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Bacillus sp. TB-90 urate oxidase (BTUO) is one of the most thermostable homotetrameric enzymes. We previously reported [Hibi, T., et al. (2014) Biochemistry 53, 3879-3888] that specific binding of a sulfate anion induced thermostabilization of the enzyme, because the bound sulfate formed a salt bridge with two Arg298 residues, which stabilized the packing between two ß-barrel dimers. To extensively characterize the sulfate-binding site, Arg298 was substituted with cysteine by site-directed mutagenesis. This substitution markedly increased the protein melting temperature by ~20 °C compared with that of the wild-type enzyme, which was canceled by reduction with dithiothreitol. Calorimetric analysis of the thermal denaturation suggested that the hyperstabilization resulted from suppression of the dissociation of the tetramer into the two homodimers. The crystal structure of R298C at 2.05 Å resolution revealed distinct disulfide bond formation between the symmetrically related subunits via Cys298, although the Cβ distance between Arg298 residues of the wild-type enzyme (5.4 Å apart) was too large to predict stable formation of an engineered disulfide cross-link. Disulfide bonding was associated with local disordering of interface loop II (residues 277-300), which suggested that the structural plasticity of the loop allowed hyperstabilization by disulfide formation. Another conformational change in the C-terminal region led to intersubunit hydrogen bonding between Arg7 and Asp312, which probably promoted mutant thermostability. Knowledge of the disulfide linkage of flexible loops at the subunit interface will help in the development of new strategies for enhancing the thermostabilization of multimeric proteins. [ABSTRACT FROM AUTHOR]

Research has been conducted on the steroid sulfatase. The inhibition of estrone sulfatase activity has been investigated via the synthesis of estrone 3-O-sulfate analogues and the evaluation of these analogues as estrone sulfotase inhibitors, and the details are reported.

Research has been conducted on chemokine-heparin sulfate interactions. The binding features and the mechanism of these interactions have been investigated via the analysis of heparin binding to regulated on activation, normal T-cell expressed and secreted N-terminally truncated form of the chemokine group with two adjacent cysteine residues and the details are reported.

Research has been conducted on adenosine 5'-phosphosulfate kinase from the filamentous fungus Penicillium chrysogenum. The crystallization of this kinase has been carried out in the presence of ADP and adenosine 5'-phosphosulfate in investigating the structural basis of the substrate inhibition and the residues involved in substrate binding and the details are reported.

To date, little work has been conducted on the relationship between solute and buffer molecules and conformational exchange motion in enzymes. This study uses solution NMR to examine the effects of phosphate, sulfate, and acetate in comparison to MES- and HEPES-buffered references on the chemical shift perturbation and millisecond, chemical, or conformational exchange motions in the enzyme ribonuclease A (RNase A), triosephosphate isomerase (TIM) and HisF. The results indicate that addition of these solutes has a small effect on 1H and 15N chemical shifts for RNase A and TIM but a significant effect for HisF. For RNase A and TIM, Carr-Purcell-Meiboom-Gill relaxation dispersion experiments, however, show significant solute-dependent changes in conformational exchange motions. Some residues show loss of millisecond motions relative to the reference sample upon addition of solute, whereas others experience an enhancement. Comparison of exchange parameters obtained from fits of dispersion data indicates changes in either or both equilibrium populations and chemical shifts between conformations. Furthermore, the exchange kinetics are altered in many cases. The results demonstrate that common solute molecules can alter observed enzyme millisecond motions and play a more active role than what is routinely believed. [ABSTRACT FROM AUTHOR]

Human SULT2AI is one of two predominant sulfotransferases in liver and catalyzes transfer of the sulfuryl moiety (-SO3) from activated sulfate (PAPS, 3'-phosphoadenosine 5-phosphosulfate) to hundreds of acceptors (metabolites and xenobiotics). Sulfation recodes the biologic activity of acceptors by altering their receptor interactions. The molecular basis on which these enzymes select and sulfonate specific acceptors from complex mixtures of competitors in vivo is a long-standing issue in the SULT field. Raloxifene, a synthetic steroid used in the prevention of osteoporosis, and dehydroepiandrosterone (DHEA), a ubiquitous steroid precusor, are reported to be sulfated efficiently by SULT2AI in vitro, yet unlike DHEA, raloxifene is not sulfated in vivo. This selectivity was explored in initial rate and equilibrium binding studies that demonstrate pronounced binding antisynergy (21-fold) between PAPS and raloxifene, but not DHEA. Analysis of crystal structures suggests that PAP binding restricts access to the acceptor-binding pocket by restructuring a nine-residue segment of the pocket edge that constricts the active site opening, or "pore", that sieves substrates on the basis of their geometries. In silico docking predicts that raloxifene, which is considerably larger than DHEA, can bind only to the unliganded (open) enzyme, whereas DHEA binds both the open and dosed forms. The predictions of these structures with regard to substrate binding are tested using equilibrium and pre-steady-state ligand binding studies, and the results confirm that a nudeotide-driven isomerization controls access to the acceptor-binding pocket and plays an important role in substrate selection by SULT2AI and possibly other sulfotransferases. [ABSTRACT FROM AUTHOR]

Heparan sulfate is a highly sulfated polysaccharide that exhibits important physiological and pathological functions. The glucosamine residue of heparan sulfate can carry sulfo groups at the 2-N, 3-O, and 6-O positions, leading to diverse polysaccharide structures. 6-O-Sulfation at the glucosamine residue contributes to a wide range of biological functions. Here, we report a method for controlling the positioning of 6-O-sulfo groups in oligosaccharides. This was achieved by synthesizing oligosaccharide backbones from a disaccharide building block utilizing glycosyltransferases followed by modifications using heparan sulfate N-sulfotransferase and 6-O-sulfotransferases. This method offers a viable approach for preparing heparan sulfate oligosaccharides with precisely located 6-O-sulfo groups. [ABSTRACT FROM AUTHOR]

ABSTRACT: Methanobactins (mb) are low-molecular mass, copper-binding molecules secreted by mosi methanotrophic bacteria. These molecules have been identified for a number of methanotrophs, but only the one produced by Meihv/osinus irichosporiam OB3b (mb-OB3b) has to date been chemically characterized. Here we report the chemical characterization and copper binding properties of a second methanobactin. which is produced by Mei/irloctsüs strain SB2 (mb-SB2). mb-SB2 shows some significant similarities to mb-OB3b, including its spectral and metal binding properties, and its ability to bind and reduce Cu(lI) to Cu(I). Like mb-OB3b. mb-S82 contains two five-member heterocyclic rings with associated enethiol groups, which together form the copper ion binding site. mb-S~2 also displays some significant differences compared to mb-OB3b, including the number and types of amino acids used to complete the structure of the molecule, the presence of an imidazolone ring in place of one of the oxazolone rings found in mb-OB3b. and the presence of a sulfate group not found in mb-OB3b. The sulfate is bonded to a threonine-like side-chain that associated with one of the heterocyclic rings and may represent the first example of this type of sulfate group found in a bacterially derived peptide. Acid-catalyzed hydrolysis and decarboxylation of the oxazolone rings found in mb-OB3b and mb-SB2 produce pairs of amino acid residues and suggest that both mb-OB3b and mb-SB2 are derived from peptides. In support of this the gene for a ribosomally produced peptide precursor for mb-OB3b has been identified in the genome of M. tricho.sporium 083b. The gene sequence indicates that the oxazolone rings in mb-OB3b are derived from the combination of a cysteinc residue and the carbonyl from the preceding residue in the peptide sequence. Taken together, the results suggest methanobactins make up a structurally diverse group of ribosomally produced, peptide-derived molecules, which share a common pair of five- member rings with associated enethiol groups that are able to bind, reduce, and stabilize copper ions in an aqueous environment. [ABSTRACT FROM AUTHOR]

Sulfite oxidase (SO) catalyzes the physiologically critical conversion of sulfite to sulfate. Enzymatic activity is dependent on the presence of the metal molybdenum complexed with a pyranopterin- dithiolene cofactor termed molybdopterin. Comparison of the amino acid sequences of SOs from a variety of sources has identified a single conservedCys residue essential for catalytic activity. The crystal structure of chicken liver sulfite oxidase indicated that this residue, Cys 185 in chicken SO, coordinates the Mo atom in the active site. To improve our understanding of the role of this residue in the catalytic mechanism of sulfite oxidase, serine and alanine variants at position 185 of recombinant chicken SO were generated. Spectroscopic and kinetic studies indicate that neither variant is capable of sulfite oxidation. The crystal structure of the C185S variant was determined to 1.9 Å resolution and to 2.4 Å resolution in the presence of sulfite, and the CI85A variant to 2.8 Å resolution. The structures of the C185S and C185A variants revealed that neither the Ser or Ala side chains appeared to closely interact with the Mo atom and that a third oxo group replaced the usual cysteine sulfur ligand at the Mo center, confirming earlier extended X-ray absorption fine structure spectroscopy (EXAFS) work on the human C2075 mutant. An unexpected result was that in the C 185S variant, tn the absence of sulfite, the active site residue Tyr322 became disordered as did the 1oop region flanking it. In the C185S variant crystallized in the presence of sulfite, the Tyr322 residue relocalized to the active site. The Cl 85A variant structure also indicated the presence ofa third oxygen ligand; however, Tyr322 remained in the active site. EXAFS studies of the Mo coordination environment indicate the Mo atom is in the oxidized Mo"1 state in both the C185S and C185A variants of chicken SO and show the expected trioxo- dithiolene active site. Density functional theory calculations of the trioxo form of the cofactor reasonably reproducd the MoO distances of the complex; however, the calculated Mo-S distances were slightly longer than either crystallographic or EXAFS measurements. Taken together, these results indicate that the active Sites of the C1855 and Cl 85A variants are essentially catalytically inactive, the crystal structures ofCl 85S and C185A variants contain a fully oxidized, trioxo form of the cofactor, and Tyr322 can undergo a confor- mational change that is relevant to the reaction mechanism. Additional DFl' calculations demonstrated that such methods can reasonably reproduce the geometry and bond lengths of the active site. [ABSTRACT FROM AUTHOR]

Infection with Plasmodium falciparum during pregnancy results in the adherence of infected red blood cells (IRBCs) in placenta, causing pregnancy-associated malaria with severe health complications in mothers and fetuses. The chondroitin 4-sulfate (C4S) chains of very low sulfated chondroitin sulfate proteoglycans (CSPG5) in placenta mediate the IRBC adherence. While it is known that partially sulfated but not fully sulfated C4S effectively binds IRBCs, structural interactions involved remain unclear and are incompletely understood. In this study, structurally defined C4S oligosaccharides of varying sulfate contents and sizes were evaluated for their ability to inhibit the binding of IRBCs from different P. falciparum strains to CSPG purified from placenta. The results clearly show that, with all parasite strains studied, dodecasaccharide is the minimal chain length required for the efficient adherence of IRBCs to CSPG and two 4-sulfated disaccharides within this minimal structural motif are sufficient for maximal binding. Together, these data demonstrate for the first time that the C4S structural requirement for IRBC adherence is parasite strain-independent. We also show that the carboxyl group on nonreducing end glucuronic acid in dodecasaccharide motif is important for IRBC binding. Thus, in oligosaccharides containing terminal 4,5-unsaturated glucuronic acid, the nonreducing end disaccharide moiety does not interact with IRBCs due to the altered spatial orientation of carboxyl group. In such C4S oligosaccharides, 14-mer but not I 2-mer constitutes the minimal motif for inhibition of IRBC binding to placental CSPG. These data have important implications for the development and evaluation of therapeutics and vaccine for placental malaria. [ABSTRACT FROM AUTHOR]

Heparan sulfate (HS) is a sulfated glycosaminoglycan attached to a core protein on the cell surface. Protein binding to cell surface HS is a key regulatory event for many cellular processes such as blood coagulation, cell proliferation, and migration. The concept whereby protein binding to HS is not random but requires a limited number of sulfation patterns is becoming clear. Here we describe a hydrophobic trapping assay for screening a library of heparin hexasaccharides for binders to antithrombin Ill (ATIII). The hexasaccharide compositions are defined with their building block content in the following format: (ΔHexA:HexA:GIcN:SO3:Ac). Of five initial compositions present in the library, (1:2:3:6:1), (1:2:3:7:1), (1:2:3:7:0), (1:2:3:8:0), and (1:2:3:9:0), only two are shown to bind ATIII, namely, (1:2:3:8:0) and (1:2:3:9:0). The use of amide hydrophilic interaction (HILIC) liquid chromatography-mass spectrometry permitted reproducible quantitative analysis of the composition of the initial library as well as that of the binding fraction. The specificity of the hexasaccharides binding ATIII was confirmed by assaying their ability to enhance ATIII-mediated inhibition of Factor Xa in vitro. [ABSTRACT FROM AUTHOR]

Flavodoxins catalyze redox reactions using the isoalloxazine moiety of the ravin mononucleotide (FMN) cofactor stacked between two aromatic residues located in two peptide loops. At high FMN concentrations that favor stacked FMN dimers in solution, isothermal titration calorimetric studies show that these dimers bind strongly to apo-flavodoxin from Desulfovibrio desulfuricans (30 °C, 20 mM Hepes, pH 7, K[SUBD] = 5.8 μM). Upon increasing the temperature so the FMN dimers dissociate (as shown by [SUP1]H NMR), only one-to-one (FMN-to-protein) binding is observed. Calorimetric titrations result in one-toone binding also in the presence of phosphate or sulfate (30 °C, 13 mM anion, pH 7, K[SUBD] = 0.4 μM). FMN remains dimeric in the presence of phosphate and sulfate, suggesting that specific binding of a divalent anion to the phosphate-binding site triggers ordering of the peptide loops so only one isoalloxazine can fit. Although the physiological relevance of FMN and other nucleotides as dimers has not been explored, our study shows that high-affinity binding to proteins of such dimers can occur in vitro. This emphasizes that the cofactor-binding site in flavodoxin is more flexible than previously expected. [ABSTRACT FROM AUTHOR]

Reports on the expression of biological activities of squid cartilage chondroitin sulfate E (CS-E), including anticoagulant activities, lymphoid regulatory activities and neuroregulatory activities, through molecular interactions with specific proteins. Isolation of hexasaccharides to obtain more detailed sequence information; Occurrence of an unreported Glc 3-0-sulfotransferase specific for chondroitin sulfate; Usefulness of the obtained sequence information for investigating the structure-function relationship and biosynthesis of CS-E.

Studies the characterization of the allosteric anion-binding site of O-acetylserine sulfhydrylase-A with chloride and sulfate as inhibitors. Formation of external aldimines; External aldimine dissociation constants; Allosteric site binding; Active site binding; Behavior of sulfide in preventing the formation of external aldimine; pH dependence of chloride and sulfide.

Examines the interaction of heparan sulfate with different ligand proteins. Formation of heparin and heparan sulfate (HS); Influence of HS on a variety of biological processes; Steps in polysaccharide assembly.

Examines the role of electrostatic interactions in the mechanism of polypeptide folding caused by sodium dodecyl sulfate (SDS) binding. Design of two hydrophilic, charged amphipathic alpha-helical peptides; Analysis of the peptide structure by circular dichroism and nuclear magnetic resonance spectroscopy; Model for the peptide-SDS complex.

Investigates whether N-deacetylase/N-sulfotransferase (NDST) isoforms influenced differently the structure of heparan sulfate (HS). Comparison of the structure of the HS produced from NDST-1 overexpressed in human kidney cells to HS from NDST-2 overexpressing cells; Importance of the NDST isoform in determining the N-sulfation pattern in HS.

Presents structural evidence of the location of sulfate anion binding sites of the ovotransferrin N-lobe via the 1.9 angstrom resolution of the apo crystal structure. Presence of three nonsynergistic sulfate anion binding sites in the interdomain cleft that is wide open as compared to the holo cleft structure.

Describes the reactions of glutamate semialdehyde aminomutase with R- and S-enantiomers of a novel compound, diaminopropyl sulfate, designed as a mechanism-based inhibitor of the enzyme. Transamination of the S-enantiomer without inactivating the enzyme; Rapid inactivation of the enzyme by the R-enantiomer.

Analyzes the effect of glycosaminoglycans (GAG) on the activity of bone and cartilage-resident cathepsins K and L and MMP-1. Cathepsins as predominant cysteine protease in osteoclast-mediated bone remodeling; Finding that GAG, particularly chondroitin sulfates, specifically and selectively increased the stability of cathepsin K.

Studies the conformations of human apolipoprotein E(263-286) and E(267-289) in aqueous solutions of sodium dodecyl sulfate by CD and 1H nuclear magnetic resonance. Resonance assignments and secondary shifts; Fingerprint and amide regions; Plots of temperature coefficients of amide protons; Side view of average structures and side chain orientations.

Investigates the physicochemical consequences of epimerization of glucuronate to iduronate by examining choindroitin-4-sulfate and dermatan sulfate in D2O solution. Nuclear Overhauser effects and spin-lattice relaxation times following selective and nonselective inversion; Tetrasaccharide fragment of typical beta-1,4;beta-1,3 linked anionic glycosaminoglycan.

Describes the energetic linkage of the hydrolysis of GTP and the synthesis of activated sulfate and the events that couple them. Steady-state and single-turnover experiments; Stoichiometry of GTP hydrolysis.

Presents a kinetic examination of sulfate activation to characterize the interaction between sulfurylase and kinase in complex organisms. Biosynthesis of the activated sulfate donor PAPS; Velocity of the adenosine 5'-phosphosulfate (APS) kinase reaction; APS' channeling between the active sites of ATP sulfurylase and APS kinase.

Chondroitin sulfate proteoglycans (CS-PG) are involved in the regulation of the central nervous system in vertebrates due to their presence on cell surfaces and in the extracellular matrix of tissues. The CS moieties are built up from repeating -4)GlcA(beta1-3)GalNAc(beta1- disaccharide units, partly O-sulfated at different positions. The presence of the disulfated disaccharide D-unit, GlcA2S(beta1-3)GalNAc6S, in the CS moiety of the proteoglycan DSD-1-PG/phosphacan, correlates with neurite outgrowth promotion. The binding of monoclonal antibody (mAb) 473HD to DSD-1-PG, reducing neuronal stimulation, is inhibited by shark cartilage CS-D. CS-D is also recognized by two other mAbs, MO-225 and CS-56. Conformational studies were performed using NMR spectroscopy and molecular modeling on five octasaccharides isolated from shark cartilage CS-D. These octasaccharides present different binding properties toward the three mAbs. The combination of the experimental and theoretical approaches revealed that the sulfate group at position 2 of GlcA in disaccharide D and the presence of an exocyclic negative tail in disaccharides C [GlcA(beta1-3)GalNAc6S] and DeltaC [Delta4,5HexA(alpha1-3)GalNAc6S] are important for antibody recognition.

High-density lipoproteins (HDL) remove cholesterol from peripheral tissues and thereby help to prevent atherosclerosis. Nascent HDL are discoidal complexes composed of a phospholipid bilayer surrounded by protein alpha-helices that are thought to form extensive stabilizing interhelical salt bridges. Earlier we showed that HDL stability, which is necessary for HDL functions, is modulated by kinetic barriers. Here we test the role of electrostatic interactions in the kinetic stability by analyzing the effects of salt, pH, and point mutations on model discoidal HDL reconstituted from human apolipoprotein C-1 (apoC-1) and dimyristoyl phosphatidylcholine (DMPC). Circular dichroism, Trp fluorescence, and light scattering data show that molar concentrations of NaCl or Na(2)SO(4) increase the apparent melting temperature of apoC-1:DMPC complexes by up to 20 degrees C and decelerate protein unfolding. Arrhenius analysis shows that 1 M NaCl stabilizes the disks by deltaDeltaG* approximately equal 3.5 kcal/mol at 37 degrees C and increases the activation energy of their denaturation and fusion by deltaE(a) approximately equal deltaDeltaH* approximately equal 13 kcal/mol, indicating that the salt-induced stabilization is enthalpy-driven. Denaturation studies in various solvent conditions (pH 5.7-8.2, 0-40% sucrose, 0-2 M trimethylamine N-oxide) suggest that the salt-induced disk stabilization results from ionic screening of unfavorable short-range Coulombic interactions. Thus, the dominant electrostatic interactions in apoC-1:DMPC disks are destabilizing. Comparison of the salt effects on the protein:lipid complexes of various composition reveals an inverse correlation between the lipoprotein stability and the salt-induced stabilization and suggests that short-range electrostatic interactions significantly contribute to lipoprotein stability: the better-optimized these interactions are, the more stable the complex is.

The pK(a) values of most histidines in small peptides and in myoglobin increase on average by 0.30 unit between 0.02 and 1.5 M NaCl [Kao et al. (2000) Biophys. J. 79, 1637]. The DeltapK(a) values reflect primarily the ionic strength dependence of the solvation energy; screening of Coulombic interactions contributes only in a minor way. This implies that Coulombic interactions are weak, or that attractive and repulsive contributions to the pK(a) values are balanced. To distinguish experimentally between these two possibilities, and to further characterize the magnitude and salt sensitivity of surface electrostatic interactions in proteins, the salt dependence of pK(a) values of histidines in staphylococcal nuclease was measured by (1)H NMR spectroscopy. Three of the four histidines titrated with significantly depressed pK(a) values, and the salt sensitivity of all histidine pK(a) values was substantial. In three cases, the pK(a) values increased by a full unit between 0.01 and 1.5 M KCl. Anion-specific effects were found; the pK(a) values measured under equivalent ionic strengths in SCN(-) and SO(4)(2-) were higher than in Cl(-); the order of the sensitivity of pK(a) values to anions was SCN(-) > Cl(-) > SO(4)(2-). Structure-based pK(a) calculations with continuum methods were performed to interpret the measured effects structurally and to test their ability to capture the experimental behavior. Calculations in which the protein interior was treated empirically with a dielectric constant of 20 reproduced the pK(a) values and their dependence on the concentration of Cl(-). According to the calculations, the pK(a) values are depressed because of unfavorable self-energies and repulsive Coulombic interactions. Their striking salt sensitivity reflects screening of weak, repulsive, Coulombic interactions among charges separated by more than 10 A. Long-range Coulombic interactions on the surfaces of proteins are weak, but they can add up to produce substantial electrostatic effects when positive and negative charges are not balanced.

Structural analysis of the initial steps in protein folding is difficult because of the swiftness with which these steps occur. Hence, the link between initial polypeptide chain collapse and formation of secondary and other specific structures remains poorly understood. Here, an equilibrium model has been developed for characterizing the initial steps of folding of the small protein barstar, which lead to the formation of a productive molten globule in the folding pathway. In this model, the high-pH-unfolded form (D form) of barstar, which is shown to be as unstructured as the urea-denatured form, is transformed progressively into a molten globule B form by incremental addition of the salt Na(2)SO(4) at pH 12. At very low concentrations of Na(2)SO(4), the D form collapses into a pre-molten globule (P) form, whose volume exceeds that of the native (N) state by only 20%, and which lacks any specific structure as determined by far- and near-UV circular dichroism. At higher concentrations of Na(2)SO(4), the P form transforms into the molten globule (B) form in a highly noncooperative transition populated by an ensemble of at least two intermediates. The B form is a dry molten globule in which water is excluded from the core, and in which secondary structure develops to 65% and tertiary contacts develop to 40%, relative to that of the native protein. Kinetic refolding experiments carried out at pH 7 and at high Na(2)SO(4) concentrations, in which the rate of folding of the D form to the N state is compared to that of the B form to the N state, indicate conclusively that the B form is a productive intermediate that forms on the direct pathway of folding from the D form to the N state.

The protein phosphatase encoded by bacteriophage lambda (lambda PP) belongs to a family of Ser/Thr phosphatases (Ser/Thr PPases) that includes the eukaryotic protein phosphatases 1 (PP1), 2A (PP2A), and 2B (calcineurin). These Ser/Thr PPases and the related purple acid phosphatases (PAPs) contain a conserved phosphoesterase sequence motif that binds a dinuclear metal center. The mechanisms of phosphoester hydrolysis by these enzymes are beginning to be unraveled. To utilize lambda PP more effectively as a model for probing the catalytic mechanism of the Ser/Thr PPases, we have determined its crystal structure to 2.15 A resolution. The overall fold resembles that of PP1 and calcineurin, including a conserved beta alpha beta alpha beta structure that comprises the phosphoesterase motif. Substrates and inhibitors probably bind in a narrow surface groove that houses the active site dinuclear Mn(II) center. The arrangement of metal ligands is similar to that in PP1, calcineurin, and PAP, and a bound sulfate ion is present in two novel coordination modes. In two of the three molecules in the crystallographic asymmetric unit, sulfate is coordinated to Mn2 in a monodentate, terminal fashion, and the two Mn(II) ions are bridged by a solvent molecule. Two additional solvent molecules are coordinated to Mn1. In the third molecule, the sulfate ion is triply coordinated to the metal center with one oxygen coordinated to both Mn(II) ions, one oxygen coordinated to Mn1, and one oxygen coordinated to Mn2. The sulfate in this coordination mode displaces the bridging ligand and one of the terminal solvent ligands. In both sulfate coordination modes, the sulfate ion is stabilized by hydrogen bonding interactions with conserved arginine residues, Arg 53 and Arg 162. The two different active site structures provide models for intermediates in phosphoester hydrolysis and suggest specific mechanistic roles for conserved residues.

Novel aryl derivatives of benzamidine were synthesized and tested for their inhibitory potency against bovine trypsin, rat skin tryptase, human recombinant granzyme A, human thrombin, and human plasma kallikrein. All compounds show competitive inhibition against these proteases with Ki values in the micromolar range. X-ray structures were determined to 1.8 A resolution for trypsin complexed with two of the para-substituted benzamidine derivatives, 1-(4-amidinophenyl)-3-(4-chlorophenyl)urea (ACPU) and 1-(4-amidinophenyl)-3-(4-phenoxyphenyl)urea (APPU). Although the inhibitors do not engage in direct and specific interactions outside the S1 pocket, they do form intimate indirect contacts with the active site of trypsin. The inhibitors are linked to the enzyme by a sulfate ion that forms an intricate network of three-centered hydrogen bonds. Comparison of these structures with other serine protease structures with noncovalently bound oxyanions reveals a pair of highly conserved oxyanion-binding sites in the active site. The positions of noncovalently bound oxyanions, such as the oxygen atoms of sulfate, are distinct from the positions of covalent oxyanions of tetrahedral intermediates. Noncovalent oxyanion positions are outside the "oxyanion hole." Kinetics data suggest that protonation stabilizes the ternary inhibitor/oxyanion/protease complex. In sum, both cations and anions can mediate Ki. Cation mediation of potency of competitive inhibitors of serine proteases was previously reported by Stroud and co-workers [Katz, B. A., Clark, J. M., Finer-Moore, J. S., Jenkins, T. E., Johnson, C. R., Ross, M. J., Luong, C., Moore, W. R., and Stroud, R. M. (1998) Nature 391, 608-612].

Measurements of 15N NMR relaxation parameters have been used to characterize the backbone dynamics of folded and denatured states of the N-terminal SH3 domain from the adapter protein drk, in high salt or guanidinium chloride, respectively. Values of the spectral density function evaluated at a number of frequencies are compared. The levels of backbone dynamics in the folded protein show little variation across the molecule and are of similar magnitude to those determined previously for the folded state of the protein in exchange with an unfolded state at low salt concentrations [Farrow et al. (1995) Biochemistry 34, 868-878]. The denatured state of the domain exhibits both more extensive and more heterogeneous dynamics than the folded state. In particular the profile of the spectral density function evaluated at zero-frequency for the unfolded state of the domain indicates that residues in the middle of the protein sequence are considerably less mobile than those at the termini. These data suggest that the molecule is not behaving as an extended polymer and that concerted motions of the central portions of the molecule are occurring, consistent with a reasonably compact conformation in this region. The backbone dynamics of the folded and unfolded states were studied at two temperatures. The level of high-frequency motions in the folded molecule is largely unaffected by changes in temperature, whereas an increase in temperature results in increased high-frequency motion in the unfolded state.

The covalent attachment of a managanese-tris(methylpyridiniumyl)porphyrin entity to an antisense oligonucleotide allowed sequence-selective oxidative cleavage of DNA when the metalloporphyrin was activated by potassium monopersulfate (KHSO5). We prepared several structurally modified metallo-porphyrin-oligonucleotide conjugates in order to find out the most efficient compound for in vitro DNA cleavage. The nature and the length of the tether were modulated, the metalloporphyrin entity was modified (metal, ligand), and different ways of activation of the metalloporphyrin were assayed. We noticed that the location of the peptidic bond within the linker could greatly affect the cleavage efficiency of the different conjugates. We showed that the most efficient conjugate for oxidative DNA cleavage was a manganese tetracationic porphyrin-oligonucleotide compound. When the metalloporphyrin moiety was activated by a reducing agent in the presence of molecular oxygen, DNA cleavage was efficient at suitable concentrations of the reducing agent, in order to avoid the reduction of the activated DNA cleaver, a putative high-valent metal-oxo species, by the excess of reducing agent.

Chemically modified heparins were tested for their activities in (i) inhibiting HIV-1 replication in vitro and (ii) inhibiting the binding to recombinant HIV-1 gp120 of monoclonal antibodies specific for the V3 loop. The results reveal that N-desulfation reduces activity, although this is largely restored on N-acetylation. Selective O-desulfation also markedly reduces activity, whereas carboxyl reduction has little effect. Overall these results show that the anti-HIV-1 activity of heparin does not depend simply on negative density, and indicate instead that particular structures, notably O-sulfates, are involved. Our studies reveal that for chemically modified heparins and heparin-derived fragments there is a striking correlation between anti-HIV-1 activity in vitro and binding to the V3 loop of gp120 in solid phase ELISA. This strongly suggests that the heparin exerts its anti-HIV-1 activity by binding to the V3 loop of gp120.

Carbonic anhydrase III, found predominantly in skeletal muscle, is the least efficient of the mammalian carbonic anhydrases in catalyzing the hydration of CO2. Phenylalanine-198 is located on the hydrophobic side of the active-site cavity with its phenyl ring in the proximity of the catalytically active zinc-bound water. We replaced phenylalanine-198 in human carbonic anhydrase III with seven other amino acids (Ala, Asn, Asp, His, Leu, Tyr, Val) using site-directed mutagenesis. The catalytic properties of these enzymes were determined by stopped-flow spectrophotometry, and the exchange of 18O between CO2 and water was measured by mass spectrometry. All of the mutants had maximal values of kcat/Km for the hydration of CO2 enhanced, and five of the mutants had the pKa of the zinc-bound water increased compared with the wild-type enzyme. The largest effects were observed with the replacement Phe-198-->Asp which increased the maximal kcat/Km 140-fold and increased the pKa of the zinc-bound water from near 5 to 9.2. A Brønsted correlation was observed between log(kcat/Km) for hydration of CO2 and the pKa of the zinc-bound water (correlation coefficient r = 0.92); in addition, this pKa was inversely correlated with hydrophobicity of the residue at 198 (correlation coefficient r = -0.83). A direct correlation between the logarithm of the maximal kcat/Km for hydration and the logarithm of the pH-independent value of Ki for inhibition by cyanate (r = 0.95) indicated that the effect of the mutations at residue 198 occurred in large part by enhancement of the rate of dissociation of the enzyme-bicarbonate complex.

Leucine zipper proteins comprise a recently identified class of DNA binding proteins that contain a bipartite structural motif consisting of a "leucine zipper" dimerization domain and a segment rich in basic residues responsible for DNA interaction. Protein fragments encompassing the zipper plus basic region domains (bZip) have previously been used to determine the conformational and dynamic properties of this motif. In the absence of DNA, the coiled-coil portion is alpha-helical and dimeric, whereas the basic region is flexible and partially disordered. Addition of DNA containing a specific recognition sequence induces a fully helical conformation in the basic regions of these fragments. However, the question remained whether the same conformational change would be observed in native bZip proteins where the basic regions might be stabilized in an alpha-helical conformation even in the absence of DNA, through interactions with portions of the protein not included in the bZip motif. We have now examined the DNA-induced conformational transition for an intact bZip protein, GCN4, and for the bZip fragment of C/EBP with two enhancers that are differentially symmetric. Our results are consistent with the induced helical fork model wherein the basic regions are largely flexible in the absence of DNA and become fully helical in the presence of the specific DNA recognition sequence.

Mixtures of histones H3 and H4 were examined by analytical ultracentrifugation and circular dichroism to determine their association behavior and secondary structure content in high and low ionic strength solvents containing chloride, phosphate, or sulfate. H3 and H4 were also cross-linked by using DSP in order to directly trap any intermolecular interactions occurring in solution. While H3 and H4 can exist as an H3-H4 dimer under limited conditions, they behave as a stable (H3-H4)2 tetramer under most conditions, particularly those which are physiologically relevant. In chloride-containing solutions, the equilibrium between H3-H4 and (H3-H4)2 is responsive to changes in ionic strength and paralleled by large changes in alpha-helicity. In sulfate- and phosphate-containing solutions, the equilibrium is again governed by ionic strength, but there are no significant changes in secondary structure accompanying shifts in the equilibrium. Small oligomers can be formed in the presence of sulfate and phosphate and trapped by the cross-linking reagent; these oligomers are much smaller than those formed in chloride-containing solutions. However, addition of the H2A-H2B dimer into the system prevents aggregation of the (H3-H4)2 tetramer by acting as a "molecular cap" and thus regulating the assembly pathway toward the formation of tripartite octamers. The observed assembly of H3 and H4 into a stable, tetrameric complex supports the concept of the core histone octamer having a tripartite organization in solution rather than being organized as two heterotypic tetramers.

A model procedure for removing salt from relatively fragile salt-induced protein crystals is proposed. The procedure is based on physical principles and is validated by using millimeter-size crystals of rabbit muscle phosphoglucomutase grown from a 2.1 M solution of ammonium sulfate. Three types of operations are included in the procedure: initial transfer to salt solutions of reduced concentration; transfer to the organic-rich phase of an equilibrium biphasic mixture obtained with aqueous solutions of polyoxyethylene and the salt; and addition of various replacement cosolutes in aqueous solutions of polyoxyethylene to reduce osmotic stress on the crystal as the remaining salt is removed. A critical feature of the overall procedure is maintenance of near equilibrium throughout by using a large number of steps involving small changes in solute concentration. The conditions used in the actual transfer were adjusted to eliminate the fracturing of crystals by visually distinguishing between two opposing types of fracture patterns: those produced by osmotic crushing as opposed to osmotic expansion. Basic requirements for a successful procedure with other protein crystals are a high permeability toward small solutes and a relatively slow dissolution rate at salt concentrations for which biphasic mixtures can be obtained. Desalted crystals of phosphoglucomutase have no visible fractures, are stable in the final solution for at least a week, and exhibit no noticeable change in the resolution of their X-ray diffraction pattern. In fact, desalted crystals can be rapidly cooled to 160 K, whereas untreated crystals are almost completely disordered by the same cooling procedure. The component of the desalting mixture whose presence is crucial to the success of the cooling process is polyoxyethylene, which apparently impedes the formation of ice within the protein crystal. Diffraction data obtained with an area-detector diffractometer did not differ significantly, either in terms of quality or resolution range, between crystals in 2.3 M ammonium sulfate at room temperature and crystals at 160 K in which ammonium sulfate had been replaced by glycine. The successful use of the following replacement solutes, instead of glycine, also is documented: sucrose, glycerol, and a low molecular weight poly(ethylene glycol) (PEG-400).

Raman spectra of ribonuclease A in D2O solution at various pD values have been studied with special attention to the N-deuterated imidazolium ring vibration at 1408 cm-1, the SO4(2-) symmetric stretching vibration at 984 cm-1, the amide I' band, and the tyrosine doublet. Concomitant decrease and increase in the intensities of the 1408- and 984-cm-1 bands in the pD range between 5 and 7 indicate that a sulfate anion is actually hydrogen bonded to an imidazolium ring of a histidine residue located in the interior of the molecule. The mechanism of the sulfate desorption has been compared with that on heat denaturation.

Glycogen synthase from swine adipose tissue was purified to apparent homogeneity using ethanol precipitation, DEAE chromatography, and affinity chromatography utilizing glucosamine 6-phosphate as the ligand. The purified enzyme migrated as a single protein component during electrophoresis on polyacrylamide gels at pH 7.3 although some protein failed to enter the running gel. Enzyme incubated with sodium dodecyl sulfate (SDS) migrated as one component (mol wt similar to 90,000) on SDS-polyacrylamide gel electrophoresis. The enzyme was relatively unstable at all stages of the purification procedure, but stability was increased in the presence of glucose 6-phosphate, UDPG, or glycerol. The isoelectric point of the purified enzyme and of enzyme activity in crude homogenates was pH 4.8. The sedimentation coefficient of the enzyme in crude homogenates was 8.5 S. The pH-activity profile showed an optimum at pH 7.8 in the absence of glucose 6-phosphate but no definable optimum between pH 7.0 and 9.2 in its presence. The Km of glycogen synthase I for UDPG was 250 muM in the absence and 37 muM in the presence of glucose 6-phosphate; the K-a for glucose 6-phosphate was 18 mu-M. The K-m of glycogen synthase D for UDPG was 130 mu-M in the presence of glucose 6-phosphate; the Ka for glucose 6-phosphate was 1 mM. The anions sulfate and phosphate activated the enzyme when assays were performed in the absence of glucose 6-phosphate. Fluoride produced activation of enzyme assayed either in the presence or in the absence of glucose 6-phosphate.

Reported studies indicate that the association of potassium monopersulfate with [Mn(TMPyP)](OAc)5, a water-soluble manganese porphyrin complex, leads to an efficient reagent for the oxidative cleavage of DNA. Single-strand breaks (SSBs) are observed on double-stranded DNA at manganese porphyrin concentrations as low as 0.5 nM with a short incubation time of 1 min. The number of SSBs linearly varies with the concentration of the manganese complex, and potassium monopersulfate is at least 3 orders of magnitude more efficient as oxygen source than hydrogen peroxide. Cleavage efficiency is optimal in the pH range 7.5-9.0 for a NaCl concentration between 80 and 150 mM or for a MgCl2 concentration of 10 mM. At very low manganese porphyrin concentration and by increasing the incubation time a catalytic cleavage activity of the complex is evidenced: up to 5 SSBs per manganese porphyrin are observed. The high cleavage activity of the monopersulfate-manganese porphyrin system makes it a good candidate for DNA-footprinting experiments.

Circular dichroism, absorbance, hypochromicity, and the formation of Mg2+ and Zn2+ complexes have been measured for a series of bisnucleoside oligophosphates that contain adenosine, guanosine, and mixed guanosine/adenosine, guanosine/cytidine, and guanosine/uridine, as well as 7-methylguanosine and ribose-methylated purine nucleosides. All of the metal complex ions have stacking interactions at 2 degrees C, 10 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 8.0. There is a measurable degree of base stacking for all unsubstituted purine nucleotides that differs, however, from that of bases in nucleic acids. The degree of base stacking varies with the length of oligophosphate chains and the state of methylation. The effect of 7-methylation of guanosine is interpreted as causing a switch of nucleic acid base stacking from an atypical to a typical mode, which could be important for cap function in mRNA. The Mg2+ and Zn2+ complexes give rise to characteristic circular dichroism. In all instances excepting 7-methylated bisguanosine oligophosphates, the active secondary structures are disrupted, and in this regard, Zn2+ is more effective than Mg2+. At least two sets of binding sites are involved. A single metal ion is bound tightly. Stability, in terms of equilibrium constants, increases by more than 1000-fold as a function of chain length varying from two to six phosphates. The consequences of methylation are only minor. Electrostatic attraction between metal ions and phosphates is the most likely mechanism of these phenomena as judged by the effect of high ionic strength.

Preferential interactions of bovine serum albumin were measured with guanidine sulfate, guanidine acetate, and guanidine hydrochloride. The results showed an increasing preferential hydration with increasing salt concentration for the sulfate, positive preferential salt binding for the hydrochloride, and an intermediate situation for the acetate. These results correlate well with the known effects of the three salts on protein stability, namely, the stabilizing effect of guanidine sulfate and the denaturing effect of guanidine hydrochloride. Comparison of guanidinium and magnesium salts indicated that the substitution of guanidinium ion for Mg2+ decreases the preferential hydration and increases the preferential salt binding, suggesting that the perturbation by guanidinium ion binding of the surface free energy is greater than that by Mg2+ ion. It was concluded that guanidine salts are not a special class, but their activity toward proteins is modulated by the same fine balance between hydration and salt binding to protein as in the case of other salts, with the second factor being stronger in guanidine salts.

The preferential interactions of proteins with solvent components were studied in concentrated aqueous solutions of the sulfate, acetate, and chloride salts of Mg2+, Ba2+, Ca2+, Mn2+ and Ni2+ [except for CaSO4, BaSO4, Mn-(OAc)2, and Ni(OAc)2], and results were compared with those of the Na+ salts. It was found that, for all the salts, the preferential hydration increased in the order of Cl- less than CH3-COO- less than SO42- regardless of the cationic species used, in agreement with the anionic lyotropic series, and that the same parameter exhibited a tendency to increase in the order of Mn2+, Ni2+ less than Ca2+, Ba2+ less than Mg2+ less than Na+. The salting-out and stabilizing or salting-in and destabilizing effectiveness of the salts were interpreted in terms of the observed preferential interactions. The surface tension increment of salts, which is a major factor responsible for the preferential interactions of the Na+ salts, had no correlation with those of the divalent cation salts. It was shown that the binding of divalent cations to the proteins overcomes the salt exclusion due to the surface tension increase, leading to a decrease in the preferential hydration. In conformity with this mechanism, the preferential interaction of MgCl2 was strongly pH dependent, because of the protein charge-dependent affinity of Mg2+ for proteins, while NaCl showed no pH dependence of the preferential interaction. The proposed mechanism was supported by a strong correlation between the preferential interaction results and the interaction of these salts with the model peptide compound acetyltetraglycine ethyl ester, described by Robinson and Jencks.