Well, Obviously

Discussion in 'Science and Technology' started by wiggles, May 16, 2007.

  1. wiggles
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    wiggles Active Member

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    Enzymatic analysis of a rhomboid intramembrane protease implicates transmembrane helix 5 as the lateral substrate gate

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    Rosanna P. Baker*, Keith Young*, Liang Feng, Yigong Shi, and Sinisa Urban*

    *Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 507 Preclinical Teaching Building, 725 North Wolfe Street, Baltimore, MD 21205; and {dagger}Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, NJ 08544

    Edited by Douglas C. Rees, California Institute of Technology, Pasadena, CA, and approved March 19, 2007 (received for review January 30, 2007)

    Intramembrane proteolysis is a core regulatory mechanism of cells that raises a biochemical paradox of how hydrolysis of peptide bonds is accomplished within the normally hydrophobic environment of the membrane. Recent high-resolution crystal structures have revealed that rhomboid proteases contain a catalytic serine recessed into the plane of the membrane, within a hydrophilic cavity that opens to the extracellular face, but protected laterally from membrane lipids by a ring of transmembrane segments. This architecture poses questions about how substrates enter the internal active site laterally from membrane lipid. Because structures are static glimpses of a dynamic enzyme, we have taken a structure–function approach analyzing >40 engineered variants to identify the gating mechanism used by rhomboid proteases. Importantly, our analyses were conducted with a substrate that we show is cleaved at two intramembrane sites within the previously defined Spitz substrate motif. Engineered mutants in the L1 loop and active-site region of the GlpG rhomboid protease suggest an important structural, rather than dynamic, gating function for the L1 loop that was first proposed to be the substrate gate. Conversely, three classes of mutations that promote transmembrane helix 5 displacement away from the protease core dramatically enhanced enzyme activity 4- to 10-fold. Our functional analyses have identified transmembrane helix 5 movement to gate lateral substrate entry as a rate-limiting step in intramembrane proteolysis. Moreover, our mutagenesis also underscores the importance of other residue interactions within the enzyme that warrant further scrutiny.

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  2. Ninja
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    Ninja Senior Member

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    PNAS = Post Nature And Science ;)
     
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  3. Dirt McGirt
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    Dirt McGirt Bad Mother****er

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    I know, right? Wiggles should probably finish the 8th grade before posting a basic study like that and acting like nobody would understand it. I mean what's so difficult to understand- it's just molecular biology. Her intellectual inferiority is embarrassing to the collective board sometimes. Thanks for making everyone here look stupid Wiggles...all the other message boards are laughing at us now.
     
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  4. Shattered
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    Ow.
    :eusa_doh:
     

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