neal williams`

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Papers by neal williams`

Riley, L.G. etal. Cloning, expression, and spectroscopic studies of the Jun leucine zipper domain. Eur. J. Biochem. 219, 877 -886

European Journal of Biochemistry

Inhibitors of dihydro-orotase, amidophosphoribosyltransferase and IMP cyclohydrolase as potential drugs

Biochemical Society transactions, 1995

The Catalytic Mechanism of Hamster Dihydroorotase

Advances in Experimental Medicine and Biology, 1994

Mitochondrial Deletions in Normal and Degenerating Rat Retina

Advances in Experimental Medicine and Biology, 2003

Photoreceptor death by apoptosis is the central pathology of most forms of retinal degeneration. ... more Photoreceptor death by apoptosis is the central pathology of most forms of retinal degeneration. Mitochondria play key roles in apoptosis, releasing both signals which induce apoptosis (cytochrome c, caspases) and signals which inhibit apoptosis (Bcl-2). Because mitochondria are the site of oxidative metabolism they are also a major site of formation of the toxic oxygen intermediates which form as oxygen is recruited into the oxidative phosphorylation pathway. Previous studies have shown that deletions in mtDNA accumulate in postmitotic tissues (central nervous, muscle) and that their accumulation is accelerated by oxidative stress (such as hypoxia) (Takeda et al. 1996; Lee et al. 1994; Merril et al. 1996; Englander et al. 1999). It seems possible therefore that mitochondria are a site at which oxidative stress induces the death of retinal neurones. This study investigates the accumulation of mtDNA deletions in the rat retina, in both normal (non-degenerative) and degenerative strains. Deletions were undetectable in Sprague-Dawley albino rats (24 months) but were detected at 15 months in the rapidly degenerating RCS strain. The appearance of deletions in the RCS strain, in which retinal oxygen tension is known to rise as the degeneration progresses, gives support to the ideas that oxidative stress is a factor in mtDNA deletions, and in the progress of the late stages of the degeneration.

Publications Central Nervous System Stability and Degeneration

Cloning, expression, and spectroscopic studies of the Jun leucine zipper domain

European Journal of Biochemistry, 1994

Association of the human c-Jun and c-Fos proteins depends upon interactions involving their leuci... more Association of the human c-Jun and c-Fos proteins depends upon interactions involving their leucine zipper domains. We are interested in elucidating the tertiary structure of the Jun and Fos leucine zipper domains with a view to understanding the precise intermolecular interactions which govern the affinity and specificity of interaction in these proteins, which have the unusual capacity to form either homodimeric or heterodimeric zipper pairs. With this goal in mind, we have developed a bacterial expression system for the efficient production of both unlabelled and isotopically labelled c-Jun leucine zipper domain. A synthetic junLZ gene was created by annealing, ligation, and polymerase-chain-reaction amplification of overlapping synthetic oligonucleotides which comprised 132 bp of coding sequence encompassing residues Arg276-Asn314 of cJun plus a total of five engineered non-native residues at the N-and C-termini. The junLZ gene was cloned into the pGEX-2T vector from which recombinant c-Jun leucine zipper domain (rJunLZ; 46 residues, 5.1 kDa) was overexpressed (~1 5 % total cell protein) in Escherichia coli as a fusion protein of 31.4 kDa, consisting of rJunLZ fused to the carbuxy-terminal portion of Schistosorna japonicum glutathione Stransferase. Two markedly different expression strategies have been devised which allow purification of rJunLZ from the soluble or inclusion-body fraction of induced cells. We have used these strategies to produce unlabelled and uniformly 15N-labelled rJunLZ for NMR studies which, in combination with circular dichroic measurements, reveal that rJunLZ most likely forms a symmetric coiled-coil of parallel a-helices. We also present I5N-NMR chemical shift assignments for the backbone and sidechain amide nitrogens of rJunLZ, which should assist in determination of a high-resolution structure of the homodimeric Jun leucine zipper using heteronuclear three-dimensional NMR spectroscopy.

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Multiple oligomeric forms ofEscherichia coli DnaB helicase revealed by electrospray ionisation mass spectrometry

Rapid Communications in Mass Spectrometry, 2007

The Escherichia coli DnaB protein (DnaB(6)) is the hexameric helicase that unwinds genomic DNA so... more The Escherichia coli DnaB protein (DnaB(6)) is the hexameric helicase that unwinds genomic DNA so it can be copied by the DNA replication machinery. Loading of the helicase onto DNA requires interactions of DnaB(6) with six molecules of its loading partner protein, DnaC. Nano-electrospray ionisation mass spectrometry (nanoESI-MS) of mutant proteins was used to examine the roles of the residues Phe102 (F102) and Asp82 (D82) in the N-terminal domain of DnaB in the assembly of the hexamer. When the proteins were prepared in 1 M ammonium acetate containing magnesium and adenosine triphosphate (ATP) at pH 7.6, both hexameric and heptameric forms of wild-type and F102W, F102E and D82N mutant DnaBs were observed in mass spectra. The spectra of the D82N mutant also showed substantial amounts of a decameric species and small amounts of a dodecamer. In contrast, the F102H DnaB mutant was incapable of forming oligomers of order higher than the hexamer. Thus, although Phe102 is not the only determinant of hexamer assembly, this residue has a role in oligomerisation. NanoESI mass spectra were obtained of mixtures of DnaB(6) with DnaC. The DnaB(6)(DnaC)(6) complex (calculated M(r) 481 164) was observed only when the two proteins were present in equimolar amounts. The data are consistent with cooperative assembly of the complex. ESI mass spectra of mixtures containing DnaC and ATP showed that DnaC slowly hydrolysed ATP to ADP as indicated by ions corresponding to DnaC/ATP and DnaC/ADP complexes. These experiments show that E. coli DnaB can form a heptameric complex and that nanoESI-MS can be used to probe assembly of large (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;0.5 MDa) macromolecular complexes.

Comparison of gene expression between left atria and left ventricles from non-diseased humans

PROTEOMICS, 2004

We examine the reliability and accuracy of gene array technology in analyzing differences in gene... more We examine the reliability and accuracy of gene array technology in analyzing differences in gene expression between human non-diseased left atrium and left ventricle. We have used cDNA gene arrays and validated those data by carefully designed quantitative real-time polymerase chain reaction (PCR). We have identified pitfalls using cDNA gene array technology based on comparisons with other gene array studies and with changes reported for the levels of expression of the genes corresponding to these cDNAs. The high error rate reported here underscores the cautionary comments reported by others in this field.

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Expression of catalytically active hamster dihydroorotase domain in Escherichia coli : purification and characterization

"Protein Engineering, Design and Selection", 1993

Dihydroorotase is the central domain of trifunctional L-dihydroorotate synthetase which also cont... more Dihydroorotase is the central domain of trifunctional L-dihydroorotate synthetase which also contains carbamyl phosphate synthetase at the N-terminus and aspartate transcarbamylase at the C-terminus. The cDNA, corresponding to the active dihydroorotase domain as isolated after digestion of dihydroorotate synthetase with elastase, has been sub-cloned into the expression vector pCW12 which was then used to transform Escherichia coli S phi 1263 pyrC- lacking dihydroorotase activity. However, induction of this recombinant strain with IPTG produced large amounts of the dihydroorotase domain which were completely inactive. A number of cDNAs were expressed which were longer on the C-terminal side; all cDNAs expressed active dihydroorotase domain down to a minimal extension of 12 amino acids (-Val-Pro-Pro-Gly-Tyr-Gly-Gln-Asp-Val-Arg-Lys-Trp) into the bridge region between the dihydroorotase and aspartate transcarbamylase domains. Part of this dodecapeptide may form an amphipathic helix which in some way constrains the isolated, recombinant dihydroorotase domain to an active conformation. The recombinant hamster dihydroorotase purified from a cell-free extract of E. coli in four steps has a turnover number of 297 mol/min/(mol domain) for the conversion of L-dihydroorotate back to N-carbamyl-L-aspartate with Ks = 8.7 +/- 1.5 microM for L-dihydroorotate, a subunit molecular weight of 39,008 determined from the sequence and 37,900 +/- 400 when subjected to SDS-PAGE, and an isoelectric point of 5.7. Ultracentrifugal analysis of the recombinant domain showed a single species of s20,w = 4.1 S and a single molecular species of M(r) = 76,000 corresponding to a dimer.

Sequence limits of DNA strands in the arrested replication fork at the Bacillus subtilis chromosome terminus

Nucleic Acids Research, 1989

The DNA sequence limits of the leading and lagging strands in the arrested clockwise replication ... more The DNA sequence limits of the leading and lagging strands in the arrested clockwise replication fork at the terminus of the Bacillus subtilis chromosome have been investigated. On the basis of hybridization to synthetic oligonucleotides corresponding to known positions in the terminus region sequence it has been shown that neither the leading nor lagging strands, as they approach terC, traverse the distal inverted repeat, IRI. But a small fraction of the leading strands pass through the proximal inverted repeat, IRII. This is consistent with IRI being the functional inverted repeat in arresting the clockwise fork. But most of the forks appear to stop at least 100 nucleotides short of IRI, and at various positions extending over a distance of at least 100 nucleotides.

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The unstructured C-terminus of the subunit of Escherichia coli DNA polymerase III holoenzyme is the site of interaction with the subunit

Nucleic Acids Research, 2007

The q subunit of Escherichia coli DNA polymerase III holoenzyme interacts with the a subunit thro... more The q subunit of Escherichia coli DNA polymerase III holoenzyme interacts with the a subunit through its C-terminal Domain V, q C 16. We show that the extreme C-terminal region of q C 16 constitutes the site of interaction with a. The q C 16 domain, but not a derivative of it with a C-terminal deletion of seven residues (q C 16Á7), forms an isolable complex with a. Surface plasmon resonance measurements were used to determine the dissociation constant (K D ) of the aÀq C 16 complex to be $260 pM. Competition with immobilized q C 16 by q C 16 derivatives for binding to a gave values of K D of 7 mM for the aÀq C 16Á7 complex. Low-level expression of the genes encoding q C 16 and q C 16i7, but not q C 16Á11, is lethal to E. coli. Suppression of this lethal phenotype enabled selection of mutations in the 3 0 end of the q C 16 gene, that led to defects in a binding. The data suggest that the unstructured C-terminus of q becomes folded into a helix-loop-helix in its complex with a. An N-terminally extended construct, q C 24, was found to bind DNA in a salt-sensitive manner while no binding was observed for q C 16, suggesting that the processivity switch of the replisome functionally involves Domain IV of q.

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Relationship between mitochondrial DNA damage and photoreceptor death in developing and adult retina, assessed in normal and degenerative rat strains

Mitochondrion, 2007

Photoreceptor death by apoptosis is the central pathology of most retinal degenerations, and may ... more Photoreceptor death by apoptosis is the central pathology of most retinal degenerations, and may be induced by oxidative stress. Work in other tissues has shown that mitochondria release apoptosis-inducing signals in response to oxidative stress, and that the frequency of deletions from the mitochondrial genome is a useful index of oxidative stress. We have therefore investigated whether retinal mitochondria show increased frequencies of mtDNA deletions during periods of photoreceptor death, in normal and degenerative rat strains. Methods. Real-time PCR was used to quantify a 4,834bp deletion from the mitochondrial genome in retinal DNA from developmental series of 3 strains of rat: the non-degenerative Sprague Dawley (SD); the RCS, in which a naturally occurring mutation induces abnormal hypoxia of outer retina; and the P23H (line 3) transgenic, in which a rhodopsin mutant transgene causes photoreceptor degeneration in situ. Results: In the SD and RCS strains, mtDNA deletion frequency increased and fell during neonatal life, correlating with rates of photoreceptor death during the critical period of photoreceptor development, and into adulthood. Further, rates of mtDNA deletions were ~100 x higher in the RCS strain in neonatal life (the critical period), matching the greater rates of photoreceptor death in this strain. In the P23H-3 strain, by contrast, mtDNA deletion frequency did not increase with photoreceptor death during the critical period, but did increase slowly during adult life. Conclusions: The present data

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The 2.7 Å Crystal Structure of the Autoinhibited Human c-Fms Kinase Domain

Journal of Molecular Biology, 2007

c-Fms, a member of the Platelet-derived Growth Factor (PDGF) receptor family of receptor tyrosine... more c-Fms, a member of the Platelet-derived Growth Factor (PDGF) receptor family of receptor tyrosine kinases (RTKs), is the receptor for macrophage colony stimulating factor (CSF-1) that regulates proliferation, differentiation and survival of cells of the mononuclear phagocyte lineage. Abnormal expression of c-fms proto-oncogene is associated with a significant number of human pathologies, including a variety of cancers and rheumatoid arthritis. Accordingly, c-Fms represents an attractive therapeutic target. To further understand the regulation of c-Fms, we determined the 2.7 Å resolution crystal structure of the cytosolic domain of c-Fms that comprised the kinase domain and the juxtamembrane domain. The structure reveals the crucial inhibitory role of the juxtamembrane domain (JM) that binds to a hydrophobic site immediately adjacent to the ATP binding pocket. This interaction prevents the activation loop from adopting an active conformation thereby locking the c-Fms kinase into an autoinhibited state. As observed for other members of the PDGF receptor family, namely c-Kit and Flt3, three JM-derived tyrosine residues primarily drive the mechanism for autoinhibition in c-Fms, therefore defining a common autoinhibitory mechanism within this family. Moreover the structure provides an understanding of c-Fms inhibition by Gleevec as well as providing a platform for the development of more selective inhibitors that target the inactive conformation of c-Fms kinase.

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Dissecting Specificity in the Janus Kinases: The Structures of JAK-Specific Inhibitors Complexed to the JAK1 and JAK2 Protein Tyrosine Kinase Domains

Journal of Molecular Biology, 2009

The Janus kinases (JAKs) are a pivotal family of protein tyrosine kinases (PTKs) that play promin... more The Janus kinases (JAKs) are a pivotal family of protein tyrosine kinases (PTKs) that play prominent roles in numerous cytokine signaling pathways, with aberrant JAK activity associated with a variety of hematopoietic malignancies, cardiovascular diseases and immune-related disorders. Whereas the structures of the JAK2 and JAK3 PTK domains have been determined, the structure of the JAK1 PTK domain is unknown. Here, we report the high-resolution crystal structures of the "active form" of the JAK1 PTK domain in complex with two JAK inhibitors, a tetracyclic pyridone 2-tbutyl-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-one (CMP6) and (3R,4R)-3-[4-methyl-3-[N-methyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]piperidin-1-yl]-3-oxopropionitrile (CP-690,550), and compare them with the corresponding JAK2 PTK inhibitor complexes. Both inhibitors bound in a similar manner to JAK1, namely buried deep within a constricted ATP-binding site, thereby providing a basis for the potent inhibition of JAK1. As expected, the mode of inhibitor binding in JAK1 was very similar to that observed in JAK2, highlighting the challenges in developing JAK-specific inhibitors that target the ATP-binding site. Nevertheless, differences surrounding the JAK1 and JAK2 ATP-binding sites were apparent, thereby providing a platform for the rational design of JAK2-and JAK1-specific inhibitors.

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Stabilization of Native Protein Fold by Intein-Mediated Covalent Cyclization

Journal of Molecular Biology, 2005

A mutant version of the N-terminal domain of Escherichia coli DnaB helicase was used as a model s... more A mutant version of the N-terminal domain of Escherichia coli DnaB helicase was used as a model system to assess the stabilization against unfolding gained by covalent cyclization. Cyclization was achieved in vivo by formation of an amide bond between the N and C termini with the help of a split mini-intein. Linear and circular proteins were constructed to be identical in amino acid sequence. Mutagenesis of Phe102 to Glu rendered the protein monomeric even at high concentration. A difference in free energy of unfolding, DDG, between circular and linear protein of 2.3(G0.5) kcal mol K1 was measured at 10 8C by circular dichroism. A theoretical estimate of the difference in conformational entropy of linear and circular random chains in a three-dimensional cubic lattice model predicted DDGZ2.3 kcal mol K1 , suggesting that stabilization by protein cyclization is driven by the reduced conformational entropy of the unfolded state. Amide-proton exchange rates measured by NMR spectroscopy and mass spectrometry showed a uniform, approximately tenfold decrease of the exchange rates of the most slowly exchanging amide protons, demonstrating that cyclization globally decreases the unfolding rate of the protein. The amide proton exchange was found to follow EX1 kinetics at near-neutral pH, in agreement with an unusually slow refolding rate of less than 4 min K1 measured by stopped-flow circular dichroism. The linear and circular proteins differed more in their unfolding than in their folding rates. Global unfolding of the N-terminal domain of E. coli DnaB is thus promoted strongly by spatial separation of the N and C termini, whereas their proximity is much less important for folding.

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Crystal Structures of the Lyn Protein Tyrosine Kinase Domain in Its Apo- and Inhibitor-bound State

Journal of Biological Chemistry, 2009

The Src-family protein-tyrosine kinase (PTK) Lyn is the most important Src-family kinase in B cel... more The Src-family protein-tyrosine kinase (PTK) Lyn is the most important Src-family kinase in B cells, having both inhibitory and stimulatory activity that is dependent on the receptor, ligand, and developmental context of the B cell. An important role for Lyn has been reported in acute myeloid leukemia and chronic myeloid leukemia, as well as certain solid tumors. Although several Src-family inhibitors are available, the development of Lyn-specific inhibitors, or inhibitors with reduced off-target activity to Lyn, has been hampered by the lack of structural data on the Lyn kinase. Here we report the crystal structure of the non-liganded form of Lyn kinase domain, as well as in complex with three different inhibitors: the ATP analogue AMP-PNP; the pan Src kinase inhibitor PP2; and the BCR-Abl/ Src-family inhibitor Dasatinib. The Lyn kinase domain was determined in its "active" conformation, but in the unphosphorylated state. All three inhibitors are bound at the ATP-binding site, with PP2 and Dasatinib extending into a hydrophobic pocket deep in the substrate cleft, thereby providing a basis for the Src-specific inhibition. Analysis of sequence and structural differences around the active site region of the Src-family PTKs were evident. Accordingly, our data provide valuable information for the further development of therapeutics targeting Lyn and the important Src-family of kinases.

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In Vivo Protein Cyclization Promoted by a Circularly Permuted Synechocystis sp. PCC6803 DnaB Mini-intein

Journal of Biological Chemistry, 2002

A synthetic Synechocystis sp. PCC6803 DnaB split mini-intein gene was constructed for the in vivo... more A synthetic Synechocystis sp. PCC6803 DnaB split mini-intein gene was constructed for the in vivo cyclization of recombinant proteins expressed in Escherichia coli. The system was used to cyclize the N-terminal domain of E. coli DnaB, the structure of which had been determined previously by NMR spectroscopy. Cyclization was found to proceed efficiently, with little accumulation of precursor, and the product was purified in high yield. The solution structure of cyclic DnaB-N is not significantly different from that of linear DnaB-N and it unfolds reversibly at temperatures ~14 ˚C higher. Improved hydrogen bonding was observed in the first and last helices, and the length of the last helix was increased, while the 9 amino acid linker used to join the the N-and C-termini was found to be highly mobile. The measured thermodynamic stabilization of the structure (∆∆G ≈ 2 kcal/mol) agrees well with the value estimated from the reduced conformational entropy in the unfolded form. Simple polymer theory can be used to predict likely free energy changes resulting from protein cyclization and how the stabilization depends on the size of the protein and the length of the linker used to connect the termini. INTRODUCTION Although proteins are normally linear polymers of amino acid residues, there are now several examples of ribosomally-synthesized polypeptides that are cyclized post-translationally by formation of a normal peptide bond between amino and carboxyl ends via mechanisms that are not well understood (for examples, see Refs. 1-3). These proteins characteristically have unusually high thermal stabilities. Moreover, it has been well established that the deliberate circularization of normally-linear proteins by a covalent peptide link between the N-and Cterminal ends provides a route for stabilization of the native structure (4-6). Thermal stability is an important feature, for example, for use of enzymes as catalysts in industrial applications, and by guest on October 26, 2016 http://www.jbc.org/ Downloaded from

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EX1 hydrogen–deuterium exchange in an all-helical protein and its cyclized derivative at neutral pH

International Journal of Mass Spectrometry, 2011

The all-helical protein, DnaBN, exhibited EX1 type hydrogen exchange at pH 7.2. Approximately 45 ... more The all-helical protein, DnaBN, exhibited EX1 type hydrogen exchange at pH 7.2. Approximately 45 protons were exchanged relatively rapidly, while an additional ∼50 protons exchanged more slowly. The rates of exchange for these slowly exchanging protons were the same, demonstrating that the slowest exchange events represent global unfolding. EX1 behavior is uncommon for native proteins. The protein was cyclized by joining the N-and C-termini through peptide linkers that were three, four, five or nine amino acids long. The corresponding "linear" proteins were extended by addition of the same amino acids to give proteins of identical amino acid composition as their cyclized versions but differing by the mass of a water molecule. All of the proteins unfolded approximately five times faster in 10 mM compared with 100 mM ammonium acetate. In all cases, the cyclized proteins showed slower rates of amide proton exchange related to global unfolding than their linear counterparts by a factor of approximately 7-to 12-fold. Interestingly, the rate of exchange for the slowly exchanging protons decreased for both the linear and cyclized proteins as linker length increased, and this correlated with predictions that the C-terminal helix of the protein would be extended by addition of these extra amino acids. This indicates that lengthening of this helix leads to a modest increase in stability of DnaBN.

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Location of the dihydroorotase domain within trifunctional hamster dihydroorotate synthetase

Gene, 1990

Mammalian dihydroorotase (DHOase, EC 3.5.2.3) is part of a trifunctional protein, dihydroorotate ... more Mammalian dihydroorotase (DHOase, EC 3.5.2.3) is part of a trifunctional protein, dihydroorotate synthetase which catalyzes the first three reactions of de novo pyrimidine biosynthesis. We have subcloned a portion of the eDNA from the plasmid pCAD 142 and obtained a nucleotide sequence which extends 2.1 kb in the 5' direction from the sequence encoding the aspartate transcarbamoylase (ATCase) domain at the Y-end of the cDNA. The DHOase and ATCase domains have been purified from all elastase digest of the trifunctional protein and subjected to amino acid (aa) sequencing from their N termini. The sequence of the N-terminal 24 aa of the DHOase domain has been obtained and aligned with the cDNA sequence. The C-terminal residues of the DHOase domain have been identified as Lea followed by Val which, when taken with partial sequences of the CNBr fragments of this domain, defines the coding sequence of the active, globular,. IOase domain released by proteolysis. Prediction of protein secondary structure from the deduced aa sequence showed that the DHOase domain (Mr 37 751) is separated from the C-terminal ATCase domain (M, 34 323) by a bridging sequence (Mr ! 2 532) consisting of multiple p-turns.

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