-opsonized bacteria to human neutrophils resulted in extensive loss of GFP fluorescence; fluorescence microscopy revealed that phagocytosed bacteria were completely quenched but that bacteria remaining in the external media were unquenched. Addition of sodium azide to the medium to inhibit MPO prevented neutrophil-mediated fluorescence quenching. Because the amount of HOC! required to inhibit bacterial fluorescence was an order of magnitude greater than required to inhibit colonial growth, these results imply that sufficient HOC! was formed within the neutrophil phagosome to kill the microbe. [ABSTRACT FROM AUTHOR]

Examines the spectral tuning and evolution of short wave-sensitive cone pigments in cottoid fish from Lake Baikal in Siberia, Russia. Presence of a porphyropsin and rhodopsin mixture in Cottus gobio; Mutagenesis of the C. gobio; Alignment of deduced amino acid sequences of cottoid optsins.

Investigates the internal dynamics of a flavin-bearing protein using an oxidized flavodoxin from Cyanobacterium anabaena. Assignments of flavodoxin; Characterization of molecular tumbling; Dynamics of flavodoxin.

Investigates site-specific phosphorylation of human p53 protein determined by mass spectrometry. Differential phosphorylation status of r-p53 expressed in the pressence of OA; N-termini of control r-p53 and OA r-p53.