Thesis (Ph. D.)--Massachusetts Institute of Technology and Woods Hole Oceanographic Institution, 1981. / Vita. Includes bibliographical references (p. 239-262).

Thesis (Ph. D.)--University of Missouri-Columbia, 2005. / Title from title screen of research.pdf file (viewed on December 22, 2006). The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. "May 2005" Vita. Includes bibliographical references.

No description available.

B. anthracis alters its virulence gene expression profile in response to a number of environmental signals, including levels of bicarbonate and CO2. Virulence plasmid pXO1 is important to Bacillus anthracis pathogenicity as it carries the genes encoding the anthrax toxin and virulence regulatory factors. Induction of toxin and other virulence genes requires the pXO1-encoded AtxA regulatory protein. The cytochrome c maturation system influences the expression of virulence factors in Bacillus anthracis. B. anthracis carries two copies of the ccdA gene, encoding predicted thiol-disulfide oxidoreductases that contribute to cytochrome c maturation. Loss of both ccdA genes results in a reduction of cytochrome c production, an increase in virulence factor expression, and a reduction in sporulation efficiency. pXO1 also carries a gene encoding an Hfq-like protein, pXO1-137. Loss of pXO1-137 results in significant growth defects and reductions in toxin gene expression only when grown under toxin inducing conditions. Similarly, loss of a small RNA on pXO1, sRNA-1, results in similar growth defects and reductions in toxin gene production. Both increased and decreased expression of pXO1-137 and sRNA-1 result in growth defects suggesting narrow functional set points for Hfq and sRNA levels.

The crystal structure of yeast (Saccharomyces cerevisiae) iso-1- cytochrome c has been determined through molecular replacement techniques, and refined against X-ray diffraction data in the resolution range 6.0-1.23 Å to a crystallographic R-factor of 0.192. The yeast iso-1-cytochrome c molecule has the typical cytochrome c fold, with the polypeptide chain organized into five α-helices and a series of loops which serve to enclose almost completely the heme prosthetic group within a hydrophobic pocket Comparison of the structures of yeast iso-1-, tuna and rice cytochromes c shows that the polypeptide backbone fold, intramolecular hydrogen bonding, conformation of side chains and particularly packing within the heme crevice of protein groups against the heme moiety are very similar in the three proteins. Significant structural differences among the three cytochromes c can be explained by differences in amino acid sequence. X-ray crystallographic techniques have also been used to study the effect of single-site amino acid substitutions at Phe82 and at Arg38 in iso-1-cytochrome c. The structures of the various variant iso-1-cytochromes c have been determined at nominal resolutions in the range 2.8 to 1.76 Å. Conspicuous structural perturbations in the neighborhood of the substituted side chain are evident in all of the variant proteins. In wild-type iso-1-cytochrome c, the phenyl ring of Phe82 is positioned adjacent and approximately parallel to the heme group, and occupies a non-polar cavity within the heme crevice. In the Ser82 variant, a channel extending from the surface of the molecule down into the heme crevice is created. In the Gly82 variant, the polypeptide backbone has refolded into the space formerly occupied by the phenyl ring of Phe82. Steric conflicts prevent both the phenolic ring of Tyr82 and the side chain of Ile82 from being completely accommodated within the pocket normally occupied by a phenyl ring. Substitution of alanine at position 38 causes a slight reorganization of the hydrogen bonding network in which Arg38 normally participates, and also exposes to external solvent a normally buried propionic acid group of the heme. The altered functional properties of the position 82 variant proteins have been interpreted with respect to the observed structural perturbations. The drop in reduction potential, most notably for the Ser82 and Gly82 variants, can be explained by the elevated heme environment polarity arising from the increased access of solvent or polar protein groups to the heme pocket The reduced stability of the heme crevice, as indicated by lowered pKa's for alkaline isomerization, is likely due to the disruption of stabilizing packing forces formed by the Phe82 phenyl ring within its hydrophobic cavity. The lowered activity, in comparison to the wild-type protein and the Tyr82 variant, for electron transfer with Zn+-cytochrome c peroxidase is attributed to the loss of an aromatic group positioned adjacent to the heme group. The altered surface topography of the variant proteins (particularly the Gly82, Tyr82 and Ile82 variants) may further hinder productive complex formation between cytochrome c and its redox partners. These results suggest that the invariant Phe82 contributes in at least three ways to the proper functioning of cytochrome c. It has an important structural role in maintaining the integrity of the heme crevice and in establishing the appropriate heme environment The phenyl ring of Phe82 may also be required for efficient movement of an electron to and from the heme of cytochrome c. Finally, Phe82 may have a role in forming intermolecular interactions with enzymic redox partners of cytochrome c. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

A soluble form of cytochrome b_5, with a 413/280 nm ratio of nearly unity, has been isolated and purified from porcine kidney using fractional precipitation, ion exchange resins, and gel filtration procedures. The cytochrome b_5 shows heat stability up to 50° and is stable in the pH range 6.0 to 8.5. The oxidized protein exhibits an absorption maximum at 413 nm, while the reduced form shows three peaks: 423, 526, 556 nm. Molecular weight determinations have given variable results, suggesting the possibility of a polymer composed of four and eight units of a 13,500 molecular weight protomer. This study has used no lipases, proteases, or detergents in the solubilization of the cytochrome, and the possibility exists that the cytochrome found represents newly formed cytochrome b_5 which is as yet unattached to the microsomes, and which reflects the form found in intact cells.

Interprotein electron transfer (ET) is crucial for natural energy conversion and a fundamental reaction in the pursuit of understanding the broader problem of proteinprotein interactions and reactivity. Simplifying the complicated nature of these natural systems has driven development of biomimetic approaches. Functionalized gold nanoparticles offer simplified, tunable surfaces that can serve as a proxy to study the reactivity and dynamics of proteins. Amino-acid functionalized gold nanoparticles (Au-TX) served as a complementary partner to cytochrome c (Cyt c) and catalyzed its ET reactivity without altering the native structure. Redox mediator and EPR experiments confirmed that the redox potential and coordination environment of the heme were unaltered. Varying the functionality of Au-TX under limiting redox reagent concentrations resulted in distinct ET reactivity. These conditions reflected the collision of a small redox reagent with the Cyt c/Au-TX adduct, introducing the possibility of Cyt c/Au-TX dynamics to modulate ET. Under high ionic strength conditions, the rate enhancement ranged from 0.0870 " 1011 for Cyt c/Au-TAsp to 1.95 " 1011 M-1 s-1 for Cyt c/Au-TPhe. Au-TAsp binds to a larger surface of the front face of Cyt c than Au-TPhe, likely reducing heme access and resulting in attenuated ET reactivity.Site-directed spin-labeling characterized the dynamic interactions and motion of Cyt c with Au-TX. Several mutants of Cyt c were utilized to extract information about the different dynamics of the Cyt c/Au-TPhe and Cyt c/Au-TAsp systems. Cyt c appeared to have a highly dynamic binding interaction with the surface of Au-TPhe while binding to Au-TAsp resulted in a more rigid interface, particularly at the heme crevice. The dynamic interaction of Cyt c/Au-TX at the heme crevice could promote a gated ET mechanism between Cyt c and its redox partner. Thus, the reduced reactivity of Cyt c/Au-TAsp is likely a result of both slower global dynamics and more rigid binding near the heme crevice.

No description available.

No description available.

Lau Pui Man Irene. / Thesis submitted in: July 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 162-172). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abbreviations --- p.ii / Abstract --- p.v / Abstract in Chinese --- p.vii / List of Figures --- p.ix / List of Tables --- p.xii / Contents --- p.xiii / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.1.1. --- Therapeutic uses of nucleic acids --- p.2 / Chapter 1.1.1.1 --- Antisense oligonucleotides --- p.2 / Chapter 1.1.1.2 --- RNA interference --- p.4 / Chapter 1.1.1.3 --- Aptamer --- p.6 / Chapter 1.2 --- Selection of Aptamer --- p.7 / Chapter 1.2.1 --- SELEX 'Systematic Evolution of Ligands by Exponential enrichment' --- p.7 / Chapter 1.2.1.1 --- In vitro selection --- p.8 / Chapter 1.2.1.2 --- Amplification --- p.8 / Chapter 1.2.1.3 --- Monoclonal Aptamer --- p.10 / Chapter 1.2.2 --- Photo-SELEX --- p.10 / Chapter 1.3 --- Examples of target molecules of aptamers --- p.12 / Chapter 1.4 --- Applications of aptamer --- p.14 / Chapter 1.4.1 --- Detection of Aptamer --- p.14 / Chapter 1.4.2 --- Examples of diagnostic use Contents --- p.15 / Chapter 1.4.2.1 --- Aptamer against theophylline with high specificity --- p.15 / Chapter 1.4.2.2 --- Aptamer chip --- p.16 / Chapter 1.4.3 --- Examples of therapeutic use --- p.18 / Chapter 1.4.3.1 --- Vascular endothelial growth factor (VEGF) --- p.18 / Chapter 1.4.3.2 --- Aptamer as a reversible antagonists of coagulation factor IXa is another example to show the potential case of aptamers as therapeutic agents --- p.20 / Chapter 1.4.4 --- Problem faced by aptamer --- p.21 / Chapter 1.4.4.1 --- Stability --- p.21 / Chapter 1.4.4.2 --- Clearance from blood --- p.22 / Chapter 1.5 --- Comparison between aptamer and antibody --- p.24 / Chapter 1.5.1 --- General comparison between aptamer and antibody --- p.24 / Chapter 1.5.1.1 --- Diversity --- p.24 / Chapter 1.5.2 --- Specificity --- p.26 / Chapter 1.5.3 --- Disadvantages of antibody --- p.26 / Chapter 1.5.4 --- Advantages of aptamer --- p.27 / Chapter 1.6 --- Project Objectives --- p.29 / Chapter Chapter 2. --- Materials and Methods --- p.31 / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Chemicals --- p.32 / Chapter 2.1.2 --- Buffers --- p.36 / Chapter 2.1.2.1 --- Buffers commonly used --- p.37 / Chapter 2.1.2.2 --- Reagents for molecular work --- p.37 / Chapter 2.1.3 --- Bacterial Culture --- p.38 / Chapter 2.1.4 --- Culture of cell --- p.38 / Chapter 2.1.4.1 --- "TNF-α Sensitive Cell Line, L929" --- p.38 / Chapter 2.1.4.2 --- Medium for cell culture --- p.38 / Chapter 2.1.5 --- Reagent for Western blotting --- p.39 / Chapter 2.1.5.1 --- Protein extraction --- p.39 / Chapter 2.1.5.2 --- SDS-PAGE --- p.40 / Chapter 2.1.5.3 --- Electro-blotting --- p.41 / Chapter 2.2 --- Methods --- p.42 / Chapter 2.2.1 --- Conjugation of protein to solid support --- p.42 / Chapter 2.2.1.1 --- Conjugation of protein on PVDF membrane --- p.42 / Chapter 2.2.4.2 --- Conjugation of protein on Sepharose --- p.42 / Chapter 2.2.4.3 --- Conjugation of protein on magnetic bead --- p.42 / Chapter 2.2.2 --- SELEX --- p.43 / Chapter 2.2.2.1 --- Selection --- p.43 / Chapter 2.2.2.2 --- Photo-selection --- p.44 / Chapter 2.2.2.3 --- PCR --- p.45 / Chapter 2.2.3 --- Separation of oligonucleotides --- p.46 / Chapter 2.2.3.1 --- Separate short length double-stranded oligonucleotides by using polyacrylamide gel --- p.46 / Chapter 2.2.3.2 --- Separate short length single-stranded oligonucleotides by using denaturing polyacrylamide gel --- p.47 / Chapter 2.2.3.3 --- Extract the DNA from polyacrylamide gel --- p.48 / Chapter 2.2.3.4 --- Estimate the amount of DNA in solution after extraction --- p.49 / Chapter 2.2.3.5 --- Agarose Gel Electrophoresis --- p.49 / Chapter 2.2.4 --- Cloning of selected polyclonal aptamer --- p.50 / Chapter 2.2.4.1 --- Restriction cutting --- p.50 / Chapter 2.2.4.2 --- Ligation --- p.50 / Chapter 2.2.4.3 --- Preparation of the competent cells --- p.50 / Chapter 2.2.4.4 --- Transformation of plasmid into competent cell --- p.51 / Chapter 2.2.4.5 --- Plasmid extraction from bacterial culture --- p.51 / Chapter 2.2.5 --- Cell culture --- p.52 / Chapter 2.2.5.1 --- Cell culture of L929 --- p.52 / Chapter 2.2.5.2 --- Preservation of cells --- p.52 / Chapter 2.2.5.3 --- Treatment with TNF-α --- p.53 / Chapter 2.2.5.4 --- Fixation of cells --- p.53 / Chapter 2.2.6 --- Western blotting analysis --- p.54 / Chapter 2.2.6.1 --- Preparation of proteins from cells --- p.54 / Chapter 2.2.6.2 --- SDS polyacrylamide gel electrophoresis (SDS-PAGE) --- p.54 / Chapter 2.2.6.3 --- Electroblotting of protein --- p.55 / Chapter 2.2.6.4 --- Probing antibodies or aptamers for proteins --- p.55 / Chapter 2.2.6.5 --- Enhanced chemiluminescence (ECL) Assay --- p.56 / Chapter Chapter 3. --- Results --- p.57 / Chapter 3.1 --- Selection of aptamer against cytochrome c dotted on membrane with counter selection against BSA on membrane --- p.58 / Chapter 3.1.1 --- Selection process --- p.58 / Chapter 3.1.1.1 --- PCR cycles --- p.59 / Chapter 3.1.1.2 --- Polyclonal aptamer --- p.61 / Chapter 3.1.1.3 --- Monoclonal aptamer Contents --- p.63 / Chapter 3.1.2 --- Binding test of cy-1 to cy-4 to cytochrome c --- p.65 / Chapter 3.1.3 --- Binding of cy-3 to the cytochrome c dotted on PVDF membrane --- p.67 / Chapter 3.1.4 --- Test the binding of cy-3 with cytochrome c by ELISA --- p.68 / Chapter 3.1.5 --- Competitive binding between monoclonal aptamer cy-3 and anti-cytochrome c antibody --- p.70 / Chapter 3.1.6 --- Western blotting of pure cytochrome c by cy-3 --- p.71 / Chapter 3.1.7 --- Western blotting of pure cytochrome c from different species --- p.73 / Chapter 3.1.8 --- Cell lysate SDS-PAGE labeled with cy-3 --- p.75 / Chapter 3.1.9 --- Cell lysate labeled with cy-1 to cy-9 after SDS-PAGE --- p.77 / Chapter 3.2 --- Selection of cytochrome c-specific aptamer with counter selection against cytosolic protein --- p.79 / Chapter 3.2.1 --- Selection of aptamer against cytochrome c with counter selection against cytosolic cell lysate --- p.79 / Chapter 3.2.2 --- Selection of aptamer against cytochrome c by fixed cell followed by cytochrome c elution --- p.82 / Chapter 3.2.3 --- Selection of aptamer from cytochrome c band --- p.84 / Chapter 3.3 --- Primers Testing --- p.86 / Chapter 3.3.1 --- Cell lysate labeled with primers after SDS-PAGE --- p.86 / Chapter 3.3.2 --- Cell lysate labeled with cy-3 without primers --- p.87 / Chapter 3.3.3 --- Test the effect of sense oligonucleotide --- p.89 / Chapter 3.3.4 --- Sequence of monoclonal aptamer --- p.90 / Chapter 3.3.5 --- Cell lysate labeled with aptamers without primer ends --- p.92 / Chapter 3.3.6 --- Test of the aptamers after mutations --- p.93 / Chapter 3.3.7 --- Test for other biotinylated primers --- p.96 / Chapter 3.4 --- Elimination of non-specific binding --- p.98 / Chapter 3.4.1 --- Different types of cell lysate --- p.98 / Chapter 3.4.2 --- Heating effect on the non-specific binding --- p.99 / Chapter 3.4.3 --- Using milk as a blocking agent --- p.101 / Chapter 3.4.3.1 --- Milk blocked membrane --- p.101 / Chapter 3.4.3.2 --- Milk prevented the binding of aptamer to cytochrome c --- p.102 / Chapter 3.4.3.3 --- Cell lysate labeled with cy-3 after SDS-PAGE by using milk as blocking agent --- p.104 / Chapter 3.4.3.4 --- Aptamer selection against cytochrome c in the presence of milk --- p.105 / Chapter 3.4.4 --- Using DNA as a Blocking agent --- p.107 / Chapter 3.4.4.1 --- DNA blocked the non-specific binding --- p.107 / Chapter 3.4.4.2 --- Cell lysate labeled with cy-3 after SDS-PAGE by using DNA as blocking agent --- p.109 / Chapter 3.4.4.3 --- Selection against cytochrome c blocked by DNA --- p.110 / Chapter 3.4.4.4 --- "Labeling of cell lysate treated with DNase, RNase or both after SDS-PAGE" --- p.112 / Chapter 3.5 --- Photo-SELEX --- p.114 / Chapter 3.5.1 --- Selection process --- p.114 / Chapter 3.5.2 --- Cell lysate labeled with photo-aptamer --- p.116 / Chapter 3.5.3 --- Testing by immunoprecipitation --- p.118 / Chapter 3.6 --- Application --- p.120 / Chapter 3.6.1 --- Detection of the cytochrome c in cytosolic proteins after treatment of TNF-α --- p.120 / Chapter 3.6.2 --- Detection of the cytochrome c in total cell lysate after treatment of TNF-α --- p.123 / Chapter 3.6.3 --- Detection of cytochrome c in different cellular compartments after treatment of TNF-α --- p.125 / Chapter Chapter 4. --- Discussion --- p.130 / Chapter 4.1 --- General information --- p.131 / Chapter 4.1.1 --- The pool of oligonucleotide --- p.131 / Chapter 4.1.2 --- Design of oligonucleotides --- p.131 / Chapter 4.1.3 --- SELEX --- p.133 / Chapter 4.1.3.1 --- Buffer condition of selection --- p.133 / Chapter 4.1.3.2 --- Binding equilibrium --- p.134 / Chapter 4.1.3.3 --- Prevalence of matrix-binding species --- p.134 / Chapter 4.2 --- Selection --- p.135 / Chapter 4.2.1 --- Cycle numbers of PCR --- p.135 / Chapter 4.3 --- Assay of aptamers selected --- p.137 / Chapter 4.3.1 --- The use of biotin-streptavidin for recognition --- p.137 / Chapter 4.3.2 --- Polyclonal aptamers --- p.137 / Chapter 4.3.3 --- Monoclonal aptamer --- p.137 / Chapter 4.3.4 --- Cy-3 shows the highest affinity to cytochrome c --- p.138 / Chapter 4.3.5 --- The presence of non-specific binding --- p.138 / Chapter 4.4 --- Counter selection against cell lysate --- p.140 / Chapter 4.5 --- Primer testing --- p.143 / Chapter 4.6 --- Sequences and secondary structures of monoclonal aptamers --- p.145 / Chapter 4.7 --- Elimination of non-specific binding Contents --- p.147 / Chapter 4.7.1 --- Non-specific binding may be mediated by sequence-independent recognition --- p.147 / Chapter 4.7.2 --- Elimination of non-specific binding by milk --- p.147 / Chapter 4.7.3 --- Eliminate the non-specific binding by using DNA --- p.149 / Chapter 4.8 --- Photo-aptamer --- p.151 / Chapter 4.9 --- Application of the monoclonal aptamer cy-3 --- p.153 / Chapter 4.9.1 --- Aptamer can label cytochrome c as antibody does --- p.153 / Chapter 4.10 --- Conclusion I --- p.158 / Chapter 4.11 --- Conclusion II --- p.159 / Chapter Chapter 5 --- References --- p.161