Archives of Biochemistry and Biophysics, Nov 1, 1984
Analytical gel chromatography has been used to examine self-association of bovine neurophysins I ... more Analytical gel chromatography has been used to examine self-association of bovine neurophysins I and II under several sets of conditions. The data provide no evidence for associated species larger than the dimer. Association constants and Stokes radii of both monomer and dimer are very similar for both proteins in both 0.1 M KOAc, 0.16 M KC1 and 0.1 M KP04, 0.16 M KC1 at pH 5.6 and 25°C. The average values derived for the Stokes radii of the monomer and dimer under these conditions are 14.5 + 0.7 and 23.0 + 0.4 A, respectively. These results confirm the conclusion of Rholam and Nicolas [(1981) Biochewktry 20, 583'7-58431 that the monomer and, to a lesser extent, the dimer are highly assymmetric. The Stokes radius of the monomer calculated by Rholam and Nicolas (op cik) is-30% larger than the value derived here. This discrepancy is probably the result of end-on penetration of the gel by elongated molecules [Y. Nozaki, N. M. Schechter, J. A. Reynolds, and C. Tanford (1976) Biochemistry 15,3884-38901. In contrast to Tellam and Winzor [(1980) Arch. B&&em. Biophgs. 201, 20-241, it was found that neurophysin II does not exist solely as the dimer in 0.1 M KPOl, pH 5.6, although substitution of 0.1 M KPOl for 0.1 M KOAc does increase the association constant by a factor of seven. Addition of 1.4 M LiCl at pH 8.1 also increases the association constant sevenfold, as well as increasing the Stokes radius of the monomer-20%. The effects of ionic strength are consistent with the conclusion of Nicolas et al. [(1978) J. BioL Chm 253,2633-26391 that formation of the dimer depends upon hydrophobic bonding. 0 19% Academic press, w. 585
Flow microcalorimetry and batch microcalorimetry have been used to survey the energetics of ligan... more Flow microcalorimetry and batch microcalorimetry have been used to survey the energetics of ligand binding by bovine neurophysins I and II. Calorimetry studies were supplemented by van't Hoff analyses of binding constants determined by circular dichroism. Free energies of binding of a series of di- and tripeptides that bind to the strong hormone binding site of neurophysin were partitioned into their enthalpic and entropic components. The results indicate that, at 25 degrees C, the binding of most peptides is an enthalpy-driven reaction associated with negative entropy and heat capacity changes. Studies elsewhere, supported by evidence here, indicate that the principal component of the negative enthalpy change does not arise from the increase in neurophysin dimerization associated with peptide binding. Accordingly, the negative enthalpy change is attributed to direct bonding interactions with peptide and possibly also to peptide-induced changes in tertiary or quaternary organization. Comparison of the binding enthalpies of different peptides indicated two types of bonding interactions that contribute to the negative enthalpy change of peptide ligation. Substitution of an aromatic- or sulfur-containing side chain for an aliphatic side chain in position 1 of bound peptides led to increases in negative enthalpy of from 1 to 6 kcal/mol, demonstrating that interactions typically classified as hydrophobic can have a significant exothermic component at 25 degrees C. Similarly, loss of hydrogen bonding potential in the peptide decreased the enthalpy change upon binding, in keeping with the expected enthalpic contribution of hydrogen bonds. In particular, the data suggested that the peptide backbone between residues 2 and 3 and the phenolic hydroxyl group in position 2 participate in hydrogen bonding.(ABSTRACT TRUNCATED AT 250 WORDS)
The emergence of genomics; ongoing computational advances; and the development of large-scale seq... more The emergence of genomics; ongoing computational advances; and the development of large-scale sequence, structural, and functional databases have created important new interdisciplinary linkages between molecular evolution, molecular biology, and enzymology. The five minireviews in this series survey advances and challenges in this burgeoning field from complementary perspectives. The series has three major themes. The first is the evolution of enzyme superfamilies, in which members exhibit increasing sequence, structural, and functional divergence with increasing time of divergence from a common ancestor. The second is the evolutionary role of promiscuous enzymes, which, in addition to their primary function, have adventitious secondary activities that frequently provide the starting point for the evolution of new enzymes. The third is the importance of in silico approaches to the daunting challenge of assigning and predicting the functions of the many uncharacterized proteins in the large-scale sequence and structural databases that are now available. A recent computational advance, the use of protein similarity networks that map functional data onto proteins clustered by similarity, is presented as an approach that can improve functional insight and inference. The three themes are illustrated with several examples of enzyme superfamilies, including the amidohydrolase, metallo--lactamase, and enolase superfamilies.
The biofilms that many bacteria and fungi produce enable them to form communities, adhere tightly... more The biofilms that many bacteria and fungi produce enable them to form communities, adhere tightly to surfaces, evade host immunity, and resist antibiotics. Pathogenic microorganisms that form biofilms are very difficult to eradicate and thus are a frequent source of life-threatening, hospital-acquired infections. This series of five minireviews from the Journal of Biological Chemistry provides a broad overview of our current understanding of biofilms and the challenges that remain. The structure, biosynthesis, and biological function of the biofilms produced by pathogenic fungi are the subject of the first article, by Sheppard and Howell. Gunn, Bakaletz, and Wozniak focus on the biochemistry and structure of bacterial biofilms, how these structures enable bacteria to evade host immunity, and current and developing strategies for overcoming this resistance. The third and fourth articles present two of the best understood cell signaling pathways involved in biofilm formation. Valentini and Filloux focus on cyclic di-GMP, while Kavanaugh and Horswill discuss the quorum-sensing (agr) system and the relationship between quorum sensing and biofilm formation. Mechanisms of antibiotic resistance, particularly the role of efflux pumps and the development of persister cells, are the topics of the final article by Van Acker and Coenye. The advances described in this series guarantee that ongoing interdisciplinary and international efforts will lead to new insights into the basic biology of biofilm formation, as well as new strategies for therapeutic interventions.
A flow cell in which a protein crystal is embedded in a changeable liquid medium during X-ray dif... more A flow cell in which a protein crystal is embedded in a changeable liquid medium during X-ray diffraction studies has proved useful in investigating the crystal chemistry of ribonuclease-S. The cell consists of a small polyethylene tube held in a brass yoke which in turn is held by a standard goniometer
F1000 - Post-publication peer review of the biomedical literature, 2016
Filamentous plant pathogens deliver effector proteins to host cells to promote infection. The Phy... more Filamentous plant pathogens deliver effector proteins to host cells to promote infection. The Phytophthora infestans RXLRtype effector PexRD54 binds potato ATG8 via its ATG8 familyinteracting motif (AIM) and perturbs host-selective autophagy. However, the structural basis of this interaction remains unknown. Here, we define the crystal structure of PexRD54, which includes a modular architecture, including five tandem repeat domains, with the AIM sequence presented at the disordered C terminus. To determine the interface between PexRD54 and ATG8, we solved the crystal structure of potato ATG8CL in complex with a peptide comprising the effector's AIM sequence, and we established a model of the full-length PexRD54-ATG8CL complex using small angle x-ray scattering. Structureinformed deletion of the PexRD54 tandem domains reveals retention of ATG8CL binding in vitro and in planta. This study offers new insights into structure/function relationships of oomycete RXLR effectors and how these proteins engage with host cell targets to promote disease.
Differential scanning calorimetry measures the heat capacity of states and the excess heat associ... more Differential scanning calorimetry measures the heat capacity of states and the excess heat associated with transitions that can be induced by temperature change. The integral of the excess heat capacity is the enthalpy for this process. Despite this potentially intimidating sounding physical chemistry background, DSC has found almost universal application in studying biological macromolecules. In the case of proteins, DSC can be used to determine equilibrium thermodynamic stability and folding mechanism but can also be used in a more qualitative manner screening for thermal stability as an indicator for, ligand binding, pharmaceutical formulation or conditions conducive to crystal growth. DSC usually forms part of a wider biophysical characterisation of the biological system of interest and so the literature is diverse and difficult to categorise for the technique in isolation. This review therefore describes the potential uses of DSC in studying protein folding and stability, giving brief examples of applications from the recent literature. There have also been some interesting developments in the use of DSC to determine barrier heights for fast folding proteins and in studying complex protein mixtures such as human plasma that are considered in more detail.
Acta Crystallographica Section D Biological Crystallography, 2001
ABSTRACT In androgen-sensitive target tissues, 3 alpha-hydroxysteroid dehydrogenase regulates the... more ABSTRACT In androgen-sensitive target tissues, 3 alpha-hydroxysteroid dehydrogenase regulates the androgen receptor (AR) activity by catalyzing the inactivation of 5 alpha-dihydrotestosterone (the most natural potent androgen) to 5 alpha-androstane-3 alpha,17 beta-diol. In this report, the crystallization of a human prostatic type 3 3 alpha-hydroxysteroid dehydrogenase, a member of the aldo-keto reductase superfamily, is described. Two different crystal forms of the complex between the human type 3 3 alpha-HSD, NADP(+) and testosterone have been obtained using PEG as precipitant. Crystal form I, which diffracts to 1.6 A, belongs to the monoclinic space group P2(1), with unit-cell parameters a = 55.07, b = 87.15, c = 76.88 A, beta = 107.37 degrees and two subunits in the asymmetric unit. A complete data set has been collected at 1.8 A. Crystal form II, which diffracts to 2.6 A, belongs to the rhombohedral space group R32, with unit-cell parameters a = b = 143.59, c = 205.86 A, alpha = beta = 90, gamma = 120 degrees and two subunits in the asymmetric unit.
Ferritin is a highly conserved multisubunit protein in animals, plants and microbes which assembl... more Ferritin is a highly conserved multisubunit protein in animals, plants and microbes which assembles with cubic symmetry and transports hydrated
F1000 - Post-publication peer review of the biomedical literature, 2016
The authors declare that they have no conflicts of interest with the contents of this article. □ ... more The authors declare that they have no conflicts of interest with the contents of this article. □ S This article contains supplemental Tables S1 and Figures S1 and S2. The atomic coordinates and structure factors (codes 5IKP and 5IKO) have been deposited in the Protein Data Bank (http://wwpdb.org/). 1 Supported by Ph.D. fellowships from the French Ministry of Research (Ecole Doctorale BioSPC). 2 Supported by a Ph.D. fellowship from Ré gion Ile de France (appel hors DIM 2013). 3 Supported by a fellowship from the China Scholarship Council.
Archives of Biochemistry and Biophysics, Nov 1, 1984
Analytical gel chromatography has been used to examine self-association of bovine neurophysins I ... more Analytical gel chromatography has been used to examine self-association of bovine neurophysins I and II under several sets of conditions. The data provide no evidence for associated species larger than the dimer. Association constants and Stokes radii of both monomer and dimer are very similar for both proteins in both 0.1 M KOAc, 0.16 M KC1 and 0.1 M KP04, 0.16 M KC1 at pH 5.6 and 25°C. The average values derived for the Stokes radii of the monomer and dimer under these conditions are 14.5 + 0.7 and 23.0 + 0.4 A, respectively. These results confirm the conclusion of Rholam and Nicolas [(1981) Biochewktry 20, 583'7-58431 that the monomer and, to a lesser extent, the dimer are highly assymmetric. The Stokes radius of the monomer calculated by Rholam and Nicolas (op cik) is-30% larger than the value derived here. This discrepancy is probably the result of end-on penetration of the gel by elongated molecules [Y. Nozaki, N. M. Schechter, J. A. Reynolds, and C. Tanford (1976) Biochemistry 15,3884-38901. In contrast to Tellam and Winzor [(1980) Arch. B&&em. Biophgs. 201, 20-241, it was found that neurophysin II does not exist solely as the dimer in 0.1 M KPOl, pH 5.6, although substitution of 0.1 M KPOl for 0.1 M KOAc does increase the association constant by a factor of seven. Addition of 1.4 M LiCl at pH 8.1 also increases the association constant sevenfold, as well as increasing the Stokes radius of the monomer-20%. The effects of ionic strength are consistent with the conclusion of Nicolas et al. [(1978) J. BioL Chm 253,2633-26391 that formation of the dimer depends upon hydrophobic bonding. 0 19% Academic press, w. 585
Flow microcalorimetry and batch microcalorimetry have been used to survey the energetics of ligan... more Flow microcalorimetry and batch microcalorimetry have been used to survey the energetics of ligand binding by bovine neurophysins I and II. Calorimetry studies were supplemented by van't Hoff analyses of binding constants determined by circular dichroism. Free energies of binding of a series of di- and tripeptides that bind to the strong hormone binding site of neurophysin were partitioned into their enthalpic and entropic components. The results indicate that, at 25 degrees C, the binding of most peptides is an enthalpy-driven reaction associated with negative entropy and heat capacity changes. Studies elsewhere, supported by evidence here, indicate that the principal component of the negative enthalpy change does not arise from the increase in neurophysin dimerization associated with peptide binding. Accordingly, the negative enthalpy change is attributed to direct bonding interactions with peptide and possibly also to peptide-induced changes in tertiary or quaternary organization. Comparison of the binding enthalpies of different peptides indicated two types of bonding interactions that contribute to the negative enthalpy change of peptide ligation. Substitution of an aromatic- or sulfur-containing side chain for an aliphatic side chain in position 1 of bound peptides led to increases in negative enthalpy of from 1 to 6 kcal/mol, demonstrating that interactions typically classified as hydrophobic can have a significant exothermic component at 25 degrees C. Similarly, loss of hydrogen bonding potential in the peptide decreased the enthalpy change upon binding, in keeping with the expected enthalpic contribution of hydrogen bonds. In particular, the data suggested that the peptide backbone between residues 2 and 3 and the phenolic hydroxyl group in position 2 participate in hydrogen bonding.(ABSTRACT TRUNCATED AT 250 WORDS)
The emergence of genomics; ongoing computational advances; and the development of large-scale seq... more The emergence of genomics; ongoing computational advances; and the development of large-scale sequence, structural, and functional databases have created important new interdisciplinary linkages between molecular evolution, molecular biology, and enzymology. The five minireviews in this series survey advances and challenges in this burgeoning field from complementary perspectives. The series has three major themes. The first is the evolution of enzyme superfamilies, in which members exhibit increasing sequence, structural, and functional divergence with increasing time of divergence from a common ancestor. The second is the evolutionary role of promiscuous enzymes, which, in addition to their primary function, have adventitious secondary activities that frequently provide the starting point for the evolution of new enzymes. The third is the importance of in silico approaches to the daunting challenge of assigning and predicting the functions of the many uncharacterized proteins in the large-scale sequence and structural databases that are now available. A recent computational advance, the use of protein similarity networks that map functional data onto proteins clustered by similarity, is presented as an approach that can improve functional insight and inference. The three themes are illustrated with several examples of enzyme superfamilies, including the amidohydrolase, metallo--lactamase, and enolase superfamilies.
The biofilms that many bacteria and fungi produce enable them to form communities, adhere tightly... more The biofilms that many bacteria and fungi produce enable them to form communities, adhere tightly to surfaces, evade host immunity, and resist antibiotics. Pathogenic microorganisms that form biofilms are very difficult to eradicate and thus are a frequent source of life-threatening, hospital-acquired infections. This series of five minireviews from the Journal of Biological Chemistry provides a broad overview of our current understanding of biofilms and the challenges that remain. The structure, biosynthesis, and biological function of the biofilms produced by pathogenic fungi are the subject of the first article, by Sheppard and Howell. Gunn, Bakaletz, and Wozniak focus on the biochemistry and structure of bacterial biofilms, how these structures enable bacteria to evade host immunity, and current and developing strategies for overcoming this resistance. The third and fourth articles present two of the best understood cell signaling pathways involved in biofilm formation. Valentini and Filloux focus on cyclic di-GMP, while Kavanaugh and Horswill discuss the quorum-sensing (agr) system and the relationship between quorum sensing and biofilm formation. Mechanisms of antibiotic resistance, particularly the role of efflux pumps and the development of persister cells, are the topics of the final article by Van Acker and Coenye. The advances described in this series guarantee that ongoing interdisciplinary and international efforts will lead to new insights into the basic biology of biofilm formation, as well as new strategies for therapeutic interventions.
A flow cell in which a protein crystal is embedded in a changeable liquid medium during X-ray dif... more A flow cell in which a protein crystal is embedded in a changeable liquid medium during X-ray diffraction studies has proved useful in investigating the crystal chemistry of ribonuclease-S. The cell consists of a small polyethylene tube held in a brass yoke which in turn is held by a standard goniometer
F1000 - Post-publication peer review of the biomedical literature, 2016
Filamentous plant pathogens deliver effector proteins to host cells to promote infection. The Phy... more Filamentous plant pathogens deliver effector proteins to host cells to promote infection. The Phytophthora infestans RXLRtype effector PexRD54 binds potato ATG8 via its ATG8 familyinteracting motif (AIM) and perturbs host-selective autophagy. However, the structural basis of this interaction remains unknown. Here, we define the crystal structure of PexRD54, which includes a modular architecture, including five tandem repeat domains, with the AIM sequence presented at the disordered C terminus. To determine the interface between PexRD54 and ATG8, we solved the crystal structure of potato ATG8CL in complex with a peptide comprising the effector's AIM sequence, and we established a model of the full-length PexRD54-ATG8CL complex using small angle x-ray scattering. Structureinformed deletion of the PexRD54 tandem domains reveals retention of ATG8CL binding in vitro and in planta. This study offers new insights into structure/function relationships of oomycete RXLR effectors and how these proteins engage with host cell targets to promote disease.
Differential scanning calorimetry measures the heat capacity of states and the excess heat associ... more Differential scanning calorimetry measures the heat capacity of states and the excess heat associated with transitions that can be induced by temperature change. The integral of the excess heat capacity is the enthalpy for this process. Despite this potentially intimidating sounding physical chemistry background, DSC has found almost universal application in studying biological macromolecules. In the case of proteins, DSC can be used to determine equilibrium thermodynamic stability and folding mechanism but can also be used in a more qualitative manner screening for thermal stability as an indicator for, ligand binding, pharmaceutical formulation or conditions conducive to crystal growth. DSC usually forms part of a wider biophysical characterisation of the biological system of interest and so the literature is diverse and difficult to categorise for the technique in isolation. This review therefore describes the potential uses of DSC in studying protein folding and stability, giving brief examples of applications from the recent literature. There have also been some interesting developments in the use of DSC to determine barrier heights for fast folding proteins and in studying complex protein mixtures such as human plasma that are considered in more detail.
Acta Crystallographica Section D Biological Crystallography, 2001
ABSTRACT In androgen-sensitive target tissues, 3 alpha-hydroxysteroid dehydrogenase regulates the... more ABSTRACT In androgen-sensitive target tissues, 3 alpha-hydroxysteroid dehydrogenase regulates the androgen receptor (AR) activity by catalyzing the inactivation of 5 alpha-dihydrotestosterone (the most natural potent androgen) to 5 alpha-androstane-3 alpha,17 beta-diol. In this report, the crystallization of a human prostatic type 3 3 alpha-hydroxysteroid dehydrogenase, a member of the aldo-keto reductase superfamily, is described. Two different crystal forms of the complex between the human type 3 3 alpha-HSD, NADP(+) and testosterone have been obtained using PEG as precipitant. Crystal form I, which diffracts to 1.6 A, belongs to the monoclinic space group P2(1), with unit-cell parameters a = 55.07, b = 87.15, c = 76.88 A, beta = 107.37 degrees and two subunits in the asymmetric unit. A complete data set has been collected at 1.8 A. Crystal form II, which diffracts to 2.6 A, belongs to the rhombohedral space group R32, with unit-cell parameters a = b = 143.59, c = 205.86 A, alpha = beta = 90, gamma = 120 degrees and two subunits in the asymmetric unit.
Ferritin is a highly conserved multisubunit protein in animals, plants and microbes which assembl... more Ferritin is a highly conserved multisubunit protein in animals, plants and microbes which assembles with cubic symmetry and transports hydrated
F1000 - Post-publication peer review of the biomedical literature, 2016
The authors declare that they have no conflicts of interest with the contents of this article. □ ... more The authors declare that they have no conflicts of interest with the contents of this article. □ S This article contains supplemental Tables S1 and Figures S1 and S2. The atomic coordinates and structure factors (codes 5IKP and 5IKO) have been deposited in the Protein Data Bank (http://wwpdb.org/). 1 Supported by Ph.D. fellowships from the French Ministry of Research (Ecole Doctorale BioSPC). 2 Supported by a Ph.D. fellowship from Ré gion Ile de France (appel hors DIM 2013). 3 Supported by a fellowship from the China Scholarship Council.
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