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Salt Bridge (salt + bridge)
Selected AbstractsAmidinium Carboxylate Salt Bridges as a Recognition Motif for Mechanically Interlocked Molecules: Synthesis of an Optically Active [2]Catenane and Control of Its Structure,ANGEWANDTE CHEMIE, Issue 32 2010Yuji Nakatani Salziges Catenan: Für die Synthese eines optisch aktiven [2]Catenans wurde eine Amidinium-Carboxylat-Salzbrücke genutzt (siehe Bild). Die relative Bewegung der beiden makrocyclischen Komponenten wurde vollständig durch ein Säure/Base- oder Zn2+/Cryptand-Paar gesteuert. [source] Supramolecular Porphyrin Assemblies through Amidinium,Carboxylate Salt Bridges and Fast Intra-Ensemble Excited Energy TransferCHEMISTRY - A EUROPEAN JOURNAL, Issue 14 2004Joe Otsuki Prof. Abstract Well-defined supramolecular assemblies of Zn and free-base porphyrins are constructed through the formation of amidinium,carboxylate salt bridges. A one-to-one donor,acceptor pair and a four-to-one antenna-type assembly are investigated. The steady-state and time-resolved fluorescence measurements unequivocally showed that efficient singlet,singlet excited energy transfer from the Zn,porphyrin complex to the free-base porphyrin takes place in these assemblies. Indeed, the observed energy-transfer rates in both types of assemblies are much faster than those the Förster mechanism would suggest, implying the involvement of an intermolecular through-bond mechanism. [source] Low-voltage electroosmosis pump for stand-alone microfluidics devicesELECTROPHORESIS, Issue 1-2 2003Yuzuru Takamura Abstract Two types of low-voltage electroosmosis pumps were developed using microfabrication technology for usage in handy or stand-alone applications of the micrototal analysis systems (,-TAS) and the lab-on-a-chip. This was done by making a thin (<,1 ,m) region in the flow path and by only applying voltages near this thin region using electrodes inserted into the flow path. The inserted electrodes must be free from bubble formation and be gas-tight in order to avoid pressure leakage. For these electrodes, Ag/AgCl or a gel salt bridge was used. For patterning the gel on the chip, a hydrophilic photopolymerization gel and a photolithographic technique were optimized for producing a gel with higher electric conductivity and higher mechanical strength. For high flow rate application, wide (33.2 mm) and thin (400 nm) pumping channels were compacted into a 1 mm×6 mm area by folding. This pump achieves an 800 Pa static pressure and a flow of 415 nL/min at 10 V. For high-pressure application, a pump was designed with the thin and thick regions in series and positive and negative electrodes were inserted between them alternatively. This pump could increase the pumping pressure without increasing the supply voltage. A pump with 10-stage connections generated a pressure of 25 kPa at 10 V. [source] Roles of adenine anchoring and ion pairing at the coenzyme B12 -binding site in diol dehydratase catalysisFEBS JOURNAL, Issue 24 2008Ken-ichi Ogura The X-ray structure of the diol dehydratase,adeninylpentylcobalamin complex revealed that the adenine moiety of adenosylcobalamin is anchored in the adenine-binding pocket of the enzyme by hydrogen bonding of N3 with the side chain OH group of Ser,224, and of 6-NH2, N1 and N7 with main chain amide groups of other residues. A salt bridge is formed between the ,-NH2 group of Lys,135 and the phosphate group of cobalamin. To assess the importance of adenine anchoring and ion pairing, Ser,224 and Lys,135 mutants of diol dehydratase were prepared, and their catalytic properties investigated. The S,224A, S,224N and K,135E mutants were 19,2% as active as the wild-type enzyme, whereas the K,135A, K,135Q and K,135R mutants retained 58,76% of the wild-type activity. The presence of a positive charge at the ,135 residue increased the affinity for cobalamins but was not essential for catalysis, and the introduction of a negative charge there prevented the enzyme,cobalamin interaction. The S,224A and S,224N mutants showed a kcat/kinact value that was less than 2% that of the wild-type, whereas for Lys,135 mutants this value was in the range 25,75%, except for the K,135E mutant (7%). Unlike the wild-type holoenzyme, the S,224N and S,224A holoenzymes showed very low susceptibility to oxygen in the absence of substrate. These findings suggest that Ser,224 is important for cobalt,carbon bond activation and for preventing the enzyme from being inactivated. Upon inactivation of the S,224A holoenzyme during catalysis, cob(II)alamin accumulated, and a trace of doublet signal due to an organic radical disappeared in EPR. 5,-Deoxyadenosine was formed from the adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus considered to be a mechanism-based one. [source] Connection of transport and sensing by UhpC, the sensor for external glucose-6-phosphate in Escherichia coliFEBS JOURNAL, Issue 7 2003Christian Schwöppe UhpC is a membrane-bound sensor protein in Escherichia coli required for recognizing external glucose-6-phosphate (Glc6P) and induction of the transport protein UhpT. Recently, it was shown that UhpC is also able to transport Glc6P. In this study we investigated whether these transport and sensing activities are obligatorily coupled in UhpC. We expressed a His-UhpC protein in a UhpC-deficient E. coli strain and verified that this construct does not alter the basic biochemical properties of the Glc6P sensor system. The effects of arginine replacements, mutations of the central loop, and introduction of a salt bridge in UhpC on transport and sensing were compared. The exchanges R46C, R266C and R149C moderately affected transport by UhpC but strongly decreased the sensing ability. This suggested that the affinity for Glc6P as a transported substrate is uncoupled in UhpC from its affinity for Glc6P as an inducer. Four of the 11 arginine mutants showed a constitutive phenotype but had near wild-type transport activity suggesting that Glc6P can be transported by a molecule locked in the inducing conformation. Introduction of an intrahelical salt bridge increased the transport activity of UhpC but abolished sensing. Three conserved residues from the central loop were mutated and although none of these showed transport, one exhibited increased affinity for sensing. Taken together, these data show that transport by UhpC is not required for sensing, that conserved arginine residues are important for sensing and not for transport, and that residues located in the central hydrophilic loop are critical for transport and for sensing. [source] The role of exon 5 in fibroblast collagenase (MMP-1) substrate specificity and inhibitor selectivityFEBS JOURNAL, Issue 6 2001Vera Knäuper Interstitial collagen is degraded by members of the matrix metalloproteinase (MMP) family, including MMP-1. Previous work has shown that the region of MMP-1 coded for by exon 5 is implicated both in substrate specificity and inhibitor selectivity. We have constructed a chimeric enzyme, the exon 5 chimera, consisting primarily of MMP-1, with the region coded for by exon 5 replaced with the equivalent region of MMP-3, a noncollagenolytic MMP. Unlike MMP-3, the exon 5 chimera is capable of cleaving type I collagen, but the activity is only 2.2% of trypsin-activated MMP-1. ,Superactivation' of the chimera has no discernible effect, suggesting that the salt bridge formed in ,superactive' MMP-1 is not present. The kinetics for exon 5 chimera cleavage of two synthetic substrates display an MMP-3 phenotype, however, cleavage of gelatin is slightly impaired as compared to the parent enzymes. The Kiapp values for the exon 5 chimera complexed with synthetic inhibitors and N-terminal TIMP-2 also show a more MMP-3-like behaviour. However, the kon values for N-terminal TIMP-1 and N-terminal TIMP-2 are more comparable to those for MMP-1. These data show that the region of MMP-1 coded for by exon 5 is involved in both substrate specificity and inhibitor selectivity and the structural basis for our findings is discussed. [source] Can the calculation of ligand binding free energies be improved with continuum solvent electrostatics and an ideal-gas entropy correction?JOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 12 2002Sonja M. Schwarzl Abstract The prediction of a ligand binding constant requires generating three-dimensional structures of the complex concerned and reliably scoring these structures. Here, the scoring problem is investigated by examining benzamidine-like inhibitors of trypsin, a system for which errors in the structures are small. Precise and consistent binding free energies for the inhibitors are determined experimentally for this test system. To examine possible improvement of scoring methods, we test the suitability of continuum electrostatics to account for solvation effects and use an ideal-gas entropy correction to account for the changes in the degrees of freedom of the ligand. The small observed root-mean-square deviation of 0.55 kcal/mol of the calculated relative to the experimental values indicates that the essentials of the binding process have been captured. Even though all six ligands make the same salt bridge and H-bonds to the protein, the electrostatic contribution varies among the ligands by as much as 2 kcal/mol. Moreover, although the ligands are rigid and similar in size, the entropic terms also significantly affect the relative binding affinities (by up to 2.7 kcal/mol). The present approach to solvation and entropy may allow the ranking of the ligands to be considerably improved at a cost that makes the method applicable to the optimization of lead compounds or to the screening of small collections of ligands. © 2002 Wiley Periodicals, Inc. J Comput Chem 23: 1143,1149, 2002 [source] Investigations into the development of catalytic activity in anti-acetylcholinesterase idiotypic and anti-idiotypic antibodiesJOURNAL OF MOLECULAR RECOGNITION, Issue 3 2009Glynis Johnson Abstract We have previously described anti-acetylcholinesterase antibodies that display acetylcholinesterase-like catalytic activity. No evidence of contaminating enzymes was found, and the antibodies are kinetically and apparently structurally distinct from both acetylcholinesterase (AChE) and butyrylcholinesterase. We have also mimicked the antibody catalytic sites in anti-anti-idiotypic (Ab3) antibodies. Independently from us, similar acetylcholinesterase-like antibodies have been raised as anti-idiotypic (Ab2) antibodies against a non-catalytic anti-acetylcholinesterase antibody, AE-2. In this paper, we describe an epitope analysis, using synthetic peptides in ELISA and competition ELISA, and a peptide array, of five catalytic anti-acetylcholinesterase antibodies (Ab1s), three catalytic Ab3s, as well as antibody AE-2 and a non-catalytic Ab2. The catalytic Ab1s and Ab3s recognized three Pro- and Gly-containing sequences (40PPMGPRRFL, 78PGFEGTE, and 258PPGGTGGNDTELVAC) on the AChE surface. As these sequences do not adjoin in the AChE structure, recognition would appear to be due to cross-reaction. This was confirmed by the observation that the sequences superimpose structurally. The non-catalytic antibodies, AE-2 and the Ab2, recognized AChE's peripheral anionic site (PAS), in particular, the sequence 70YQYVD, which contains two of the site's residues. The crystal structure of the AChE tetramer (Bourne et al., 1999) shows direct interaction and high complementarity between the 257CPPGGTGGNDTELVAC sequence and the PAS. Antibodies recognizing the sequence and the PAS may, in turn, be complementary; this may account for the apparent paradox of catalytic development in both Ab1s and Ab2s. The PAS binds, but does not hydrolyze, substrate. The catalytic Ab1s, therefore, recognize a site that may function as a substrate analog, and this, together with the presence of an Arg-Glu salt bridge in the epitope, suggests mechanisms whereby catalytic activity may have developed. In conclusion, the development of AChE-like catalytic activity in anti-AChE Ab1s and Ab2s appears to be the result of a combination of structural complementarity to a substrate-binding site, charge complementarity to a salt bridge, and specific structural peculiarities of the AChE molecule. Copyright © 2008 John Wiley & Sons, Ltd. [source] Dynamics Change of Phoborhodopsin and Transducer by Activation: Study Using D75N Mutant of the Receptor by Site-directed Solid-state 13C NMR,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Izuru Kawamura Pharaonis phoborhodopsin (ppR or sensory rhodopsin II) is a negative phototaxis receptor of Natronomonas pharaonis, and forms a complex, which transmits the photosignal into cytoplasm, with its cognate transducer (pHtrII). We examined a possible local dynamics change of ppR and its D75N mutant complexed with pHtrII, using solid-state 13C NMR of [3- 13C]Ala- and [1- 13C]Val-labeled preparations. We distinguished Ala C,13C signals of relatively static stem (Ala221) in the C-terminus of the receptors from those of flexible tip (Ala228, 234, 236 and 238), utilizing a mutant with truncated C-terminus. The local fluctuation frequency at the C-terminal tip was appreciably decreased when ppR was bound to pHtrII, while it was increased when D75N, that mimics the signaling state because of disrupted salt bridge between C and G helices prerequisite for the signal transfer, was bound to pHtrII. This signal change may be considered with the larger dissociation constant of the complex between pHtrII and M-state of ppR. At the same time, it turned out that fluctuation frequency of cytoplasmic portion of pHtrII is lowered when ppR is replaced by D75N in the complex with pHtrII. This means that the C-terminal tip partly participates in binding with the linker region of pHtrII in the dark, but this portion might be released at the signaling state leading to mutual association of the two transducers in the cytoplasmic regions within the ppR/pHtrII complex. [source] Coupling of Protonation Switches During Rhodopsin Activation,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2007Reiner Vogel Recent studies of the activation mechanism of rhodopsin involving Fourier-transform infrared spectroscopy and a combination of chromophore modifications and site-directed mutagenesis reveal an allosteric coupling between two protonation switches. In particular, the ring and the 9-methyl group of the all- trans retinal chromophore serve to couple two proton-dependent activation steps: proton uptake by a cytoplasmic network between transmembrane (TM) helices 3 and 6 around the conserved ERY (Glu-Arg-Tyr) motif and disruption of a salt bridge between the retinal protonated Schiff base (PSB) and a protein counterion in the TM core of the receptor. Retinal analogs lacking the ring or 9-methyl group are only partial agonists,the conformational equilibrium between inactive Meta I and active Meta II photoproduct states is shifted to Meta I. An artificial pigment was engineered, in which the ring of retinal was removed and the PSB salt bridge was weakened by fluorination of C14 of the retinal polyene. These modifications abolished allosteric coupling of the proton switches and resulted in a stabilized Meta I state with a deprotonated Schiff base (Meta ISB). This state had a partial Meta II-like conformation due to disruption of the PSB salt bridge, but still lacked the cytoplasmic proton uptake reaction characteristic of the final transition to Meta II. As activation of native rhodopsin is known to involve deprotonation of the retinal Schiff base prior to formation of Meta II, this Meta ISB state may serve as a model for the structural characterization of a key transient species in the activation pathway of a prototypical G protein-coupled receptor. [source] The 2.0 Å crystal structure of the ER, ligand-binding domain complexed with lasofoxifenePROTEIN SCIENCE, Issue 5 2007Felix F. Vajdos Abstract Lasofoxifene is a new and potent selective estrogen receptor modulator (SERM). The structural basis of its interaction with the estrogen receptor has been investigated by crystallographic analysis of its complex with the ligand-binding domain of estrogen receptor , at a resolution of 2.0 Å. As with other SERMs, lasofoxifene diverts the receptor from its agonist-bound conformation by displacing the C-terminal AF-2 helix into the site at which the LXXLL motif of coactivator proteins would otherwise be able to bind. Lasofoxifene achieves this effect by occupying the space normally filled by residue Leu 540, as well as by modulating the conformation of residues of helix 11 (His 524, Leu 525). A well-defined salt bridge between lasofoxifene and Asp 351 suggests that charge neutralization in this region of the receptor may explain the some of the antiestrogenic effects of lasofoxifene. The results suggest general features of ER,/SERM recognition, and add a new dimension to efforts to rationalize differences between the biological activity profiles exhibited by these important pharmacological agents. [source] Structure of the 21,30 fragment of amyloid ,-proteinPROTEIN SCIENCE, Issue 6 2006Andrij Baumketner Abstract Folding and self-assembly of the 42-residue amyloid ,-protein (A,) are linked to Alzheimer's disease (AD). The 21,30 region of A,, A,(21,30), is resistant to proteolysis and is believed to nucleate the folding of full-length A,. The conformational space accessible to the A,(21,30) peptide is investigated by using replica exchange molecular dynamics simulations in explicit solvent. Conformations belonging to the global free energy minimum (the "native" state) from simulation are in good agreement with reported NMR structures. These conformations possess a bend motif spanning the central residues V24,K28. This bend is stabilized by a network of hydrogen bonds involving the side chain of residue D23 and the amide hydrogens of adjacent residues G25, S26, N27, and K28, as well as by a salt bridge formed between side chains of K28 and E22. The non-native states of this peptide are compact and retain a native-like bend topology. The persistence of structure in the denatured state may account for the resistance of this peptide to protease degradation and aggregation, even at elevated temperatures. [source] Sequence dependence of ,-hairpin structure: Comparison of a salt bridge and an aromatic interactionPROTEIN SCIENCE, Issue 12 2003Sarah E. Kiehna Abstract A comparison of the contributions and position dependence of cross-strand electrostatic and aromatic side-chain interactions to ,-sheet stability has been performed by using nuclear magnetic resonance in a well-folded ,-hairpin peptide of the general sequence XRTVXVdPGOXITQX. Phe,Phe and Glu,Lys pairs were varied at the internal and terminal non,hydrogen-bonded position, and the resulting stability was measured by the effects on ,-hydrogen and aromatic hydrogen chemical shifts. It was determined that the introduction of a Phe,Phe pair resulted in a more folded peptide, regardless of position, and a more tightly folded core. Substitution of the Glu,Lys pair at the internal position results in a less folded peptide and increased fraying at the terminal residues. Upfield shifting of the aromatic hydrogens provided evidence for an edge-face aromatic interaction, regardless of position of the Phe,Phe pair. In peptides with two Phe,Phe pairs, substitution with Glu,Lys at either position resulted in a weakening of the aromatic interaction and a subsequent decrease in peptide stability. Thermal denaturation of the peptides containing Phe,Phe indicates that the aromatic interaction is enthalpically favored, whereas the folding of hairpins with cross-strand Glu,Lys pairs was less enthalpically favorable but entropically more favorable. [source] Mechanisms by which atrial fibrillation-associated mutations in the S1 domain of KCNQ1 slow deactivation of IKs channelsTHE JOURNAL OF PHYSIOLOGY, Issue 17 2008Lioara Restier The slow delayed rectifier K+ current (IKs) is a major determinant of action potential repolarization in the heart. IKs channels are formed by coassembly of pore-forming KCNQ1 ,-subunits and ancillary KCNE1 ,-subunits. Two gain of function mutations in KCNQ1 subunits (S140G and V141M) have been associated with atrial fibrillation (AF). Previous heterologous expression studies found that both mutations caused IKs to be instantaneously activated, presumably by preventing channel closure. The purpose of this study was to refine our understanding of the channel gating defects caused by these two mutations located in the S1 domain of KCNQ1. Site-directed mutagenesis was used to replace S140 or V141 with several other natural amino acids. Wild-type and mutant channels were heterologously expressed in Xenopus oocytes and channel function was assessed with the two-microelectrode voltage clamp technique. Long intervals between voltage clamp pulses revealed that S140G and V141M KCNQ1-KCNE1 channels are not constitutively active as previously reported, but instead exhibit extremely slow deactivation. The slow component of IKs deactivation was decreased 62-fold by S140G and 140-fold by the V141M mutation. In addition, the half-point for activation of these mutant IKs channels was ,50 mV more negative than wild-type channels. Other substitutions of S140 or V141 in KCNQ1 caused variable shifts in the voltage dependence of activation, but slowed IKs deactivation to a much lesser extent than the AF-associated mutations. Based on a published structural model of KCNQ1, S140 and V141 are located near E160 in S2 and R237 in S4, two charged residues that could form a salt bridge when the channel is in the open state. In support of this model, mutational exchange of E160 and R237 residues produced a constitutively open channel. Together our findings suggest that altered charge-pair interactions within the voltage sensor module of KCNQ1 subunits may account for slowed IKs deactivation induced by S140 or V141. [source] The apo structure of sucrose hydrolase from Xanthomonas campestris pv. campestris shows an open active-site grooveACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2009Elise Champion Glycoside hydrolase family 13 (GH-13) mainly contains starch-degrading or starch-modifying enzymes. Sucrose hydrolases utilize sucrose instead of amylose as the primary glucosyl donor. Here, the catalytic properties and X-ray structure of sucrose hydrolase from Xanthomonas campestris pv. campestris are reported. Sucrose hydrolysis catalyzed by the enzyme follows Michaelis,Menten kinetics, with a Km of 60.7,mM and a kcat of 21.7,s,1. The structure of the enzyme was solved at a resolution of 1.9,Å in the resting state with an empty active site. This represents the first apo structure from subfamily 4 of GH-13. Comparisons with structures of the highly similar sucrose hydrolase from X. axonopodis pv. glycines most notably showed that residues Arg516 and Asp138, which form a salt bridge in the X. axonopodis sucrose complex and define part of the subsite ,1 glucosyl-binding determinants, are not engaged in salt-bridge formation in the resting X. campestris enzyme. In the absence of the salt bridge an opening is created which gives access to subsite ,1 from the `nonreducing' end. Binding of the glucosyl moiety in subsite ,1 is therefore likely to induce changes in the conformation of the active-site cleft of the X. campestris enzyme. These changes lead to salt-bridge formation that shortens the groove. Additionally, this finding has implications for understanding the molecular mechanism of the closely related subfamily 4 glucosyl transferase amylosucrase, as it indicates that sucrose could enter the active site from the `nonreducing' end during the glucan-elongation cycle. [source] Removing the invariant salt bridge of parvalbumin increases flexibility in the AB -loop structureACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2009François Hoh Parvalbumins (PVs) are calcium-buffer proteins that belong to the EF-hand family. Their N-terminal domain consists of two antiparallel helices A and B that make up a flat hydrophobic surface that is associated with the opposite side of the CD and EF binding sites. A single conserved Arg75,Glu81 salt bridge is buried in this hydrophobic interface. The structure of a rat PV mutant in which Arg75 was replaced by alanine was solved by molecular replacement. Unexpectedly, a large distance deviation of 7.8,Å was observed for the AB loop but not for the residues that flank the R75A mutation. The thermal stability of the calcium-loaded form is lower (Tm = 352.0,K; ,Tm = ,11.4,K) than that of the wild-type protein and the apo mutant is unfolded at room temperature. Weaker calcium or magnesium affinities were also measured for the R75A mutant (Ca2+: K1 = 4.21 × 107,M,1, K2 = 6.18 × 106,M,1; Mg2+: K1 = 2.98 × 104,M,1, K2 = 3.09 × 103,M,1). Finally, comparison of the B factors showed an increase in the flexibility of the AB loop that is consistent with this region being more exposed to solvent in the mutant. The mutant structure therefore demonstrates the role of the salt bridge in attaching the nonbinding AB domain to the remaining protein core. Normal-mode analysis indeed indicated an altered orientation of the AB domain with regard to the CD,EF binding domains. [source] Impact of low-frequency hotspot mutation R282Q on the structure of p53 DNA-binding domain as revealed by crystallography at 1.54,Å resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2008Chao Tu Tumor suppressor p53 is a sequence-specific DNA-binding protein and its central DNA-binding domain (DBD) harbors six hotspots (Arg175, Gly245, Arg248, Arg249, Arg273 and Arg282) for human cancers. Here, the crystal structure of a low-frequency hotspot mutant, p53DBD(R282Q), is reported at 1.54,Å resolution together with the results of molecular-dynamics simulations on the basis of the structure. In addition to eliminating a salt bridge, the R282Q mutation has a significant impact on the properties of two DNA-binding loops (L1 and L3). The L1 loop is flexible in the wild type, but it is not flexible in the mutant. The L3 loop of the wild type is not flexible, whereas it assumes two conformations in the mutant. Molecular-dynamics simulations indicated that both conformations of the L3 loop are accessible under biological conditions. It is predicted that the elimination of the salt bridge and the inversion of the flexibility of L1 and L3 are directly or indirectly responsible for deactivating the tumor suppressor p53. [source] Structures of potent selective peptide mimetics bound to carboxypeptidase BACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2008Marc Adler This article reports the crystal structures of inhibitors of the functional form of thrombin-activatable fibrinolysis inhibitor (TAFIa). In vivo experiments indicate that selective inhibitors of TAFIa would be useful in the treatment of heart attacks. Since TAFIa rapidly degrades in solution, the homologous protein porcine pancreatic carboxypeptidase B (pp-CpB) was used in these crystallography studies. Both TAFIa and pp-CpB are zinc-based exopeptidases that are specific for basic residues. The final development candidate, BX 528, is a potent inhibitor of TAFIa (2,nM) and has almost no measurable effect on the major selectivity target, carboxypeptidase N. BX 528 was designed to mimic the tripeptide Phe-Val-Lys. A sulfonamide replaces the Phe-Val amide bond and a phosphinate connects the Val and Lys groups. The phosphinate also chelates the active-site zinc. The electrostatic interactions with the protein mimic those of the natural substrate. The primary amine in BX 528 forms a salt bridge to Asp255 at the base of the S1, pocket. The carboxylic acid interacts with Arg145 and the sulfonamide is hydrogen bonded to Arg71. Isopropyl and phenyl groups replace the side chains of Val and Phe, respectively. A series of structures are presented here that illustrate the evolution of BX 528 from thiol-based inhibitors that mimic a free C-terminal arginine. The first step in development was the replacement of the thiol with a phosphinate. This caused a precipitous drop in binding affinity. Potency was reclaimed by extending the inhibitors into the downstream binding sites for the natural substrate. [source] Structures of three diphtheria toxin repressor (DtxR) variants with decreased repressor activityACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2001Ehmke Pohl The diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae regulates the expression of the gene on corynebacteriophages that encodes diphtheria toxin (DT). Other genes regulated by DtxR include those that encode proteins involved in siderophore-mediated iron uptake. DtxR requires activation by divalent metals and holo-DtxR is a dimeric regulator with two distinct metal-binding sites per three-domain monomer. At site 1, three side chains and a sulfate or phosphate anion are involved in metal coordination. In the DtxR,DNA complex this anion is replaced by the side chain of Glu170 provided by the third domain of the repressor. At site 2 the metal ion is coordinated exclusively by constituents of the polypeptide chain. In this paper, five crystal structures of three DtxR variants focusing on residues Glu20, Arg80 and Cys102 are reported. The resolution of these structures ranges from 2.3 to 2.8,Å. The side chain of Glu20 provided by the DNA-binding domain forms a salt bridge to Arg80, which in turn interacts with the anion. Replacing either of the salt-bridge partners with an alanine reduces repressor activity substantially and it has been inferred that the salt bridge could possibly control the wedge angle between the DNA-binding domain and the dimerization domain, thereby modulating repressor activity. Cys102 is a key residue of metal site 2 and its substitution into a serine abolishes repressor activity. The crystal structures of Zn-Glu20Ala-DtxR, Zn-Arg80Ala-DtxR, Cd-Cys102Ser-DtxR and apo-Cys102Ser-DtxR in two related space groups reveal that none of these substitutions leads to dramatic rearrangements of the DtxR fold. However, the five crystal structures presented here show significant local changes and a considerable degree of flexibility of the DNA-binding domain with respect to the dimerization domain. Furthermore, all five structures deviate significantly from the structure in the DtxR,DNA complex with respect to overall domain orientation. These results confirm the importance of the hinge motion for repressor activity. Since the third domain has often been invisible in previous crystal structures of DtxR, it is also noteworthy that the SH3-like domain could be traced in four of the five crystal structures. [source] The Z isomer of 2,4-diaminofuro[2,3- d]pyrimidine antifolate promotes unusual crystal packing in a human dihydrofolate reductase ternary complexACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2009Vivian Cody The crystal structure of the ternary complex of human dihydrofolate reductase (hDHFR) with NADPH and the Z isomer of 2,4-diamino-5-[2-(2,-methoxyphenyl)propenyl]-furo[2,3- d]pyrimidine (Z1) shows that the Z isomer binds in the normal antifolate orientation in which the furo oxygen occupies the 8-amino position observed in the binding of 2,4-diaminopteridine antifolates such as methotrexate and with the methoxyphenyl moiety cis to and coplanar with the furo[2,3- d]pyrimidine ring. The hDHFR ternary complex crystallized in the orthorhombic space group P212121 and its structure was refined to 1.7,Å resolution. Although other hDHFR complexes crystallize in this space group, these data provide only the second example of an unusual packing arrangement in which the conserved active-site Arg70 forms a salt bridge to the side chain of Glu44 from a symmetry-related molecule. As a result, the conformations of Phe31 and Gln35 shift with respect to those observed in the structure of mouse DHFR bound to Z1, which crystallizes in the monoclinic space group P21 and shows that Gln35 interacts with Arg70. [source] Solution structure of the cyclic peptide contryphan-Vn, a Ca2+ -dependent K+ channel modulatorBIOPOLYMERS, Issue 3 2004Tommaso Eliseo Abstract The solution structure of contryphan-Vn, a cyclic peptide with a double cysteine S,S bridge and containing a D -tryptophan extracted from the venom of the cone snail Conus ventricosus, has been determined by NMR spectroscopy using a variety of homonuclear and heteronuclear NMR methods and restrained molecular dynamics simulations. The main conformational features of backbone contryphan-Vn are a type IV ,-turn from Gly 1 to Lys 6 and a type I ,-turn from Lys 6 to Cys 9. As already found in other contryphans, one of the two prolines,the Pro4,is mainly in the cis conformation while Pro7 is trans. A small hydrophobic region probably partly shielded from solvent constituted from the close proximity of side chains of Pro7 and Trp8 was observed together with a persistent salt bridge between Asp2 and Lys6, which has been revealed by the diagnostic observation of specific nuclear Overhauser effects. The salt bridge was used as a restraint in the molecular dynamics in vacuum but without inserting explicit electrostatic contribution in the calculations. The backbone of the unique conformational family found of contryphan-Vn superimposes well with those of contryphan-Sm and contryphan-R. This result indicates that the contryphan structural motif represents a robust and conserved molecular scaffold whose main structural determinants are the size of the intercysteine loop and the presence and location in the sequence of the D -Trp and the two Pro residues. © 2004 Wiley Periodicals, Inc. Biopolymers, 2004 [source] Zwitterionic States in Gas-Phase Polypeptide Ions Revealed by 157-nm Ultra-Violet PhotodissociationCHEMISTRY - A EUROPEAN JOURNAL, Issue 30 2006Frank Kjeldsen Dr. Abstract A new method of detecting the presence of deprotonation and determining its position in gas-phase polypeptide cations is described. The method involves 157-nm ultra-violet photodissociation (UVPD) and is based on monitoring the losses of CO2 (44 Da) from electronically excited deprotonated carboxylic groups relative to competing COOH losses (45 Da) from neutral carboxylic groups. Loss of CO2 is a strong indication of the presence of a zwitterionic [(+),,,(,),,,(+)] salt bridge in the gas-phase polypeptide cation. This method provides a tool for studying, for example, the nature of binding within polypeptide clusters. Collision-activated dissociation (CAD) of decarboxylated cations localizes the position of deprotonation. Fragment abundances can be used for the semiquantitative assessment of the branching ratio of deprotonation among different acidic sites, however, the mechanism of the fragment formation should be taken into account. Cations of Trp-cage proteins exist preferentially as zwitterions, with the deprotonation position divided between the Asp9 residue and the C terminus in the ratio 3:2. The majority of dications of the same molecule are not zwitterions. Furthermore, 157-nm UVPD produces abundant radical cations M.+ from protonated molecules through the loss of a hydrogen atom. This method of producing M.+ ions is general and can be applied to any gas-phase peptide cation. The abundance of the molecular radical cations M.+ produced is sufficient for further tandem mass spectrometry (MS/MS), which, in the cases studied, yielded side-chain loss of a basic amino acid as the most abundant fragmentation channel together with some backbone cleavages. [source] X-ray crystallography and structural stability of digestive lysozyme from cow stomachFEBS JOURNAL, Issue 8 2009Yasuhiro Nonaka In ruminants, some leaf-eating animals, and some insects, defensive lysozymes have been adapted to become digestive enzymes, in order to digest bacteria in the stomach. Digestive lysozyme has been reported to be resistant to protease and to have optimal activity at acidic pH. The structural basis of the adaptation providing persistence of lytic activity under severe gastric conditions remains unclear. In this investigation, we obtained the crystallographic structure of recombinant bovine stomach lysozyme 2 (BSL2). Our denaturant and thermal unfolding experiments revealed that BSL2 has high conformational stability at acidic pH. The high stability in acidic solution could be related to pepsin resistance, which has been previously reported for BSL2. The crystal structure of BSL2 suggested that negatively charged surfaces, a shortened loop and salt bridges could provide structural stability, and thus resistance to pepsin. It is likely that BSL2 loses lytic activity at neutral pH because of adaptations to resist pepsin. [source] Solution structure of a hydrophobic analogue of the winter flounder antifreeze proteinFEBS JOURNAL, Issue 4 2002Edvards Liepinsh The solution structure of a synthetic mutant type I antifreeze protein (AFP I) was determined in aqueous solution at pH 7.0 using nuclear magnetic resonance (NMR) spectroscopy. The mutations comprised the replacement of the four Thr residues by Val and the introduction of two additional Lys-Glu salt bridges. The antifreeze activity of this mutant peptide, VVVV2KE, has been previously shown to be similar to that of the wild type protein, HPLC6 (defined here as TTTT). The solution structure reveals an ,,helix bent in the same direction as the more bent conformer of the published crystal structure of TTTT, while the side chain ,1 rotamers of VVVV2KE are similar to those of the straighter conformer in the crystal of TTTT. The Val side chains of VVVV2KE assume the same orientations as the Thr side chains of TTTT, confirming the conservative nature of this mutation. The combined data suggest that AFP I undergoes an equilibrium between straight and bent helices in solution, combined with independent equilibria between different side chain rotamers for some of the amino acid residues. The present study presents the first complete sequence-specific resonance assignments and the first complete solution structure determination by NMR of any AFP I protein. [source] Computational alanine scanning of the 1:1 human growth hormone,receptor complexJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 1 2002Shuanghong Huo Abstract The MM-PBSA (Molecular Mechanics,Poisson,Boltzmann surface area) method was applied to the human Growth Hormone (hGH) complexed with its receptor to assess both the validity and the limitations of the computational alanine scanning approach. A 400-ps dynamical trajectory of the fully solvated complex was simulated at 300 K in a 101 Å×81 Å×107 Å water box using periodic boundary conditions. Long-range electrostatic interactions were treated with the particle mesh Ewald (PME) summation method. Equally spaced snapshots along the trajectory were chosen to compute the binding free energy using a continuum solvation model to calculate the electrostatic desolvation free energy and a solvent-accessible surface area approach to treat the nonpolar solvation free energy. Computational alanine scanning was performed on the same set of snapshots by mutating the residues in the structural epitope of the hormone and the receptor to alanine and recomputing the ,Gbinding. To further investigate a particular structure, a 200-ps dynamical trajectory of an R43A hormone,receptor complex was simulated. By postprocessing a single trajectory of the wild-type complex, the average unsigned error of our calculated ,,Gbinding is ,1 kcal/mol for the alanine mutations of hydrophobic residues and polar/charged residues without buried salt bridges. When residues involved in buried salt bridges are mutated to alanine, it is demonstrated that a separate trajectory of the alanine mutant complex can lead to reasonable agreement with experimental results. Our approach can be extended to rapid screening of a variety of possible modifications to binding sites. © 2002 Wiley Periodicals, Inc. J Comput Chem 23: 15,27, 2002 [source] The structure of a putative S-formylglutathione hydrolase from Agrobacterium tumefaciensPROTEIN SCIENCE, Issue 10 2009Karin E. van Straaten Abstract The structure of the Atu1476 protein from Agrobacterium tumefaciens was determined at 2 Å resolution. The crystal structure and biochemical characterization of this enzyme support the conclusion that this protein is an S-formylglutathione hydrolase (AtuSFGH). The three-dimensional structure of AtuSFGH contains the ,/, hydrolase fold topology and exists as a homo-dimer. Contacts between the two monomers in the dimer are formed both by hydrogen bonds and salt bridges. Biochemical characterization reveals that AtuSFGH hydrolyzes CO bonds with high affinity toward short to medium chain esters, unlike the other known SFGHs which have greater affinity toward shorter chained esters. A potential role for Cys54 in regulation of enzyme activity through S-glutathionylation is also proposed. [source] The L49F mutation in alpha erythroid spectrin induces local disorder in the tetramer association region: Fluorescence and molecular dynamics studies of free and bound alpha spectrinPROTEIN SCIENCE, Issue 9 2009Yuanli Song Abstract The bundling of the N-terminal, partial domain helix (Helix C,) of human erythroid ,-spectrin (,I) with the C-terminal, partial domain helices (Helices A, and B,) of erythroid ,-spectrin (,I) to give a spectrin pseudo structural domain (triple helical bundle A,B,C,) has long been recognized as a crucial step in forming functional spectrin tetramers in erythrocytes. We have used apparent polarity and Stern,Volmer quenching constants of Helix C, of ,I bound to Helices A, and B, of ,I, along with previous NMR and EPR results, to propose a model for the triple helical bundle. This model was used as the input structure for molecular dynamics simulations for both wild type (WT) and ,I mutant L49F. The simulation output structures show a stable helical bundle for WT, but not for L49F. In WT, four critical interactions were identified: two hydrophobic clusters and two salt bridges. However, in L49F, the region downstream of Helix C, was unable to assume a helical conformation and one critical hydrophobic cluster was disrupted. Other molecular interactions critical to the WT helical bundle were also weakened in L49F, possibly leading to the lower tetramer levels observed in patients with this mutation-induced blood disorder. [source] Combinatorial engineering to enhance thermostability of amylosucrasePROTEIN SCIENCE, Issue 6 2008Stéphane Emond Abstract Amylosucrase is a transglucosidase that catalyzes amylose-like polymer synthesis from sucrose substrate. About 60,000 amylosucrase variants from two libraries generated by the MutaGen random mutagenesis method were submitted to an in vivo selection procedure leading to the isolation of more than 7000 active variants. These clones were then screened for increased thermostability using an automated screening process. This experiment yielded three improved variants (two double mutants and one single mutant) showing 3.5- to 10-fold increased half-lives at 50°C compared to the wild-type enzyme. Structural analysis revealed that the main differences between wild-type amylosucrase and the most improved variant (R20C/A451T) might reside in the reorganization of salt bridges involving the surface residue R20 and the introduction of a hydrogen-bonding interaction between T451 of the B, domain and D488 of flexible loop 8. This double mutant is the most thermostable amylosucrase known to date and the only one usable at 50°C. At this temperature, amylose synthesis by this variant using high sucrose concentration (600 mM) led to the production of amylose chains twice as long as those obtained by the wild-type enzyme at 30°C. [source] Design of a minimal protein oligomerization domain by a structural approachPROTEIN SCIENCE, Issue 12 2000Peter Burkhard Abstract Because of the simplicity and regularity of the ,-helical coiled coil relative to other structural motifs, it can be conveniently used to clarify the molecular interactions responsible for protein folding and stability. Here we describe the de novo design and characterization of a two heptad-repeat peptide stabilized by a complex network of inter- and intrahelical salt bridges. Circular dichroism spectroscopy and analytical ultracentrifugation show that this peptide is highly ,-helical and 100% dimeric under physiological buffer conditions. Interestingly, the peptide was shown to switch its oligomerization state from a dimer to a trimer upon increasing ionic strength. The correctness of the rational design principles used here is supported by details of the atomic structure of the peptide deduced from X-ray crystallography. The structure of the peptide shows that it is not a molten globule but assumes a unique, native-like conformation. This de novo peptide thus represents an attractive model system for the design of a molecular recognition system. [source] Molecular design and synthesis of artificial double helicesTHE CHEMICAL RECORD, Issue 1 2007Yoshio Furusho Abstract This account describes novel artificial double helices recently developed by our group. We have designed and synthesized the double helices consisting of two complementary, m -terphenyl-based strands that are intertwined through chiral amidinium,carboxylate salt bridges. Due to the chiral substituents on the amidine groups, the double helices adopted an excess one-handed helical conformation in solution as well as in the solid state. By extending the modular strategy, we have synthesized double helices bearing Pt(II) linkers, which underwent the double helix-to-double helix transformations through the chemical reactions of the Pt(II) complex moieties. In addition, artificial double-stranded metallosupramolecular helical polymers were constructed by combining the salt bridges and metal coordination. In contrast to the design-oriented double helices based on salt bridges, we have serendipitously developed a spiroborate-based double helicate bearing oligophenol strands. The optical resolution of the helicate was successfully attained by a diastereomeric salt formation. We have also unexpectedly found that oligoresorcinols consisting of a very simple repeating unit self-assemble into double helices with the aid of aromatic interactions in water. Furthermore, a bias in the twist sense of the double helices can be achieved by incorporating chiral substituents at both ends of the strands. © 2007 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 7: 1,11; 2007: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.20097 [source] | ||||||||||||||||||||||||||||