FUNCTIONS OF THE SEVENTEEN KILODALTON PROTEIN IN ESCHERICHIA COLI PROTEOSTASIS
Sandlin, Clifford Wade
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Outer membrane (OM) biogenesis is critical for the survival of Gram-negative bacteria. This process depends on periplasmic protein homeostasis, or proteostasis. Proteostasis depends on quality control (QC) networks. However, the periplasmic network is dense with aggregation-prone uOMPs and lacks ATP for disaggregases. To combat this, Seventeen Kilodalton Protein (Skp) is believed to provide chaperone activity (CA) and antiaggregation activity (AA). The Skp CA is proposed to work by enclosing uOMPs within a cavity of trimeric Skp. This cavity is smaller than the dimensions of free uOMPs. To resolve this paradox, I used SANS to measure the Rg of apo- and holo-Skp. SANS and modeling suggest that Skp adapts differently to uOmpW and uOmpA. Next, I revisited the evidence for CA and sought to verify Skp CA with OMPs. SDS-PAGE delayed-folding assays with uOmpW, uOmpLa and uBamA only showed Skp CA with uOmpW. This suggested that Skp CA does not apply to all uOMPs. In addition, I asked whether Skp is always a trimer. AUC sedimentation equilibrium (SE) reveals that some Skp is monomeric at physiological concentrations. This link between Skp trimerization and concentration led to a “switching hypothesis”, that functional Skp trimers are upregulated by stress. In support of switching, sedimentation velocity (SV) shows a monomeric variant of Skp lacks AA. Skp AA and CA varied between uOmpA, uOmpLa, and uBamA-TM. Surprisingly, I found that AA did not depend on uOMP structure or correlate with CA. ￼ii Finally, with fluorescence and SV I show that free uOmpLa and uBamA aggregate at ~10 nM in 150 mM NaCl, 80 mM urea, and pH 8.0. This suggests that the nanomolar affinity of Skp is well-suited to prevent uOMP aggregation in cells. I conclude that Skp is mainly a quality control antiaggregation factor, and has only a minor function as an OMP chaperone. I propose a shift away from the chaperone paradigm for Skp, and suggest that Skp evolved to serve as a “buffer”, for uOMPs, absorbing excess uOMPs when periplasmic conditions do not favor folding.