Dataset Information

Protein transport across membranes: Comparison between lysine and guanidinium-rich carriers.

ABSTRACT: The mechanism(s) by which certain small peptides and peptide mimics carry large cargoes across membranes through exclusively non-covalent interactions has been difficult to resolve. Here, we use the droplet-interface bilayer as a platform to characterize distinct mechanistic differences between two such carriers: Pep-1 and a guanidinium-rich peptide mimic we call D9. While both Pep-1 and D9 can carry an enzyme, horseradish peroxidase (HRP) across a lipid bilayer, we found that they do so by different mechanisms. Specifically, Pep-1 requires voltage or membrane asymmetry while D9 does not. In addition, D9 can facilitate HRP transport without pre-forming a complex with HRP. By contrast, complex formation is required by Pep-1. Both carriers are capable of forming pores in membranes but our data hints that these pores are not responsible for cargo transport. Overall, D9 appears to be a more potent and versatile transporter when compared with Pep-1 because D9 does not require an applied voltage or other forces to drive transport. Thus, D9 might be used to deliver cargo across membranes under conditions where Pep-1 would be ineffective.

SUBMITTER: Lein M

PROVIDER: S-EPMC4704449 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Protein transport across membranes: Comparison between lysine and guanidinium-rich carriers.

Lein Max M deRonde Brittany M BM Sgolastra Federica F Tew Gregory N GN Holden Matthew A MA

Biochimica et biophysica acta 20150902 11 Pt A

The mechanism(s) by which certain small peptides and peptide mimics carry large cargoes across membranes through exclusively non-covalent interactions has been difficult to resolve. Here, we use the droplet-interface bilayer as a platform to characterize distinct mechanistic differences between two such carriers: Pep-1 and a guanidinium-rich peptide mimic we call D9. While both Pep-1 and D9 can carry an enzyme, horseradish peroxidase (HRP) across a lipid bilayer, we found that they do so by diff ...[more]

PMID: 26342679

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Project description:Under the shell of a chicken egg are two opposed proteinaceous disulfide-rich membranes. They are fabricated in the avian oviduct using fibers formed from proteins that are extensively coupled by irreversible lysine-derived crosslinks. The intractability of these eggshell membranes (ESM) has slowed their characterization and their protein composition remains uncertain. In this work, reductive alkylation of ESM followed by proteolytic digestion led to the identification of a cysteine rich ESM protein (abbreviated CREMP) that was similar to spore coat protein SP75 from cellular slime molds. Analysis of the cysteine repeats in partial sequences of CREMP reveals runs of remarkably repetitive patterns. Module a contains a C-X(4)-C-X(5)-C-X(8)-C-X(6) pattern (where X represents intervening non-cysteine residues). These inter-cysteine amino acid residues are also strikingly conserved. The evolutionarily-related module b has the same cysteine spacing as a, but has 11 amino acid residues at its C-terminus. Different stretches of CREMP sequences in chicken genomic DNA fragments show diverse repeat patterns: e.g. all a modules; an alternation of a-b modules; or an a-b-b arrangement. Comparable CREMP proteins are found in contigs of the zebra finch (Taeniopygia guttata) and in the oviparous green anole lizard (Anolis carolinensis). In all these cases the long runs of highly conserved modular repeats have evidently led to difficulties in the assembly of full length DNA sequences. Hence the number, and the amino acid lengths, of CREMP proteins are currently unknown. A 118 amino acid fragment (representing an a-b-a-b pattern) from a chicken oviduct EST library expressed in Escherichia coli is a well folded, highly anisotropic, protein with a large chemical shift dispersion in 2D solution NMR spectra. Structure is completely lost on reduction of the 8 disulfide bonds of this protein fragment. Finally, solid state NMR spectra suggest a surprising degree of order in intact ESM fibers.

Dataset Information

Protein transport across membranes: Comparison between lysine and guanidinium-rich carriers.

Publications

Protein transport across membranes: Comparison between lysine and guanidinium-rich carriers.

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