Folding Kinetics and Structure of OEP16

Abstract

The chloroplast outer membrane contains different, specialized pores that are involved in highly specific traffic processes from the cytosol into the chloroplast and vice versa. One representative member of these channels is the outer envelope protein 16 (OEP16), a cation-selective high conductance channel with high selectivity for amino acids. Here we study the mechanism and kinetics of the folding of this membrane protein by fluorescence and circular dichroism spectroscopy, using deletion mutants of the two single tryptophanes Trp-77-->Phe-77 and Trp-100-->Phe-100. In addition, the wild-type spectra were deconvoluted, depicting the individual contributions from each of the two tryptophan residues. The results show that both tryptophan residues are located in a completely different environment. The Trp-77 is deeply buried in the hydrophobic part of the protein, whereas the Trp-100 is partially solvent exposed. These results were further confirmed by studies of fluorescence quenching with I(-). The kinetics of the protein folding are studied by stopped flow fluorescence and circular dichroism measurements. The folding process depends highly on the detergent concentration and can be divided into an ultrafast phase (k > 1000 s(-1)), a fast phase (200-800 s(-1)), and a slow phase (25-70 s(-1)). The slow phase is absent in the W100F mutant. Secondary structure analysis and comparision with closely related proteins led to a new model of the structure of OEP16, suggesting that the protein is, in contrast to most other outer membrane proteins studied so far, purely alpha-helical, consisting of four transmembrane helices. Trp-77 is located in helix II, whereas the Trp-100 is located in the loop between helices II and III, close to the interface to helix III. We suggest that the first, very fast process corresponds to the formation of the helices, whereas the insertion of the helices into the detergent micelle and the correct folding of the II-III loop may be the later, rate-limiting steps of the folding process.

Solute channels of the outer membrane: from bacteria to chloroplasts

“…Furthermore, the OEP16 protein shows sequence and secondary structure similarity to components of the protein translocase of the inner mitochondrial membrane (Tim17, Tim22, and Tim23) and to a lesser extent to LivH, an amino acid transporter in the cytosolic membrane of E. coli (Rassow et al, 1999). Like these membrane transporters, OEP16 forms four a-helical transmembrane domains according to circular dichroism studies ( Figure 2A, Linke et al, 2004). This is rather unusual, since the channel proteins in the outer membranes of mitochondria (VDAC) and Gram-negative bacteria (see above) are exclusively built by b-barrels.…”

“…Angemeldet | 129.187.254.47 Heruntergeladen am | 18.11.13 10:46 Linke et al (2004), OEP16 from pea contains four a-helical transmembrane domains. Helices II and IV are determined to be three to five amino acids longer than helices I and III.…”

Origin of mitochondria by intracellular enslavement of a photosynthetic purple bacterium

“…The even more closely related OM protein OEP16 of chloroplasts probably evolved from Tim22 after it diverged from Tim23/17 (they group on a tree: Rassow et al 1999). OEP16, unlike bacterial OM proteins, is not a b-barrel, but has four membrane-spanning a-helices (Linke et al 2004). It probably evolved from Tim22 by gene duplication and insertion into the OM from the cytosol, another example of recruitment of a pre-existing mitochondrial import component for chloroplast import, as postulated (Cavalier-Smith 1982).…”

A search for factors influencing etioplast–chloroplast transition

“…Subsequent studies on the phylogeny and secondary structure revealed that OEP16 belongs to the superfamily of preprotein and amino acid transporters (PRAT) including the protein translocating channels Tim17, Tim22, and Tim23 of the inner mitochondrial membrane (16,37). Like these transporters, OEP16 forms 4 ␣-helical transmembrane domains (38). In Arabidopsis 3 genes are coding for OEP16 isoforms, named OEP16.1, OEP16.2, and OEP16.4, respectively (16,39).…”