Visualizing mitochondrial FoF1-ATP synthase as the target of the immunomodulatory drug Bz-423

Targeting the mitochondrial enzyme FoF1-ATP synthase and modulating its catalytic activities with small molecules is a promising new approach for treatment of autoimmune diseases. The immuno-modulatory compound Bz-423 is such a drug that binds to subunit OSCP of the mitochondrial FoF1-ATP synthase and induces apoptosis via increased reactive oxygen production in coupled, actively respiring mitochondria. Here we review the experimental progress to reveal the binding of Bz-423 to the mitochondrial target and discuss how subunit rotation of FoF1-ATP synthase is affected by Bz-423. Briefly, we report how F\"orster resonance energy transfer (FRET) can be employed to colocalize the enzyme and the fluorescently tagged Bz-423 within the mitochondria of living cells with nanometer resolution.Comment: 10 pages, 2 figure

RNA Folding Pathways

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143692/1/cpnc1100.pd

Going forward

No abstract.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55863/1/20632_ftp.pd

Engineering Disulfide Cross‐Links in RNA Via Air Oxidation

This unit presents protocols for the synthesis of alkylthiol‐modified ribonucleosides, their incorporation into synthetic RNA, and the formation of intramolecular disulfide bonds in RNA by air oxidation. The disulfide bonds can be formed in quantitative yields between thiols positioned in close proximity by virtue of either the secondary or tertiary structure of the RNA. Disulfide cross‐links are useful tools to probe solution structures of RNA, to monitor dynamic motion, to stabilize folded RNAs, and to study the process of tertiary structure folding.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143806/1/cpnc0504.pd

36 degree step size of proton-driven c-ring rotation in FoF1-ATP synthase

Synthesis of the biological "energy currency molecule" adenosine triphosphate ATP is accomplished by FoF1-ATP synthase. In the plasma membrane of Escherichia coli, proton-driven rotation of a ring of 10 c subunits in the Fo motor powers catalysis in the F1 motor. While F1 uses 120 degree stepping, Fo models predict a step-by-step rotation of c subunits 36 degree at a time, which is here demonstrated by single-molecule fluorescence resonance energy transfer.Comment: 8 pages, 1 figur

Chemical sequencing of disulfide-crosslinked oligodeoxyribonucleotides

Oligodeoxyribonucleotides containing a novel disulfide crosslink are shown to be amenable to chemical sequencing using standard sequencing reagents following reduction of the disulfide with DTT and selective alkylation of the free thiols with N-ethylmaleimide. This strategy will facilitate conformational and molecular recognition studies of DNA stabilized by crosslinking.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30570/1/0000205.pd

Incorporation of alkylthiol chains at C-5 of deoxyuridine

A series of alkylthiol-tether homologs at C-5 of 2'-deoxyuridine have been synthesized and incorporated into DNA oligomers through solid-phase DNA phosphoramidite synthesis. DNA-ligand disulfide crosslinks have been initially addressed through formation of an n-butyl-DNA disulfide conjugate.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30630/1/0000271.pd

Effect of somatic mutation on DNA binding properties of anti-DNA autoantibodies

Autoantibodies that bind DNA are a hallmark of systemic lupus erythematosus. A subset of autoantibody•DNA complexes localize to kidney tissue and lead to damage and even death. 11F8, 9F11, and 15B10 are clonally related anti-DNA autoantibodies isolated from an autoimmune mouse. 11F8 binds ssDNA in a sequence-specific manner and causes tissue damage, while 9F11 and 15B10 bind ssDNA non-specifically and are benign. Among these antibodies, DNA binding properties are mediated by five amino acid differences in primary sequence. Thermodynamic and kinetic parameters associated with recognition of structurally different DNA sequences were determined for each antibody to provide insight toward recognition strategies, and to explore a link between binding properties and disease pathogenesis. A model of 11F8 bound to its high affinity consensus sequence provides a foundation for understanding the differences in thermodynamic and kinetic parameters between the three mAbs. Our data suggest that 11F8 utilizes the proposed ssDNA recognition motif including Y32 V L , a hydrogen bonding residue at 91 V L , and an aromatic residue at the tip of the third heavy chain complementarity determining region. Interestingly, a somatic mutation to arginine at 31 V H in 11F8 may afford additional binding site contacts including R31 V H , R96 V H , and R98 V H that could determine specificity. © 2007 Wiley Periodicals, Inc. Biopolymers 85: 471–480, 2007. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected] Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55984/1/20691_ftp.pd

Application of the gel shift assay to study the affinity and specificity of anti-DNA autoantibodies

We have demonstrated that the gel shift assay, a powerful method to study protein [middle dot] DNA interactions under equilibrium conditions, is both an accurate and precise method to measure the affinity of anti-DNA [middle dot] DNA immune complexes. One difficulty in performing gel shift assays is disruption of protein [middle dot] DNA equilibria during the time needed for complexes to enter the gel matrix. However, we have found that highly cross-linked polyacrylamide gels, which are known to form non-restrictive matrices, do not perturb anti-DNA[middle dot]DNA complexes. Using anti-ssDNA BV04-01 as a model antibody, we find good agreement between the dissociation constants (Kd) measureed in the gel shift assay using a 5.4% polyacrylamide gel cross-linked with 0.6% (bis)acrylamide, and those obtained previously by fluorescence quenching. Because gel shift assays require only nanogram quantities of analyte and can be performed in several hours, it is well suited for a range of anti-DNA binding studies.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31125/1/0000022.pd