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http://voh.chem.ucla.edu/classes/159A/

Lectures can be downloaded at the dept. voh site at:. http://voh.chem.ucla.edu/classes/159A/. From last time - activation zone up from -40. -40. up. +20. and oxyR binds in this zone to activate various members of its regulon in response to peroxide and related oxidants. References for oxyR.

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  1. Lectures can be downloaded at the dept. voh site at: http://voh.chem.ucla.edu/classes/159A/

  2. From last time - activation zone up from -40 -40 up +20 and oxyR binds in this zone to activate various members of its regulon in response to peroxide and related oxidants.

  3. References for oxyR Zheng, M., F. Åslund, and G. Storz. (1998). Science 279:1718-1721 Kim et. al (2002), Cell, 109:383-396 Choi H. et. al. Cell, 105:103-113 Lee et al, Nat Struct Mol Biol. (2004) 11:1179-85 Tao et. al (1995) J. Bact. 177. pp. 6740-44 (later)

  4. Purification history No change in amount + or - peroxide Purify oxyR Western blot + or - peroxide Purify oxyR Activity assay Not reproducible High and reproducible Activity assay Purify oxyR without reductants like dtt The active form of oxyR may require it to be oxidized Interpretation: A little history of peroxide challenge and oxyR purification Genetic pathway: Various anti-stress proteins Peroxide treatment of cells oxyS oxyR induction Some other regulators

  5. These forms of oxyR were used in the Storz lab exps 4 of 6 cysteines inactivated by mutation. (others not important for transcription - next slide) DNA-binding domain 199 C C C CC C 208 structure biochemistry

  6. Zheng, M., F. Åslund, and G. Storz. 1998. Science 279:1718-1721 4 cys gone conclusion: only 199/208 needed

  7. The oxidized form, when fragmented, shows a disulfide between 199 and 208 by mass spec. Next: Is the redox system Grx1/glutathione involved in controlling activity?

  8. oxyR-dependent txn. is inactivated by the GrxI protein and reducing form of glutathione (GSH) titration conclusion - the oxidation state of glutathione can determine whether oxyR will be active. (and Grx1 acts as a catalyst for the reaction)

  9. Transcription starting with reduced oxyR Reduced oxyR can be re-activated by either peroxide or the GRX1/glutathione system. So either direct or Grx1/glutathione oxidation contributes to the net state.

  10. Model: Oxidants work directly or indirectly via glutathione oxidation. The net effect is that oxyR monitors the cells’ oxidation status. inactive active

  11. Choi H. et. al. Cell, 105:103-113 They went on to determine the structure of the oxidized and reduced forms. Oxidized form: A fragment missing the DNA-binding domain and in which 4 (non-essential) cysteines had been replaced by site-directed mutagenesis Reduced form: Same as above except C199 has also been replaced to prevent oxidation.

  12. reduced oxidized superpositon disulfide

  13. A close-up of the region around the 2 cysteines This arginine may enhance reactivity by stabilizing the thiolate anion at cys-199 disulfide Long distance between cysteines in reduced, inactive form Active, oxidized form

  14. They also found large changes in contacts between monomers and speculated about how these could lead to activation. But this is very far from clear because the DNA-binding domain and the DNA are both absent. Moreover, a subsequent paper must be taken into account.

  15. Kim et. al, Cell, Vol 109, 383-396, May 2002 • They were skeptical of the oxyR model for several reasons including: • They say only cys199, not its partner cys208, is absolutely essential for activity. • They previously found a form of oxyR in which only cys199 was modified, • in this case to SNO (S-nitrosothiol) • cys199 and cys208 are so far apart in the structure that it would seem to be • difficult for them to react readily in vivo. • it didn't fit with their prior speculation about the redox changes leading • to a graded response, rather than acting via an on-off switch (see next slide).

  16. Annual Review of Genetics December 2003, Vol. 37, pp. 91-121 Observed oxidized forms of cysteine Only this matters to Storz

  17. Storz model Kim model Additional possible oxidants (at cys199?) nitroso sulfenic glutathio A graded response depending on redox conditions?

  18. Essentially, they repeated the mass-spec.analysis using the native protein and found: "we conclude that C25 and C199 are solvent-accessible thiols; that C143 and C208 are free thiols, albeit buried in the protein; and that C180 and C259 form a disulfide, which is accessible to reductants in the native (aerobically purified) state… Second, the finding that residues C180 and C259 (alanines in the crystal) participate in a disulfide bond is supported by their very close proximity (5.9 Å). Third, the crystallographic data places distantly located C199 and C208 in a disulfide (despite being 18 Å apart in the truncated 4CysAla mutant protein). We find no evidence for a disulfide bond between C199 and C208 in the full-length wild-type OxyR." Uh-oh!

  19. Summary of their results DNA-binding domain Solvent accessible 199 C C C CC C Disulfide in wt protein

  20. They made various oxidized forms of the solvent accessible cysteine 199, (reduced with dtt for comparison), and tested katG gene activation. Conclusion: all forms are active.

  21. Docking (right) shows that GSSG (disulfide created by glutathione oxidation) fits perfectly in a site near cys199. If GSSH were created by oxidation after redox challenge it might go there and produce cys199-S-G, which is one of their active forms.

  22. CD spectra of the various forms imply a graded degree of structure change. And there is some evidence (see the paper) that each form binds DNA with a unique affinity.

  23. A (very recent) reply to Kim from the Storz/Ryu groups Nat Struct Mol Biol. 2004 Dec;11(12):1179-85. Revised protocol: Use full length wt protein (like Kim et al study). Differs in didn’t use air-oxidized protein (Kim purified as usual). Instead reduce it fully with dtt and then oxidize with peroxide.

  24. Mass spec to see which disulfides form Reduced - all free CSH Re-oxidized - 199-208 disulfide Interpretation: they were correct before. Other group erred in using air-oxidized protein.

  25. Now look for role of cysteines in pathway of oxyR oxidation. Protocol: Modify mutants with a thiol oxidation reagent. 2. Detect modification via UV spectroscopy Mutants used: 4CA: C208 and C199 left 4CA-C208S: only C199 left 4CA-C199S: only C208 left

  26. Only when C199 is present alone does S-OH form 350nm known to come from sulfenic acid S-OH 3 Interpretation: C199 is modified to S-OH. It is unstable and then reacts with C208 to form a disulfide. 1 2

  27. Interpretation: See this reaction scheme no S-OH forms if rxn occurs in presence of gluathione

  28. Proposal (includes rate data) If glutathione is present GSH C199-SSG form

  29. Synthesis/cop-out in review from Georgiou (2002) Cell 111, 607-610. Based on the work presented suggest a plausible model for the role of glutathione in redox sensing. 250 words or less, please.

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