Self-association and modification of a genetically engineered polypeptide

A genetically synthesized polypeptide and polyethylene glycol (5 kDa or 10 kDa) functionalized forms of its alanine-rich helical domain were characterized. The polypeptide composed of an N-terminal histidine tag, and an alanine-rich domain, denoted as 17H6, has a sequence of: MGH10 SSGHIHM(AAAQEAAAAQAAAQAEAAQAAQ)6AGGYGGMG. 17H6 was originally designed as a scaffold to investigate multivalent interactions after glycosylation through reactive glutamic acid residues. We speculated that the protonation of the glutamic acid residues in these sequences would afford facile opportunities to manipulate their folding and assembly behavior considering the beta-sheet propensities of similar polypeptides at acidic pH. Thus, in the first part of this study, thermal unfolding, reversible self-association, and irreversible aggregation of 17H6 were investigated. Dynamic light scattering, and thermal unfolding measurements indicate that 17H6 spontaneously and reversibly self-associates at an acidic pH and ambient temperature. The resulting multimers have an average hydrodynamic radius of ∼ 10-20 nm and reversibly dissociate to monomers upon an increase to pH 7.4. Both free monomer and 17H6 chains within the multimers are beta-helical and folded at ambient and sub-ambient temperatures. Reversible unfolding of the monomer occurs upon heating of solutions at pH 7.4. At pH 2.3, heating first causes incomplete dissociation and unfolding of the constituent chains. Further incubation at an elevated temperature (80°C) induces additional structural and morphological changes and results in fibrils with a beta-sheet structure and a width of 5-10 nm (7 nm mean) as observed via transmission electron microscopy (TEM). In the second part, the histidine tag, which imparts solubility to the alanine-rich domain at acidic pH was cleaved. Propionaldehyde-functionalized poly(ethylene glycol) (PEG) molecules (5 kDa or 10 kDa) were attached to the N-terminus of the cleaved polypeptide, c17H6, as a hydrophilic block to compare the effect of these solubility tags on the aggregation and conformation behavior of the alanine-rich domain. Circular dichroic spectroscopy showed that the alpha-helical conformation of the alanine-rich domain was conserved in the conjugates below and near ambient temperatures independent of pH. Similarly, no significant difference between the thermal denaturation behavior of the polypeptide and that of the conjugates was observed at neutral pH. At acidic pH, on the other hand, 17H6 exhibited ∼ 25% loss in the initial alpha-helical structure upon incubation at 80°C for 3 hours followed by the refolding experiments, whereas the initial alpha-helical content of the conjugates did not change. Comparison of the apparent melting temperatures (∼ 54°C for 17H6 and PEG5K-c17H6 and ∼ 51°C for PEG10K-c17H6) indicated that high temperature stability of the conjugates is not due to the stabilization of the native alpha-helical conformation upon PEGylation. Kinetic experiments at 80 ¡aC for prolonged intervals indicated that PEGylation slowed down the rate of beta-sheet formation and reduced apparent cooperativity. These findings suggest that improved stability of the conjugates at acidic pH is due to the stabilization of the intermediate structures (which is likely to be random coil or early stage of beta-sheet structures) that form prior to the aggregation by reducing the interactions between these intermediates. In contrast to the polypeptide fibrils with ∼ 7 nm width, TEM images of the conjugates incubated at 80°C for 18 hours showed fibrillar structures with a width of ∼ 20-30 nm. Thus, it is likely that PEG conjugation also interferes to the arrangement of the polypeptide chains during or prior to the fibril formation. In the third part of this study, the aggregates of the polypeptide and the PEG-polypeptide conjugates that form at the physiological or sub-physiological temperatures and an acidic pH were characterized in detail via dynamic light scattering (DLS), small angle neutron scattering (SANS) and cryogenic transmission electron microscopy (cryo-TEM). DLS experiments showed that pH responsiveness is also operative for the conjugates clearly indicating that the aggregation state of the polypeptide and the conjugates is controlled by the charged state of the glutamic acid residues. Thus, it is likely that when the glutamic acid residues are functionalized with hydrophobic molecules, self-association of the alanine-rich domain can be triggered independent of pH. It was also shown that the aggregates could be detected at polypeptide/conjugate concentrations as low as 20 muM (∼ 0.3-0.5 mg/ml) at physiological and ambient temperatures. Cryo-TEM images indicated that 17H6 and PEG5K-c17H6 form elongated structures in contrast to the spherical morphology of PEG10K-c17H6 conjugate. These scattering and microscopy results showed that the size, the aggregation number, and the morphology of the aggregates could be tuned by the size and the nature of the hydrophilic tag. Thus, this tunable nature of the morphology of the aggregates along with their low critical aggregation concentration suggests that PEG-c17H6 conjugates can be used as a drug delivery vehicle where c17H6 serves as a drug attachment domain.