The Skp Chaperone Helps Fold Soluble Proteinsin Vitroby Inhibiting Aggregation

Abstract

The periplasmic seventeen kilodalton protein (Skp) chaperone has been characterized primarily for its role in outer membrane protein (OMP) biogenesis, during which the jellyfish-like trimeric protein encapsulates partially folded OMPs, protecting them from the aqueous environment until delivery to the BAM outer membrane protein insertion complex. However, Skp is increasingly recognized as a chaperone that also assists in folding soluble proteins in the bacterial periplasm. In this capacity, Skp co-expression increases the active yields of many recombinant proteins and bacterial virulence factors. Using a panel of single-chain antibodies and a single-chain T-cell receptor (collectively termed scFvs) possessing varying stabilities and biophysical characteristics, we performed in vivo expression, and in vitro folding and aggregation assays in the presence or absence of Skp. For Skp-sensitive scFvs, the presence of Skp during in vitro refolding assays reduced aggregation but did not alter the observed folding rates, resulting in a higher overall yield of active protein. Of the proteins analyzed, Skp sensitivity in all assays correlated with the presence of folding intermediates, as observed with urea denaturation studies. These results are consistent with Skp acting as a holdase, sequestering partially folded intermediates and thereby preventing aggregation. Because not all soluble proteins are sensitive to Skp co-expression, we hypothesize that the presence of a long-lived protein folding intermediate renders a protein sensitive to Skp. Improved understanding of the bacterial periplasmic protein folding machinery may assist in high-level recombinant protein expression and may help identify novel approaches to block bacterial virulence.

Life Stage-specific Proteomes of Legionella pneumophila Reveal a Highly Differential Abundance of Virulence-associated Dot/Icm effectors

“…S3, supplemental Table S6). Although it forms trimeric periplasmic complexes, its ability to directly interact with membrane lipids and LPS has been described (71)(72)(73). TolB is a periplasmic constituent of TolB-Pal cell envelope complex linked to the outer membrane with function in colicin import in E. coli (74,75).…”

Deuterium Labeling Together with Contrast Variation Small-Angle Neutron Scattering Suggests How Skp Captures and Releases Unfolded Outer Membrane Proteins

“…It can also inhibit the aggregation and assist the folding of a number of soluble proteins. Notable examples include single chain antibodies (Entzminger, Chang, Myhre, McCallum, & Maynard, 2012) and lysozyme (Walton & Sousa, 2004). …”

Skp Trimer Formation Is Insensitive to Salts in the Physiological Range

“…This binding underlies the Skp function as a holding chaperone (5, 6), an activity accomplished by the enclosure of uOMPs within a cavity formed by a trimer of Skp monomers (5, 9, 10). The trimer is thus accepted as the relevant species in vivo (5, 7–21).…”

“…If trimers possess the bulk of holding activity (6), then regulation of Skp concentration regulates this activity. Such regulation of functional multimers by gene expression has been observed before (33, 34).…”