94 research outputs found

    Functional Consequences of Protein Dynamics

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    This thesis explores the possible uses of dynamical fluctuations in protein structure for ligand binding and catalysis. Particular emphasis is placed on the dynamic interpretation of allosteric interactions and a statistical mechanical model of dynamic allostery is presented. This model shows that changes in either low frequency collective motions or random uncorrelated atomic fluctuations of the protein induced by the binding of a ligand can alter the binding properties of other remote ligand binding sites giving rise to allostery. Increases in the frequency of vibrational modes and reductions in uncorrelated motions gives rise to positive cooperativity and is equivalent to a stiffening of the protein structure. Small changes in the dynamics can be treated classically with many changes being required to give observed cooperative free energies. Large shifts in low frequency vibrational modes give much larger contributions to the cooperativity and the use of quantum mechanics is required. The dynamic allostery model predicts that cooperativity arising from dynamic changes is predominantly entropic in origin and complements the more conventional models of allostery which invoke changes in the static conformation of the protein involving domain movement, bond rearrangements and electrostatic effects with consequent effects on the enthalpy. The predictions of this model are tested using laser Raman spectroscopy of solid samples to study low frequency modes in proteins and an allosteric model compound. The small organic molecule which displays positive cooperativity between its two binding sites, shows sizeable shifts to higher frequencies in the low frequency spectrum in agreement with the model. The vibrational shifts seen require only the classical version of the model which when combined with changes in the uncorrelated motions of the atoms in the molecule can account for the observed cooperative free energy. The cooperativity is solely entropic in origin in agreement with published results. High frequency spectra of the molecule in various states of ligation are presented and analysed in terms of localised vibrations of atoms and groups of atoms. The low frequency Raman spectra of lysozyme and its complex with the small inhibitor tri-N-acetyl glucosamine, and of trypsin and its complex with pancreatic trypsin inhibitor all displayed a broad band at 20cm. This band is a superposition of a large number of low frequency modes of the protein and the expected shift in frequency of some modes on inhibitor binding is not visible within such a broad band. The allosteric enzyme glyceraldehyde 3-phosphate dehydrogenase and its complex with the cofactor NAD also shows no changes in its low frequency spectrum. These results and their implications are discussed. High frequency Raman spectra of these enzymes are also presented and analysed

    DNA translocation by type III restriction enzymes: a comparison of current models of their operation derived from ensemble and single-molecule measurements

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    Much insight into the interactions of DNA and enzymes has been obtained using a number of single-molecule techniques. However, recent results generated using two of these techniques—atomic force microscopy (AFM) and magnetic tweezers (MT)—have produced apparently contradictory results when applied to the action of the ATP-dependent type III restriction endonucleases on DNA. The AFM images show extensive looping of the DNA brought about by the existence of multiple DNA binding sites on each enzyme and enzyme dimerisation. The MT experiments show no evidence for looping being a requirement for DNA cleavage, but instead support a diffusive sliding of the enzyme on the DNA until an enzyme–enzyme collision occurs, leading to cleavage. Not only do these two methods appear to disagree, but also the models derived from them have difficulty explaining some ensemble biochemical results on DNA cleavage. In this ‘Survey and Summary’, we describe several different models put forward for the action of type III restriction enzymes and their inadequacies. We also attempt to reconcile the different models and indicate areas for further experimentation to elucidate the mechanism of these enzymes

    Generation of tools for expression and purification of the phage-encoded Type I restriction enzyme inhibitor, Ocr

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    DNA manipulation is an essential tool in molecular microbiology research that is dependent on the ability of bacteria to take up and preserve foreign DNA by horizontal gene transfer. This process can be significantly impaired by the activity of bacterial restriction modification systems; bacterial operons comprising paired enzymatic activities that protectively methylate host DNA, while cleaving incoming unmodified foreign DNA. Ocr is a phage-encoded protein that inhibits Type I restriction modification systems, the addition of which significantly improves bacterial transformation efficiency. We recently established an improved and highly efficient transformation protocol for the important human pathogen group A Streptococcus using commercially available recombinant Ocr protein, manufacture of which has since been discontinued. In order to ensure the continued availability of Ocr protein within the research community, we have generated tools and methods for in-house Ocr production and validated the activity of the purified recombinant protein.</p

    A unique homologue of the eukaryotic protein-modifier ubiquitin present in the bacterium Bacteroides fragilis, a predominant resident of the human gastrointestinal tract

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    In the complete genome sequences of Bacteroides fragilis NCTC9343 and 638R, we have discovered a gene, ubb, the product of which has 63 % identity to human ubiquitin and cross-reacts with antibodies raised against bovine ubiquitin. The sequence of ubb is closest in identity (76 %) to the ubiquitin gene from a migratory grasshopper entomopoxvirus, suggesting acquisition by inter-kingdom horizontal gene transfer. We have screened clinical isolates of B. fragilis from diverse geographical regions and found that ubb is present in some, but not all, strains. The gene is transcribed and the mRNA is translated in B. fragilis, but deletion of ubb did not have a detrimental effect on growth. BfUbb has a predicted signal sequence; both full-length and processed forms were detected in whole-cell extracts, while the processed form was found in concentrated culture supernatants. Purified recombinant BfUbb inhibited in vitro ubiquitination and was able to covalently bind the human E1 activating enzyme, suggesting it could act as a suicide substrate in vivo. B. fragilis is one of the predominant members of the normal human gastrointestinal microbiota with estimates of up to >1011 cells per g faeces by culture. These data indicate that the gastro-intestinal tract of some individuals could contain a significant amount of aberrant ubiquitin with the potential to inappropriately activate the host immune system and/or interfere with eukaryotic ubiquitin activity. This discovery could have profound implications in relation to our understanding of human diseases such as inflammatory bowel and autoimmune diseases

    DNA bending by M.EcoKI methyltransferase is coupled to nucleotide flipping

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    The maintenance methyltransferase M.EcoKI recognizes the bipartite DNA sequence 5′-AACNNNNNNGTGC-3′, where N is any nucleotide. M.EcoKI preferentially methylates a sequence already containing a methylated adenine at or complementary to the underlined bases in the sequence. We find that the introduction of a single-stranded gap in the middle of the non-specific spacer, of up to 4 nt in length, does not reduce the binding affinity of M.EcoKI despite the removal of non-sequence-specific contacts between the protein and the DNA phosphate backbone. Surprisingly, binding affinity is enhanced in a manner predicted by simple polymer models of DNA flexibility. However, the activity of the enzyme declines to zero once the single-stranded region reaches 4 nt in length. This indicates that the recognition of methylation of the DNA is communicated between the two methylation targets not only through the protein structure but also through the DNA structure. Furthermore, methylation recognition requires base flipping in which the bases targeted for methylation are swung out of the DNA helix into the enzyme. By using 2-aminopurine fluorescence as the base flipping probe we find that, although flipping occurs for the intact duplex, no flipping is observed upon introduction of a gap. Our data and polymer model indicate that M.EcoKI bends the non-specific spacer and that the energy stored in a double-stranded bend is utilized to force or flip out the bases. This energy is not stored in gapped duplexes. In this way, M.EcoKI can determine the methylation status of two adenine bases separated by a considerable distance in double-stranded DNA and select the required enzymatic response

    Removal of a frameshift between the hsdM and hsdS genes of the EcoKI Type IA DNA restriction and modification system produces a new type of system and links the different families of Type I systems

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    The EcoKI DNA methyltransferase is a trimeric protein comprised of two modification subunits (M) and one sequence specificity subunit (S). This enzyme forms the core of the EcoKI restriction/modification (RM) enzyme. The 3′ end of the gene encoding the M subunit overlaps by 1 nt the start of the gene for the S subunit. Translation from the two different open reading frames is translationally coupled. Mutagenesis to remove the frameshift and fuse the two subunits together produces a functional RM enzyme in vivo with the same properties as the natural EcoKI system. The fusion protein can be purified and forms an active restriction enzyme upon addition of restriction subunits and of additional M subunit. The Type I RM systems are grouped into families, IA to IE, defined by complementation, hybridization and sequence similarity. The fusion protein forms an evolutionary intermediate form lying between the Type IA family of RM enzymes and the Type IB family of RM enzymes which have the frameshift located at a different part of the gene sequence

    Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI–DNA complexes

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    DNA base flipping is an important mechanism in molecular enzymology, but its study is limited by the lack of an accessible and reliable diagnostic technique. A series of crystalline complexes of a DNA methyltransferase, M.HhaI, and its cognate DNA, in which a fluorescent nucleobase analogue, 2-aminopurine (AP), occupies defined positions with respect the target flipped base, have been prepared and their structures determined at higher than 2 Å resolution. From time-resolved fluorescence measurements of these single crystals, we have established that the fluorescence decay function of AP shows a pronounced, characteristic response to base flipping: the loss of the very short (∼100 ps) decay component and the large increase in the amplitude of the long (∼10 ns) component. When AP is positioned at sites other than the target site, this response is not seen. Most significantly, we have shown that the same clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP fluorescence decay function reveals conformational heterogeneity in the DNA–enzyme complexes that cannot be discerned from the present X-ray structures

    Atomic force microscopy of the EcoKI Type I DNA restriction enzyme bound to DNA shows enzyme dimerization and DNA looping

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    Atomic force microscopy (AFM) allows the study of single protein–DNA interactions such as those observed with the Type I Restriction–Modification systems. The mechanisms employed by these systems are complicated and understanding them has proved problematic. It has been known for years that these enzymes translocate DNA during the restriction reaction, but more recent AFM work suggested that the archetypal EcoKI protein went through an additional dimerization stage before the onset of translocation. The results presented here extend earlier findings confirming the dimerization. Dimerization is particularly common if the DNA molecule contains two EcoKI recognition sites. DNA loops with dimers at their apex form if the DNA is sufficiently long, and also form in the presence of ATPγS, a non-hydrolysable analogue of the ATP required for translocation, indicating that the looping is on the reaction pathway of the enzyme. Visualization of specific DNA loops in the protein–DNA constructs was achieved by improved sample preparation and analysis techniques. The reported dimerization and looping mechanism is unlikely to be exclusive to EcoKI, and offers greater insight into the detailed functioning of this and other higher order assemblies of proteins operating by bringing distant sites on DNA into close proximity via DNA looping

    The structure of M.EcoKI Type I DNA methyltransferase with a DNA mimic antirestriction protein

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    Type-I DNA restriction–modification (R/M) systems are important agents in limiting the transmission of mobile genetic elements responsible for spreading bacterial resistance to antibiotics. EcoKI, a Type I R/M enzyme from Escherichia coli, acts by methylation- and sequence-specific recognition, leading to either methylation of DNA or translocation and cutting at a random site, often hundreds of base pairs away. Consisting of one specificity subunit, two modification subunits, and two DNA translocase/endonuclease subunits, EcoKI is inhibited by the T7 phage antirestriction protein ocr, a DNA mimic. We present a 3D density map generated by negative-stain electron microscopy and single particle analysis of the central core of the restriction complex, the M.EcoKI M2S1 methyltransferase, bound to ocr. We also present complete atomic models of M.EcoKI in complex with ocr and its cognate DNA giving a clear picture of the overall clamp-like operation of the enzyme. The model is consistent with a large body of experimental data on EcoKI published over 40 years
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