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Chirascan™ References
The last 25 references for the Chirascan circular dichroism spectrometer are listed below. A complete searchable database with all known circular dichroism and stopped-flow CD references can be accessed by logging into the APL members area.
| Authors | Title | Year | Keywords | Journal/Proceedings | |
|---|---|---|---|---|---|
| Frédéric Girard, Vincent Vachon, Gabrielle Préfontaine, Lucie Marceau, Jean-Louis Schwartz, Luke Masson, and Raynald Laprade | Helix α4 of the Bacillus thuringiensis Cry1Aa Toxin Plays a Critical Role in the Postbinding Steps of Pore Formation | 2009 | Membrane binding, pore formation. | Applied and Environmental Microbiology, 2009, p. 359-365, Vol. 75, No. 2. | |
| Abstract: Helix α4 of Bacillus thuringiensis Cry toxins is thought to play a critical role in the toxins' mode of action. Accordingly, single-site substitutions of many Cry1Aa helix {alpha}4 amino acid residues have previously been shown to cause substantial reductions in the protein's pore-forming activity. Changes in protein structure and formation of intermolecular disulfide bonds were investigated as possible factors responsible for the inactivity of these mutants. Incubation of each mutant with trypsin and chymotrypsin for 12 h did not reveal overt structural differences with Cry1Aa, although circular dichroism was slightly decreased in the 190- to 210-nm region for the I132C, S139C, and V150C mutants. The addition of dithiothreitol stimulated pore formation by the E128C, I132C, S139C, T142C, I145C, P146C, and V150C mutants. However, in the presence of these mutants, the membrane permeability never reached that measured for Cry1Aa, indicating that the formation of disulfide bridges could only partially explain their loss of activity. The ability of a number of inactive mutants to compete with wild-type Cry1Aa for pore formation in brush border membrane vesicles isolated from Manduca sexta was also investigated with an osmotic swelling assay. With the exception of the L147C mutant, all mutants tested could inhibit the formation of pores by Cry1Aa, indicating that they retained receptor binding ability. These results strongly suggest that helix {alpha}4 is involved mainly in the postbinding steps of pore formation. | |||||
| Leanne Kelly, Michael A. McDonough, Mathew L. Coleman, Peter J. Ratcliffe and Christopher J. Schofield | Asparagine β-hydroxylation stabilizes the ankyrin repeat domain fold | 2009 | Ankyrin repeats, Hypoxia inducible factor, protein hydroxylation | Mol. BioSyst., 2009, 5, pp. 52 - 58, | |
| Abstract: Ankyrin repeats (ARs) are one of the most common structural motifs among eukaryotic proteins. Recent analyses have shown that factor inhibiting hypoxia-inducible factor (FIH) catalyses the hydroxylation of highly conserved Asn-residues within ankyrin repeat domains (ARDs). However, the effect of Asn-hydroxylation on ARD structure is unknown. Supporting the proposal that FIH-mediated ARD hydroxylation is ubiquitous we report that consensus ARD proteins are FIH substrates both in vitro and in vivo. X-ray diffraction analyses revealed that hydroxylation does not alter the archetypical ARD conformation in the crystalline state. However, other biophysical analyses revealed that hydroxylation significantly stabilizes the ARD fold in solution. We propose that intracellular protein hydroxylation is much more common than previously thought and that one of its roles is stabilization of localized regions of ARD folds. | |||||
| Khondaker M. Rahman, Vanelssa Mussa, Mathangi Narayanaswamy, Colin H. James, Philip W. Howard and David E. Thurston | Observation of a dynamic equilibrium between DNA hairpin and duplex forms of covalent adducts of a minor groove binding agent. | 2009 | DNA stabilisation, minor groove binding, pyrrolobenzodiazepine | Chem. Commun., 2009, pp. 227 - 229 | |
| Abstract: A dynamic equilibrium between covalent 1:1 hairpin and 2:1 duplex DNA adducts of a pyrrolobenzodiazepine (PBD) minor groove binding agent ( 1) has been observed for the first time. The equilibrium, which establishes over 1 hour and must require unfolding of both types of adducts, is surprising given that PBDs normally require DNA minor groove structure for binding and take 24 hours for complete reaction with duplex DNA. The equilibrium is interesting from an energetics perspective due to the well known DNA stabilizing effect of PBDs. This observation could have significance for the in vitro and in vivo biological activity of PBDs, as DNA hairpin and loop structures are known to be important in cellular processes such as transcription and replication. | |||||
| Neeliyath A. Ramakrishnan, Marian J. Drescher, and Dennis G. Drescher | Direct Interaction of Otoferlin with Syntaxin 1A, SNAP-25, and the L-type Voltage-gated Calcium Channel CaV1.3 | 2009 | synaptic exocytosis, otoferlin, Calcium binding | J. Biol. Chem., Vol. 284, Issue 3, pp. 1364-1372, 2009. | |
| Abstract: The molecular mechanisms underlying synaptic exocytosis in the hair cell, the auditory and vestibular receptor cell, are not well understood. Otoferlin, a C2 domain-containing Ca2+-binding protein, has been implicated as having a role in vesicular release. Mutations in the OTOF gene cause nonsyndromic deafness in humans, and OTOF knock-out mice are deaf. In the present study, we generated otoferlin fusion proteins containing two of the same amino acid substitutions detected in DFNB9 patients (P1825A in C2F and L1011P in C2D). The native otoferlin C2F domain bound syntaxin 1A and SNAP-25 in a Ca2+-dependent manner (with optimal 61 µM free Ca2+ required for binding). These interactions were greatly diminished for C2F with the P1825A mutation, possibly because of a reduction in tertiary structural change, induced by Ca2+, for the mutated C2F compared with the native C2F. The otoferlin C2D domain also bound syntaxin 1A, but with weaker affinity (Kd = 1.7 x 10–5 M) than for the C2F interaction (Kd = 2.6 x 10–9 M). In contrast, it was the otoferlin C2D domain that bound the Cav1.3 II-III loop, in a Ca2+-dependent manner. The L1011P mutation in C2D rendered this binding insensitive to Ca2+ and considerably diminished. Overall, we demonstrated that otoferlin interacts with two main target-SNARE proteins of the hair-cell synaptic complex, syntaxin 1A and SNAP-25, as well as the calcium channel, with the otoferlin C2F and C2D domains of central importance for binding. Because mutations in the otoferlin C2 domains that cause deafness in humans impair the ability of otoferlin to bind syntaxin, SNAP-25, and the Cav1.3 calcium channel, it is these interactions that may mediate regulation by otoferlin of hair cell synaptic exocytosis critical to inner ear hair cell function. | |||||
| Bhunia, A., Domadia, P., Xu, X., Gingras, R., Ni, F. & Bhattacharjya, S. | Equilibrium Unfolding of the Dimeric SAM Domain of MAPKKK Ste11 from the Budding Yeast: Role of the Interfacial Residues in Structural Stability and Binding | 2008 | Unfolding, Structural Stability, Binding | Biochemistry, 47 (2), 651 -659, 2008. 10.1021/bi701941z S0006-2960(70)01941-4 | |
| Abstract: The sterile motifs or SAM domains are small (~70 amino acids) protein-protein interaction modules that are involved in diverse functions ranging from cell signaling, transcription regulation, and scaffolding. The Ste11 protein kinase in the mitogen-activated protein kinase (MAPK) signaling cascades of the budding yeast is regulated by a SAM domain located at the N-terminus of the full-length protein. The Ste11 SAM domain forms a symmetrical dimeric structure with an interface stabilized presumably by hydrophobic and ionic interactions. Here, we investigated urea-induced unfolding, using NMR and other optical spectroscopic methods, of the dimeric Ste11 SAM domain and two of the variants, namely, L57R and L60R, each containing a point mutation at the interfacial region. Our results demonstrate that the residue-specific or global unfolding of the Ste11 SAM is highly cooperative without any evidence for folded monomeric or partially folded species. However, replacement of hydrophobic residues with basic residues in the interface caused considerable changes in the stability and folding of the Ste11 SAM domain. The native dimeric structure of the L60R mutant protein is severely affected as indicated by a high propensity toward aggregation. On the other hand, the L57R mutant, although retaining the native structure, shows a dramatic decrease in the conformational stability as revealed by urea-induced denaturation and amide proton exchange studies. Furthermore, isothermal titration calorimetry and intrinsic tryptophan fluorescence experiments demonstrate that the L57R interacts with the cognate SAM domain from Ste50 with reduced affinity, while the L60R protein is devoid of any detectable binding activity. These results demonstrate that the interfacial residues of the dimeric SAM domain of Ste11 are critically involved in its structural stability and binding to the Ste50 SAM domain. | |||||
| Chakraborty, S., Nemeria, N., Yep, A., McLeish, M. J., Kenyon, G. L. & Jordan, F. | Mechanism of Benzaldehyde Lyase Studied via Thiamin Diphosphate-Bound Intermediates and Kinetic Isotope Effects | 2008 | Enzyme kinetics, kinetic isotope effects, enzyme benzaldehyde lyase, thiamin diphosphateintermediates. | Biochemistry, 47 (12), 3800–3809, 2008. 10.1021/bi702302u | |
| Abstract: Direct spectroscopic observation of thiamin diphosphate-bound intermediates was achieved on the enzyme benzaldehyde lyase, which carries out reversible and highly enantiospecific conversion of (R)-benzoin to benzaldehyde. The key enamine intermediate could be observed at λmax 393 nm in the benzoin breakdown direction and in the decarboxylase reaction starting with benzoylformate. With benzaldehyde as substrate, no intermediates could be detected, only formation of benzoin at 314 nm. To probe the rate-limiting step in the direction of (R)-benzoin synthesis, the 1H/2H kinetic isotope effect was determined for benzaldehyde labeled at the aldehyde position and found to be small (1.14 ± 0.03), indicating that ionization of the C2αH from C2α-hydroxybenzylthiamin diphosphate is not rate limiting. Use of the alternate substrates benzoylformic and phenylpyruvic acids (motivated by the observation that while a carboligase, benzaldehyde lyase could also catalyze the slow decarboxylation of 2-oxo acids) enabled the observation of the substrate-thiamin covalent intermediate via the 1′,4′-iminopyrimidine tautomer, characteristic of all intermediates with a tetrahedral C2 substituent on ThDP. The reaction of benzaldehyde lyase with the chromophoric substrate analogue (E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid and its decarboxylated product (E)-3-(pyridine-3-yl)acrylaldehyde enabled the detection of covalent adducts with both. Neither adduct underwent further reaction. An important finding of the studies is that all thiamin-related intermediates are in a chiral environment on benzaldehyde lyase as reflected by their circular dichroism signatures. | |||||
| Cremades, N., Bueno, M., Neira, J. L., Velázquez-Campoy, A. & Sancho, J. | Conformational Stability of Helicobacter pylori Flavodoxin: FIT TO FUNCTION AT pH 5 | 2008 | Flavodoxin, Helicobacter pylori, conformational stability, unfolding, stability, cofactor binding. | J. Biol. Chem., Vol. 283, Issue 5, 2883-2895, February 1, 2008 | |
| Abstract: Flavodoxin is an essential protein for Helicobacter pylori, a pathogen living in the very acidic environment of the gastric tract and responsible for several diseases. We report the conformational stability of the protein in neutral and acidic pH. The apoprotein remains native between pH 12 and 5 and adopts a monomeric molten globule conformation at more acidic pH values. The equilibrium unfolding in urea appears two-state for either conformation, but the native one coexists with a hidden equilibrium intermediate of very similar properties. The stability of H. pylori apoflavodoxin is higher than that of the Anabaena homologue throughout the entire pH interval, which may be related to better charge compensation. H. pylori apoflavodoxin is strongly stabilized by its FMN cofactor. A global analysis of apo- and holoflavodoxin equilibrium unfolding, with and without excess FMN, indicates that the cofactor only binds to the native state. Some physical-chemical properties of the protein may represent an adaptation to the acidic environment. Unlike the apoflavodoxin from Anabaena, which becomes highly insoluble at pH 5.0, that from H. pylori remains soluble to at least 40 μM. This fact, together with the high stability of the apoprotein at this low pH that can arise in the bacteria cytoplasm, seems useful to allow newly synthesized apoflavodoxin molecules to fold and remain soluble to accomplish cofactor binding, which in turn increases the stability. Also, whenever the cytoplasmic pH drops to 5, preexisting flavodoxin molecules will remain folded and soluble and will retain the FMN cofactor, thus remaining functional. | |||||
| Hafner-Bratkovič, I., Gašperšič, J., Šmid, L. M., Bresjanac, M. & Jerala, R. | Curcumin binds to the α-helical intermediate and to the amyloid form of prion protein - a new mechanism for the inhibition of PrPSc accumulation | 2008 | amyloid; Congo red; curcumin; intermediate; oligomer; prion | Journal of Neurochemistry, Volume 104, Number 6, March 2008 , pp. 1553-1564(12) | |
| Abstract: Conversion of the native, predominantly α-helical conformation of prion protein (PrP) into the β-stranded conformation is characteristic for the transmissible spongiform encephalopathies such as Creutzfeld-Jakob disease. Curcumin, an extended planar molecule and a dietary polyphenol, inhibits in vitro conversion of PrP and formation of protease resistant PrP in neuroblastoma cell lines. Curcumin recognizes the converted β-form of the PrP both as oligomers and fibrils but not the native form. Curcumin binds to the prion fibrils in the left-handed chiral arrangement as determined by circular dichroism. We show that curcumin labels the plaques of the brain sections of variant Creutzfeld-Jakob disease cases and stains the same structures as antibodies against the PrP. In contrast to thioflavin T, curcumin also binds to the α-helical intermediate of PrP present at acidic pH at stoichiometry of 1:1. Congo red competes with curcumin for binding to the α-intermediate as well as to the β-form of PrP but is toxic and binds also to the native form of PrP. We therefore show that the partially unfolded structural intermediate of the PrP can be targeted by non-toxic compound of natural origin. | |||||
| Ivanyi-Nagy, R., Lavergne, J., Gabus, C., Ficheux, D. & Darlix, J. | RNA chaperoning and intrinsic disorder in the core proteins of Flaviviridae | 2008 | Flaviviridae, RNA chaperone proteins, viruses, folding and structural rearrangements. | Nucleic Acids Research, Volume 36, Number 3, Pp. 712-725. | |
| Abstract: RNA chaperone proteins are essential partners of RNA in living organisms and viruses. They are thought to assist in the correct folding and structural rearrangements of RNA molecules by resolving misfolded RNA species in an ATP-independent manner. RNA chaperoning is probably an entropy-driven process, mediated by the coupled binding and folding of intrinsically disordered protein regions and the kinetically trapped RNA. Previously, we have shown that the core protein of hepatitis C virus (HCV) is a potent RNA chaperone that can drive profound structural modifications of HCV RNA in vitro. We now examined the RNA chaperone activity and the disordered nature of core proteins from different Flaviviridae genera, namely that of HCV, GBV-B (GB virus B), WNV (West Nile virus) and BVDV (bovine viral diarrhoea virus). Despite low-sequence similarities, all four proteins demonstrated general nucleic acid annealing and RNA chaperone activities. Furthermore, heat resistance of core proteins, as well as far-UV circular dichroism spectroscopy suggested that a well-defined 3D protein structure is not necessary for core-induced RNA structural rearrangements. These data provide evidence that RNA chaperoning—possibly mediated by intrinsically disordered protein segments—is conserved in Flaviviridae core proteins. Thus, besides nucleocapsid formation, core proteins may function in RNA structural rearrangements taking place during virus replication. | |||||
| Yan Jin, PeiXue Ling, YanLi Heb, Lei Chen, JianYing Chen and TianMin Zhang | Preparation, characterization and anti-Helicobacter pylori activity of Bi3+-hyaluronate complex | 2008 | Bi3+; NaHA; Complex; Characterization; Anti-Helicobacter pylori activity | Carbohydrate Polymers Volume 74, Issue 1, 1 October 2008, Pages 50-58 | |
| Abstract: Bi3+-hyaluronate complexes were prepared at pH 11.0–12.0. The complexes were characterized by elemental analysis, 13C NMR, FT-IR, CD, XRD, TGA and XPS. Participation of N and O atoms in coordination with Bi3+ has been confirmed. The anti-Helicobacter pylori activity in vitro of the complexes are similar with that of CBS, the most utilized bismuth preparation clinically. | |||||
| Klewpatinond, M., Davies, P., Bowen, S., Brown, D. R. & Viles, J. H. | Deconvoluting the Cu2+ Binding Modes of Full-length Prion Protein | 2008 | prion protein, copper binding, glycoprotein, misfolding, transmissible spongiform encephalopathies. | J. Biol. Chem., Vol. 283, Issue 4, 1870-1881, January 25, 2008 | |
| Abstract: The prion protein (PrP) is a cell-surface Cu2+-binding glycoprotein that when misfolded is responsible for a number of transmissible spongiform encephalopathies. Full-length PrP-(23231) and constructs in which the octarepeat region has been removed, or His95 and His110 is replaced by alanine residues, have been used to elucidate the order and mode of Cu2+ coordination to PrP-(23231). We have built on our understanding of the appearance of visible CD spectra and EPR for various PrP fragments to characterize Cu2+ coordination to full-length PrP. At physiological pH, Cu2+ initially binds to full-length PrP in the amyloidogenic region between the octarepeats and the structured domain at His95 and His110. Only subsequent Cu2+ ions bind to single histidine residues within the octarepeat region. Ni2+ ions are used to further probe metal binding and, like Cu2+, Ni2+ will bind individually to His95 and His110, involving preceding main chain amides. Competitive chelators are used to determine the affinity of the first mole equivalent of Cu2+ bound to full-length PrP; this approach places the affinity in the nanomolar range. The affinity and number of Cu2+ binding sites support the suggestion that PrP could act as a sacrificial quencher of free radicals generated by copper redox cycling. | |||||
| Klocek, G. & Seelig, J. | Melittin Interaction with Sulfated Cell Surface Sugars | 2008 | Melittin, cationic peptide, cell membrane, glycosaminoglycans. | Biochemistry, 47 (9), 2841–2849, 2008. 10.1021/bi702258z | |
| Abstract: Melittin is a 26-residue cationic peptide with cytolytic and antimicrobial properties. Studies on the action mechanism of melittin have focused almost exclusively on the membrane-perturbing properties of this peptide, investigating in detail the melittin−lipid interaction. Here, we report physical−chemical studies on an alternative mechanism by which melittin could interact with the cell membrane. As the outer surface of many cells is decorated with anionic (sulfated) glycosaminoglycans (GAGs), a strong Coulombic interaction between the two oppositely charged molecules can be envisaged. Indeed, the present study using isothermal titration calorimetry reveals a high affinity of melittin for several GAGs, that is, heparan sulfate (HS), dermatan sulfate, and heparin. The microscopic binding constant of melittin for HS is 2.4 × 105 M−1, the reaction enthalpy is ΔHmelittin0 = −1.50 kcal/mol, and the peptide-to-HS stoichiometry is ~11 at 10 mM Tris, 100 mM NaCl at pH 7.4 and 28 °C. ΔHmelittin0 is characterized by a molar heat capacity of ΔCP0 = −227 cal mol−1 K−1. The large negative heat capacity change indicates that hydrophobic interactions must also be involved in the binding of melittin to HS. Circular dichroism spectroscopy demonstrates that the binding of the peptide to HS induces a conformational change to a predominantly α-helical structure. A model for the melittin−HS complex is presented. Melittin binding was compared with that of magainin 2 and nisin Z to HS. Magainin 2 is known for its antimicrobial properties, but it does not cause lysis of the eukaryotic cells. Nisin Z shows activity against various Gram-positive bacteria. Isothermal titration calorimetry demonstrates that magainin 2 and nisin Z do not bind to HS (5–50 °C, 10 mM Tris, and 100 mM NaCl at pH 7.4). | |||||
| Levi, S., Rawet, M., Kliouchnikov, L., Parnis, A. & Cassel, D. | Topology of amphipathic motifs mediating golgi localization in ArfGAP1 and its splice isoforms. | 2008 | folding, amphipathic helices. | J. Biol. Chem., Vol. 283, Issue 13, 8564-8572, March 28, 2008 | |
| Abstract: The interaction of the Arf1-directed GTPase-activating protein ArfGAP1 with the Golgi apparatus depends on motifs in its non-catalytic part that are unstructured in solution but are capable of folding into amphipathic helices in vitro upon interaction with poorly packed lipids. In previous studies a few hydrophobic residues that are critical for lipid binding and Golgi localization were identified, but the precise topology of the amphipathic motifs has not been determined. Here we present a detailed analysis of the Golgi targeting and in vitro folding features of the region encompassing the amphipathic motifs (residues 199-294). Point mutation analysis revealed that most hydrophobic residues within this region contribute to Golgi localization, whereas analysis by proline replacements and alanine insertions revealed that Golgi interaction depends on folding into two amphipathic helices with a short interrupting sequence. Analysis of splice isoforms containing a 10 residue in-frame insertion within the first amphipathic motif revealed that the insertion causes a truncation of the amphipathic helix which does not extend beyond the insertion sequence. Lastly, a lysine replacement mutant recently reported to bind to negatively-charged liposomes in a curvature-independent manner showed normal cellular distribution, suggesting that Golgi targeting of ArfGAP1 may involve factors other than sensing lipid packing. | |||||
| Lim, E., Tu, G., Schwartz, E., Cornelissen, J., Rowan, A., Nolte, R. & Huck, W. | Synthesis and Characterization of Surface-Initiated Helical Polyisocyanopeptide Brushes | 2008 | surface-initiated polymerization, isocyanide monomers, atomic force microscopy, ellipsometry, circular dichroism. | Macromolecules, 41 (6), 1945 -1951, 2008. 10.1021/ma702531u S0024-9297(70)02531-7 | |
| Abstract: This article describes the first surface-initiated polymerization of isocyanide monomers onto various surfaces in a controlled manner. Brushes up to 200 nm could be easily obtained within 3 h of polymerization, and the polymer growth was studied as a function of reaction time, monomer concentration, and growth conditions, using atomic force microscopy (AFM) and ellipsometry. The monomers used in this study contain dipeptide (L,L-Ala-Ala) side groups; circular dichroism (CD) measurements, Fourier transform infrared spectroscopy (FTIR), and AFM confirmed that the well-defined helical conformation was retained in the polyisocyanide brushes with hydrogen bonds along the polymer chains. | |||||
| Patel, S., Bui, T. T., Drake, A. F., Fraternali, F. & Nikolova, P. V | The p73 DNA Binding Domain Displays Enhanced Stability Relative to Its Homologue, the Tumor Suppressor p53, and Exhibits Cooperative DNA Binding. | 2008 | p53 protein, cancer, homologue p73. | Biochemistry, 47 (10), 3235–3244, 2008. 10.1021/bi7023207 | |
| Abstract: The p53 protein family is involved in the control of an intricate network of genes implicated in cell cycle, through to germ line integrity and development. Although the role of p53 is well-established, the intrinsic nature of its homologue p73 has yet to be fully elucidated. Here, the biochemical characterization and homology-based modeling of the p73 protein is presented and the implications for its function(s) examined. The DNA binding domains (DBDs) of p53, p63, and p73 bind to the specific target site of a 30-mer gadd45 dsDNA, as tested by EMSA. The monomeric DBDs bind cooperatively forming tetrameric complexes. However, a larger construct consisting of p73 DBD plus TET domain (p73 CT) and the corresponding p53 DBD plus TET domain (p53 CT) bind gadd45 differently than the respective DBDs. Significantly, p73 DBD exhibited enhanced thermodynamic stability relative to the p53 DBD but not compared to p63 DBD as shown by DSC, CD, and equilibrium unfolding. The p73 CT is less stable than p73 DBD. The modeling data show distinct electrostatic surfaces of p73 and p53 dimers when bound to DNA. Specifically, the p73 surface is less complementary for DNA binding, which may account for the differences in affinity and specificity for p53 REs. These stability and DNA binding data for p73 in vitro enhance and complement our understanding of the role of the p73 protein in vivo and could be exploited in designing strategies for cancer therapy in places where p53 is mutated. | |||||
| Christian Beyschau Andersen, Daniel Otzen, Gunna Christiansen, and Christian Rischel | Glucagon Amyloid-like Fibril Morphology Is Selected via Morphology-Dependent Growth Inhibition | 2007 | Protein structure and biophysics, peptide, hormone, glucagon, fibrillation, circular dichroism, kinetics. | BIOCHEMISTRY-USA, 2007, Vol 46, Iss 24, pp 7314-7324 | |
| Abstract: The 29-residue peptide hormone glucagon readily fibrillates at low pH, but the structure and morphology of the fibrils are very sensitive to the environmental conditions. Here we have investigated the mechanism behind the differences in morphology observed when glucagon fibrils are formed at different peptide concentrations. Electron microscopy shows that fibrils formed at low glucagon concentration (0.25 mg/mL) are twisted, while fibrils formed at high concentration (8 mg/mL) are straight. Monitoring the fibrillation kinetics at different concentrations, we find that the lag time has an unexpected maximum at a concentration of 1 mg/mL, with faster fibrillation at both lower and higher concentrations. Seeding experiments show that small amounts of straight fibril seeds can accelerate fibril growth at both low and high glucagon concentration, while twisted fibril seeds cannot grow at high concentrations. We conclude that there exists a morphology-dependent mechanism for inhibition of glucagon fibril growth. Light scattering experiments indicate that glucagon is mainly monomeric below 1 mg/mL and increasingly trimeric above this concentration. We propose that the glucagon trimer is able to specifically inhibit growth of the twisted fibril morphology. Such inhibitory binding of molecules in an unproductive conformation could also play a role in the selection of morphologies for other fibril-forming peptides and proteins. | |||||
| Andersen, C., Otzen, D., Christiansen, G. & Rischel, C. | Glucagon Amyloid-like Fibril Morphology Is Selected via Morphology-Dependent Growth Inhibition | 2007 | Glucagon Amyloid-like Fibril Morphology | Biochemistry, 46 (24), 7314 -7324, 2007. | |
| Abstract: The 29-residue peptide hormone glucagon readily fibrillates at low pH, but the structure and morphology of the fibrils are very sensitive to the environmental conditions. Here we have investigated the mechanism behind the differences in morphology observed when glucagon fibrils are formed at different peptide concentrations. Electron microscopy shows that fibrils formed at low glucagon concentration (0.25 mg/mL) are twisted, while fibrils formed at high concentration (8 mg/mL) are straight. Monitoring the fibrillation kinetics at different concentrations, we find that the lag time has an unexpected maximum at a concentration of 1 mg/mL, with faster fibrillation at both lower and higher concentrations. Seeding experiments show that small amounts of straight fibril seeds can accelerate fibril growth at both low and high glucagon concentration, while twisted fibril seeds cannot grow at high concentrations. We conclude that there exists a morphology-dependent mechanism for inhibition of glucagon fibril growth. Light scattering experiments indicate that glucagon is mainly monomeric below 1 mg/mL and increasingly trimeric above this concentration. We propose that the glucagon trimer is able to specifically inhibit growth of the twisted fibril morphology. Such inhibitory binding of molecules in an unproductive conformation could also play a role in the selection of morphologies for other fibril-forming peptides and proteins. | |||||
| Bhattacharjya, S., Domadia, P., Bhunia, A., Malladi, S. & David, S. | High-Resolution Solution Structure of a Designed Peptide Bound to Lipopolysaccharide: Transferred Nuclear Overhauser Effects, Micelle Selectivity, and Anti-Endotoxic Activity | 2007 | Lipopolysaccharide,Transferred Nuclear Overhauser Effects, Micelle Selectivity, and Anti-Endotoxic Activity | Biochemistry, 46 (20), 5864 -5874, 2007. 10.1021/bi6025159 S0006-2960(60)02515-4 | |
| Abstract: Designing peptides that would interact with lipopolysaccharides (LPS) and acquire a specific folded conformation can generate useful structural insights toward the development of anti-sepsis agents. In this work, we have constructed a 12-residue linear peptide, YW12, rich in aromatic and aliphatic amino acid residues with a centrally located stretch of four consecutive positively charged (KRKR) residues. In absence of LPS, YW12 is predominantly unstructured in aqueous solution. Using transferred nuclear Overhauser effect (Tr-NOE) spectroscopy, we demonstrate that YW12 adopts a well-folded structure as a complex with LPS. Structure calculations reveal that YW12 assumes an extended conformation at the N-terminus followed by two consecutive β-turns at its C-terminus. A hydrophobic core is formed by extensive packing between number of aromatic and nonpolar residues, whereas the positively charged residues are segregated out to a separate region essentially stabilizing an amphipathic structure. In an in vitro LPS neutralization assay using NF-κB induction as the readout, YW12 shows moderate activity with an IC50 value of ~10 μM. As would be expected, tryptophan fluorescence studies demonstrate that YW12 shows selective interactions only with the negatively charged lipid micelles including sodium dodecyl sulfate (SDS), 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol (POPG), and LPS, and no significant interactions are detected with zwitterionic lipid micelles such as dodecyl-phosphocholine (DPC). Far-UV CD studies indicate the presence of β-turns or β-sheet-like conformations of the peptide in negatively charged micelles, whereas no structural transitions are apparent in DPC micelles. These results suggest that structural features of YW12 could be utilized to develop nontoxic antisepsis compounds. | |||||
| Biukovic, G., Rossle, M., Gayen, S., Mu, Y. & Gruber, G | Small-Angle X-ray Scattering Reveals the Solution Structure of the Peripheral Stalk Subunit H of the A1AO ATP Synthase from Methanocaldococcus jannaschii and Its Binding to the Catalytic A Subunit | 2007 | Methanocaldococcus jannaschii, Small-Angle X-ray Scattering, Binding, ATP. | Biochemistry, 46 (8), 2070 -2078, 2007. 10.1021/bi062123n S0006-2960(06)02123-4 | |
| Abstract: The H subunit of the A1AO ATP synthase is a component of one of the peripheral stalks connecting the A1 and AO domain. Subunit H of the Methanocaldococcus jannaschii A1AO ATP synthase was analyzed by small-angle X-ray scattering (SAXS) in order to determine the first low-resolution structure of this molecule in solution. Independent to the concentration used, the protein is dimeric and has a boomerang-like shape, divided into two arms of 12.0 and 6.8 nm in length. Circular dichroism (CD) spectroscopy revealed that subunit H is comprised of 78% α-helix and a coiled-coil arrangement. To understand the orientation of the helices and the localization of the N- and C-termini inside the dimer, three truncated forms of subunit H (H8-104, H1-98, and H8-98) were expressed, purified, and analyzed by CD. SAXS experiments of H1-98 show that the maximum dimension of the truncated protein dropped to 15.1 nm. Comparison of the low-resolution shapes of H and H1-98 indicates that this goes along with structural changes in the C-terminal arm of the boomerang-like structure. Together with the result of a disulfide formation of a fourth truncated form, H1-47, with a cysteine at position 47, the data suggest a parallel -helical interaction. In addition, all four truncated proteins are dimeric in solution. Tryptophan emission spectra showed specific binding of H and H8-104 to the neighboring, catalytic A subunit, which could not be detected in the presence of H1-98. Finally, the arrangement of H within the A1AO ATP synthase is presented. | |||||
| Bleijlevens, B., Shivarattan, T., Sedgwick, B., Rigby, S. & Matthews, S. | Replacement of non-heme Fe(II) with Cu(II) in the α-ketoglutarate dependent DNA repair enzyme AlkB: Spectroscopic characterization of the active site. | 2007 | AlkB; Non-heme iron; Dioxygenase; Copper; EPR | Journal of Inorganic Biochemistry Volume 101, Issue 7, July 2007, Pages 1043-1048. | |
| Abstract: The bacterial DNA repair enzyme AlkB is an α-ketoglutarate (αKG) dependent non-heme Fe(II) containing dioxygenase. Here we describe, for the first time, the preparation of a Cu(II)-reconstituted form of AlkB in various complexes. Spectroscopic characterization showed correct AlkB folding upon incorporation of Cu(II) in the active site. The Cu site was classified as a type 2 site by EPR spectroscopy. The accessibility of the active site metal was studied using imidazole as a probe. Although addition of imidazole did not change the EPR spectrum of the AlkB–Cu–αKG complex, the spectrum of the AlkB–Cu–succinate complex clearly changed, indicating binding of imidazole at the Cu site. Binding of substrate (methylated DNA) to the AlkB–Cu–αKG complex did not induce changes in the EPR spectrum, demonstrating that the substrate does not bind in the immediate vicinity of the metal centre. This work provides a basis for advanced EPR approaches aimed at studying the interactions and dynamics of AlkB complexes in solution. | |||||
| Bonod-Bidaud, C., Beraud, M., Vaganay, E., Delacoux, F., Font, B., Hulmes, D. & Ruggiero, F. | Enzymatic cleavage specificity of the proa1(V) chain processing analysed by site-directed mutagenesis | 2007 | extracellular matrix, metalloproteinase, procollagen, proteolytic processing, site-directed mutagenesis, transient cell transfection. | Biochem. J. (2007) 405 (299–306) | |
| Abstract: The proteolytic processing of procollagen V is complex and depends on the activity of several enzymes among which the BMP-1 (bone morphogenetic protein-1)/tolloid metalloproteinase and the furin-like proprotein convertases. Few of these processing interactions could have been predicted by analysing the presence of conserved consensus sequences in the proa1(V) chain. In the present study we opted for a cell approach that allows a straightforward identification of processing interactions. A construct encompassing the complete N-terminal end of the proa1(V) chain, referred to as Na1, was recombinantly expressed to be used for enzymatic assays and for antibody production. Structural analysis showed that Na1 is a monomer composed of a compact globule and an extended tail, which correspond respectively to the non-collagenous Na1 subdomains, TSPN-1 (thrombospondin-1 N-terminal domain-like) and the variable region. Na1 was efficiently cleaved by BMP-1 indicating that the triple helix is not required for enzyme activity. By mutating residues flanking the cleavage site, we showed that the aspartate residue at position P2´ is essential for BMP-1 activity. BMP-1 activity at the C-terminal end of the procollagen V was assessed by generating a furin double mutant (R1584A/R1585A). We showed that, in absence of furin activity, BMP-1 is capable of processing the C-propeptide even though less efficiently than furin. Altogether, our results provide new relevant information on this complex and poorly understood mechanism of enzymatic processing in procollagen V function. | |||||
| Anthony Bugaut and Shankar Balasubramanian | A Sequence-Independent Study of the Influence of Short Loop Lengths on the Stability and Topology of Intramolecular DNA G-Quadruplexes | 2007 | circular dichroism spectroscopy | Biochemistry, 47 (2), 689 -697, 2008. 10.1021/bi701873c S0006-2960(70)01873-1 | |
| Abstract: G-Rich sequences found within biologically important regions of the genome have been shown to form intramolecular G-quadruplexes with varied loop lengths and sequences. Many of these quadruplexes will be distinguishable from each other on the basis of their thermodynamic stabilities and folded conformations. It has been proposed that loop lengths can strongly influence the topology and stability of intramolecular G-quadruplexes. Previous studies have been limited to the analysis of quadruplex sequences with particular loop sequences, making it difficult to make generalizations. Here, we describe an original study that aimed to elucidate the effect of loop length on the biophysical properties of G-quadruplexes in a sequence-independent context. We employed UV melting and circular dichroism spectroscopy to examine and compare the properties of 21 DNA quadruplex libraries, each comprising partially randomized loop sequences with lengths ranging from one to three nucleotides. Our work supports a number of general predictions that can be made solely on the basis of loop lengths. In particular, the results emphasize the strong influence of single-nucleotide loops on quadruplex properties. This study provides a predictive framework that may help identify or classify biologically relevant G-quadruplex-forming sequences. | |||||
| Sylvie Campagna, Nathalie Saint, Gérard Molle, and André Aumelas | Structure and Mechanism of Action of the Antimicrobial Peptide Piscidin | 2007 | Antibacterial Peptide, Structure and Mechanism, circular dichroism, NMR, secondary structure | BIOCHEMISTRY-USA, 2007, Vol 46, Iss 7, pp 1771-1778 | |
| Abstract: Piscidin, an antibacterial peptide isolated from the mast cells of striped bass, has potent antimicrobial activity against a broad spectrum of pathogens in vitro. We investigated the mechanism of action of this 22-residue cationic peptide by carrying out structural studies and electrophysiological experiments in lipid bilayers. Circular dichroism experiments showed that piscidin was unstructured in water but had a high α-helix content in dodecylphosphocholine (DPC) micelles. 1H NMR data in water and TFE confirmed these results and demonstrated that the segment of residues 8-17 adopted an α-helical structure in a micellar environment. This molecule has a marked amphipathic character, due to well-defined hydrophobic and hydrophilic sectors. This structure is similar to those determined for other cationic peptides involved in permeabilization of the bacterial membrane. Multichannel experiments with piscidin incorporated into azolectin planar bilayers gave reproducible I-V curves at various peptide concentrations and unambiguously showed that this peptide permeabilized the membrane. This pore forming activity was confirmed by single-channel experiments, with well-defined ion channels obtained at different voltages. The characteristics of the ion channels (voltage dependence, only one or two states of conductance) clearly suggest that piscidin is more likely to permeabilize the membrane by toroidal pore formation rather than via the "barrel-stave" mechanism. | |||||
| Carruthers, N. J. & Stemmer, P. M. | Methionine Oxidation in the Calmodulin-Binding Domain of Calcineurin Disrupts Calmodulin Binding and Calcineurin Activation | 2007 | Methionine Oxidation, Binding, Activation | Biochemistry, 47 (10), 3085–3095, 2008. 10.1021/bi702044x | |
| Abstract: Calcineurin is a Ca2+/calmodulin-activated Ser/Thr phosphatase important in cellular actions resulting in memory formation, cardiac hypertrophy, and T-cell activation. This enzyme is subject to oxidative inactivation by superoxide at low micromolar concentrations and by H2O2 at low millimolar concentrations. On the basis of the hypothesis that oxidation of Met residues in calmodulin-binding domains inhibits binding to calmodulin, purified calcineurin was used to study the susceptibility of Met residues to oxidation by H2O2. The rate for oxidation of Met406 in the calmodulin-binding domain was determined to be 4.4 × 10−3 M−1 s−1, indicating a high susceptibility to oxidation. Functional repercussions of Met406 oxidation were evaluated using native enzyme and a calcineurin mutant in which Met406 was exchanged for Leu. Measurement of fluorescent calmodulin binding demonstrated that oxidation of Met406 results in a 3.3-fold decrease in the affinity of calmodulin for calcineurin. Calcineurin activation exhibited a loss in cooperativity with respect to calmodulin following Met406 oxidation as shown by a reduction in the Hill slope from 1.88 to 0.86. Maximum phosphatase activity was unaffected by Met oxidation. Changes in the calcineurin−calmodulin interaction were accompanied by a 40% loss in the ability of calmodulin to stimulate binding of immunophilin/immunosuppressant to calcineurin. All effects on calmodulin binding to the native enzyme by the treatment with H2O2 could be reversed by treating the enzyme with methionine sulfoxide reductase. These results indicate that the calmodulin-binding domain of calcineurin is susceptible to oxidation at Met406 and that oxidation disrupts calmodulin binding and enzyme activation. Oxidation-dependent decreases in the affinity of calmodulin for calcineurin can potentially modulate calmodulin-dependent signaling and calmodulin distribution. | |||||
| Castelletto, V., Krysmann, M., Kelarakis, A. & Jauregi, P. | Complex Formation of Bovine Serum Albumin with a Poly(ethylene glycol) Lipid Conjugate | 2007 | Self-assembly, bovine serum albumin, formulation, circular dichroism, small-angle x-ray scattering | BIOMACROMOLECULES, 2007, Vol 8, Iss 7, pp 2244-2249 | |
| Abstract: In this work, we report the formation of complexes by self-assembly of bovine serum albumin (BSA) with a poly(ethylene glycol) lipid conjugate (PEG2000-PE) in phosphate saline buffer solution (pH 7.4). Three different sets of samples have been studied. The BSA concentration remained fixed (1, 0.01, or 0.001 wt % BSA) within each set of samples, while the PEG2000-PE concentration was varied. Dynamic light scattering (DLS), rheology, and small-angle X-ray scattering (SAXS) were used to study samples with 1 wt % BSA. DLS showed that BSA/PEG2000-PE aggregates have a size intermediate between a BSA monomer and a PEG2000-PE micelle. Rheology suggested that BSA/PEG2000-PE complexes might be surrounded by a relatively compact PEG-lipid shell, while SAXS results showed that depletion forces do not take an important role in the stabilization of the complexes. Samples containing 0.01 wt % BSA were studied by circular dichroism (CD) and ultraviolet fluorescence spectroscopy (UV). UV results showed that at low concentrations of PEG-lipid, PEG2000-PE binds to tryptophan (Trp) groups in BSA, while at high concentrations of PEG-lipid the Trp groups are exposed to water. CD results showed that changes in Trp environment take place with a minimal variation of the BSA secondary structure elements. Finally, samples containing 0.001 wt % BSA were studied by zeta-potential experiments. Results showed that steric interactions might play an important role in the stabilization of the BSA/PEG2000-PE complexes. | |||||