Structural Characterization of Apomyoglobin Self-Associated Species in Aqueous Buffer and Urea Solution

¹ University of Wisconsin-Madison
² University of Wisconsin - Madison

^* To whom correspondence should be addressed. E-mail: cavagnero{at}chem.wisc.edu.

Submitted on July 7, 2005
Revised on August 16, 2005
Accepted on 30 September 2005

	Abstract

The biophysical characterization of non-functional protein aggregates at physiologically relevant temperatures is much needed to gain deeper insights into the kinetic and thermodynamic relationships between protein folding and misfolding. Dynamic and static laser light scattering have been employed for the detection and detailed characterization of apomyoglobin (apoMb) soluble aggregates populated at room temperature upon dissolving the purified protein in buffer at pH 6.0, both in the presence and absence of high concentrations of urea. The soluble aggregates have either -helical or random coil secondary structure, depending on solvent and solution conditions, with hydrodynamic diameters ranging from 80 to 130 nm and semi-flexible chain-like morphology. The combined use of low pH and high urea concentration leads to structural unfolding and the complete elimination of the large aggregates. Even upon starting from this virtually monomeric unfolded state, however, protein refolding leads to the formation of severely self-associated species with native-like secondary structure. Under these conditions, kinetic apoMb refolding proceeds via two parallel routes: one leading to native monomer, and the other leading to a misfolded and heavily self-associated state bearing native-like secondary structure.

Key Words: dynamic light scattering, folding, self-association, size exclusion chromatography, static light scattering