In vacuo cross-linking of proteins: A novel approach with theoretical and practical applications to the study of proteins

Abstract

This thesis describes the hypothesis and subsequent demonstration that proteins can be covalently cross-linked without use of chemical reagents by simply incubating the protein or proteins in the lyophilized state under appropriate conditions in a vacuum. This in vacuo cross-linking requires the interaction of a protonated positively-charged amino group on one molecule with a deprotonated negatively-charged carboxylate group on a second. A zerolength cross-link is formed by a condensation reaction between the two groups with the release of water. A reaction mechanism is proposed in which amide bond formation occurs by means of a proton transfer from the protonated amino group to an interacting carboxylate group followed by a nucleophilic attack by the deprotonated amino group on the carboxylic acid group (protonated). The reaction is driven by the removal of the released water by the vacuum. It was observed that proteins appeared to form trace amounts of covalently bonded products, visualized on denaturing and reducing gel electrophoresis and probed with sensitive detection methods, if the protein preparations had been subjected to a vacuum. This observation prompted the hypothesis: can proteins be covalently cross-linked by incubating lyophilized protein under vacuum? This hypothesis was tested using Ribonuclease A (RNase A) as a model protein and the in vacuo heating of RNase A demonstrated that covalently cross-linked products arise with dimer being the major product. Structural characterization of the in vacuo RNase A dimer demonstrated that the dimer was composed of a single amide cross-link at positions lysine 66 and glutamic acid 9. This dimenc protein is proposed to adopt a new conformation not previously reported as a known RNase A X-ray crystallographic structure. The in vacuo RNase A dimer also demonstrated a 2-fold increase in enzymatic activity compared to monomeric RNase A towards both double stranded and single stranded RNA, both in the presence and absence of the cytosolic ribonuclease inhibitor. The in vacuo cross-linking of protein was shown to be a general phenomenon as all proteins tested produced covalently cross-linked oligomers. The utility of this cross-linking method was then expanded to the cross-linking of proteins to functionalized solid supports and the covalent cross-linking of enzymes to antibodies to produce highly sensitive immunoconjugates for Western Blot analysis and ELISAs. A discussion of the in vacuo cross-linking of a de novo designed protein, milk bundle, and its dimeric 4-helix bundle structural characterization is also presented.