Papers by Michael Kokkinidis
... RII3/CT/2004/5060008. Expert assistance by Dr Manfred Weiss is gratefully acknowledged. We th... more ... RII3/CT/2004/5060008. Expert assistance by Dr Manfred Weiss is gratefully acknowledged. We thank Anastassia Gazi and Chrysa Meramveliotaki for assistance with data collection. References. Adams, RLP (1990). Biochem. J. 265, 309-320. ...
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Plant Pathology, 2012
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Journal of Molecular Biology, 2003
We have determined the crystal structure of the PvuII endonuclease in the presence of Mg2+. Accor... more We have determined the crystal structure of the PvuII endonuclease in the presence of Mg2+. According to the structural data, divalent metal ion binding in the PvuII subunits is highly asymmetric. The PvuII–Mg2+ complex has two distinct metal ion binding sites, one in each monomer. One site is formed by the catalytic residues Asp58 and Glu68, and has extensive similarities
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Molecular Plant Pathology, 2010
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The EMBO journal, 1989
Repressor of primer (Rop) is a small dimeric protein that participates in the mechanism that cont... more Repressor of primer (Rop) is a small dimeric protein that participates in the mechanism that controls the copy number of plasmid of the ColE1 family by increasing the affinity between two complementary RNAs. The Rop dimer is a bundle of four tightly packed alpha-helices that are held together by hydrophobic interactions. We have systematically altered, by site directed mutagenesis, most of the solvent exposed amino acids of the Rop bundle and we have identified the alterations that cause a decrease of the activity of the regulatory molecule. We conclude that Rop folding is rather insensitive to amino acid substitutions and to other mutations as drastic as deletions and insertions. Looking along the 2-fold symmetry axis the amino acid side chains whose alterations affect the function of Rop are all located on one side of the molecule. Furthermore they are clustered at the extremities of the alpha-helix bundle, the only exception being the aromatic ring of Phe-14.
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Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2006
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FEMS Microbiology Letters, 2014
Bacterial type III secretion systems (T3SSs) are specialized multicomponent nanomachines that med... more Bacterial type III secretion systems (T3SSs) are specialized multicomponent nanomachines that mediate the transport of proteins either to extracellular locations or directly into eukaryotic host cell cytoplasm. Erwinia amylovora, the main agent of rosaceous plants fireblight disease, employs an Hrp/Hrc1 T3SS to accomplish its pathogenesis. The regulatory network that controls the activation of this T3SS is largely unknown in E. amylovora. However, in Pseudomonas syringae pathovars, the HrpG/HrpV complex has been shown to directly regulate the activity of transcription factor HrpS and consequently the upregulation of the Hrp/Hrc1 T3SS related genes. In this work, we report the successful recombinant production and purification of a stable E. amylovora HrpG/HrpV complex, using pPROpET, a bicistronic expression vector. Furthermore, we present the first solution structure of this complex based on small-angle X-ray scattering data.
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Nature Structural Biology, 1994
The crystal structure of the dimeric PvuII restriction endonuclease (R.PvuII) has been determined... more The crystal structure of the dimeric PvuII restriction endonuclease (R.PvuII) has been determined at a resolution of 2.4A. The protein has a mixed alpha/beta architecture and consists of two subdomains. Despite a lack of sequence homology, extensive structural similarities exist between one R.PvuII subdomain and the DNA-binding subdomain of EcoRV endonuclease (R.EcoRV); the dimerization subdomains are unrelated. Within the similar domains, flexible segments of R.PvuII are topologically equivalent to the DNA-binding turns of R.EcoRV; potential catalytic residues can be deduced from the structural similarities to R.EcoRV. Conformational flexibility is important for the interaction with DNA. A possible classification of endonuclease structures on the basis of the positions of the scissile phosphates is discussed.
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The sequences of alpha-helical coiled-coils and bundles are characterized by a specific pattern o... more The sequences of alpha-helical coiled-coils and bundles are characterized by a specific pattern of hydrophobic and hydrophilic residues which is repeated every seven residues. Highly conserved breaks in this pattern frequently occur in segments of otherwise continuous heptad substructures. The hairpin bend of the ROP protein coincides with such a break and provides a model system for the study of the structural effects induced by heptad discontinuities. The structure of a ROP mutant which re-establishes a continuous heptad pattern, shows insignificant changes relative to the wild-type protein, as is also reflected in its conformational stability, spectroscopic properties and unfolding behaviour. Thus, formation of alpha-alpha-hairpin bends may occur both in the presence and absence of heptad breaks.
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FEBS Journal, 2007
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European Journal of Biochemistry, 2001
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European Journal of Biochemistry, 2002
In an effort to explore the role of glycine clusters on the cold adaptation of enzymes, we design... more In an effort to explore the role of glycine clusters on the cold adaptation of enzymes, we designed point mutations aiming to alter the distribution of glycine residues close to the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutagenesis targets were residues Gly261 and Gly262. The replacement of Gly262 by Ala resulted in an inactive enzyme. Substitution of Gly261 by Ala resulted to an enzyme with lower stability and increased energy of activation. The double mutant G261A/Y269A designed on the basis of side-chain packing criteria from a modelled structure of the enzyme resulted in restoration of the energy of activation to the levels of the native enzyme and in an increased stability compared to the mutant G261A. It seems therefore, that the Gly cluster in combination with its structural environment plays a significant role in the cold adaptation of the enzyme.
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Structure, 1999
Background: Conventional wisdom has it that two proteins sharing 98.4% sequence identity have nea... more Background: Conventional wisdom has it that two proteins sharing 98.4% sequence identity have nearly identical three-dimensional structures. Here we provide a counter-example to this statement by showing that a single amino acid substitution can change the topology of a homodimeric 4-α-helical bundle protein.Results: We have determined the high-resolution crystal structure of a 4-α-helical protein with a single alanine to proline
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Proteins: Structure, Function, and Genetics, 1993
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Protein Engineering Design and Selection, 2003
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Protein Engineering Design and Selection, 2001
ABSTRACT
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Photosynthesis Research, 1993
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Journal of Synchrotron Radiation, 2014
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Papers by Michael Kokkinidis