The proteolysis and enzymatic resynthesis of self -assembling proteins and the design of pH sensitive folate -targeted liposomes
Abstract
Knowledge of the secondary structural self-assembly of proteins is implicit in the de novo design of protein structures. In this work, we have investigated the self-assembling properties of both triose phosphate isomerase, an α/β barrel protein, and hen egg-white lysozyme, a protein stabilized by the presence of four disulfide bonds. Limited proteolysis was used to dissect the native proteins, and the individual proteolytic fragments of each protein were identified using SDS-PAGE electrophoresis, and characterized by N-terminal amino acid sequencing. The integrity of the non-covalent assembly of these proteins was confirmed through the ability of these assemblies to maintain biological activity comparable to that of the native enzymes. The resynthesis of both enzymes was achieved, in the presence of subtilisin, under mixed aqueous-organic solvent conditions. The resynthesis was confirmed using SDS-PAGE electrophoresis, N-terminal amino acid sequencing, circular dichroism spectroscopy, electrospray mass spectrometry, and the assay of their biological activity. We have also designed two peptides, EALA and EALA2, which exploit the mechanism of action of naturally occurring peptides to facilitate the release of liposome encapsulated molecules into the cytoplasm of KB cells following receptor mediated endocytosis. Our peptides were designed to form amphiphilic α-helices in the range of endosomal pH. The strategy involved the encapsulation of EALA or EALA2 into the aqueous interior of folate-targeted liposomes, and the covalent attachment of EALA to a maleimide derivative of phosphatidylethanolamine (PE). To quantitate the extent of peptide induced release of liposome encapsulated molecules, a new methodology was developed that exploits the dramatic increase in quantum yield of propidium iodide (PI) upon binding to DNA and RNA.
Degree
Ph.D.
Advisors
Chmielewski, Purdue University.
Subject Area
Biochemistry|Organic chemistry|Cellular biology
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