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Multinuclear Zn sites: alkaline Phosphatase. A.-F. Miller, 2008, pg 1 Structural Zn: Zn fingers The most common Zn-binding motif in the human genome is the so-called Zn-finger. Zn2+ binds tightly with Td coordination, Kd ≈ 10-9-10-11M. Binding is specific for Zn: 103 - 105 times more tighter Zn binding than binding of the next tightest binders: Co2+, Fe2+ and Ni2+. Protein folding is dependent on Zn2+ binding. The Zn finger module has a minute hydrophobic core that is not sufficient to stabilize the folded state in the absence of Zn. However when Zn is bound, the protein structure can withstand boiling. Consensus sequence is (Tyr/Phe)-X-Cys-X2,4-Cys-X3-Phe-X5-Leu-X2-His-X3,4-His-X2-6 A.-F. Miller, 2008, pg 2 Zn fingers bind DNA and RNA Zn coordination is via 2 His and 2 Cys in the first class of Zn fingers discovered (C2H2 fingers) C2C2 versions also exist, as do C6 di-Zn2+-binding proteins. Zn fingers are usually modules of larger proteins. Proteins with as many as 37 Zn fingers are known. Their role is to bind specific sequences of DNA or RNA. A number of Zn fingers occur in tandem in a protein. Each finger recognizes 3 consecutive base pairs, so a string of Zn fingers can selectively bind a unique sequence of DNA. The other modules of the protein are thus targeted to a DNA sequence where they may recruit RNA polymerase and thus increase expression of down-stream genes. A.-F. Miller, 2008, pg 3 Zn finger functions Zinc finger-containing proteins participate in DNA replication and repair, transcription and translation. Thus they coordinate metabolism, signaling, cell proliferation and apoptosis. HIV Zn fingers aid in binding and packaging viral RNA into new virions. They also play a role in reverse transcription Artificial zinc finger transcription factors have been used to target specific DNA sequence, in combination with activation or repression domains to switch genes on or off. Upon depletion of Zn2+, non-specific interactions between Znfinger proteins and other domains and proteins can result. A.-F. Miller, 2008, pg 4 Zn fingers Zn stabilizing a very small globular structure with almost no hydrophobic core, thus enabling these DNA-binding domains to be tiny yet stable. A ‘core’ of two side chains. A.-F. Miller, 2008, pg 5 1p47.pdb E.PEISACH, C.O.PABO (2003) J.MOL.BIOL. 330:1 Zn fingers Coordination to Zn holds together separate elements of structure. N and C termini face opposite sides: a string of these units will be extended. A.-F. Miller, 2008, pg 6 1p47.pdb E.PEISACH, C.O.PABO (2003) J.MOL.BIOL. 330:1 Zn fingers α helix lies in major groove A.-F. Miller, 2008, pg 7 1p47.pdb E.PEISACH, C.O.PABO (2003) J.MOL.BIOL. 330:1 Zn fingers Symmetry: helices in close, sheets on outside. A.-F. Miller, 2008, pg 8 1p47.pdb E.PEISACH, C.O.PABO (2003) J.MOL.BIOL. 330:1 Zn fingers Amino acids projecting our of α helix stick into major groove and contact edges of base pairs (outside edges). Binds to DNA to regulate transcription of 5S rRNA. Also binds to 5S RNA stabilizing it and escorting it to cytoplasm. A.-F. Miller, 2008, pg 9 1p47.pdb E.PEISACH, C.O.PABO (2003) J.MOL.BIOL. 330:1 Sample problems Metal ion and oxidation state d-electron count. High spin vs. low spin electronic configuration in Oh and Td coordination. Identities of coordinating amino acids, and modes of binding. Hard-soft series and ligand-metal preferred pairing. General schemes for metal acquisition. Non-redox cations Na+, K+, Ca2+, Mg2+. Forms in which they occur, roles they play, issues they raise. Why ? Zn2+, the ‘simple’ transition metal ion: what does it do ? Why ? A.-F. Miller, 2008, pg 10 Example Uranyl ion (UO22+) is commonly used to form heavy-atom derivatives for X-ray diffraction studies. Given that this is a relatively hard ion, what sorts of functional groups would you expect to be derivatized ? How does this behaviour compare with that expected for another heavy-metal derivatizing ion, Hg2+ ? A.-F. Miller, 2008, pg 11