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

Abstract

The biophysical characterization of nonfunctional 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. Unlike the beta-sheet self-associated aggregates previously reported for this protein at high temperatures, the soluble aggregates detected here have either alpha-helical or random coil secondary structure, depending on solvent and solution conditions. Hydrodynamic diameters range from 80 to 130 nm, with semiflexible chain-like morphology. The combined use of low pH and high urea concentration leads to structural unfolding and 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.

Delicate Balance between Functionally Required Flexibility and Aggregation Risk in a β-Rich Protein

“…Moreover, at urea concentrations higher than 3 M, the fraction of aggregated protein dropped dramatically, presumably because CRABP1 aggregates are not stable at high urea concentration – the same behavior (urea instability) has been previously observed for other amorphous aggregates. (35) These results suggest that CRABP1 aggregation is triggered by mild perturbations that cause shifts in the population of states on the CRABP1 energy landscape in the region of the native protein, rather than by global unfolding.…”

Effects of urea and acetic acid on the heme axial ligation structure of ferric myoglobin at very acidic pH

“…In fact, the absorbance of the Soret band of the native species 6cHS(1) was almost completely restored upon refolding, indicating that i) the unfolding process is completely reversible, and ii) the refolding of Mb leads essentially to the native monomer. On the contrary, the refolding process of the apoprotein under these conditions was found to proceed via two parallel routes: one leading to native monomer, and the other leading to a misfolded and heavily aggregated state bearing some native-like secondary structure [52]. Therefore the heme chromofore stabilizes the native protein.…”

“…This was rather unexpected, since the solubilization of the hydrophobic heme chromophore is unfavoured especially at low pH where the propionic groups of the porphyrin are protonated. Similarly, at neutral pH apoMb forms aggregates at high urea concentration, whereas under strongly denaturing environment the large aggregates are absent [52]. At low pH, polypeptides may acquire a net positive charge, leading to an overall increase in intermolecular electrostatic repulsion, which reduces the tendency toward intermolecular complex formation.…”

“…As far as apoMb is concerned, recent investigations have revealed that it forms a soluble monomer species under strongly denaturing conditions (highly concentrated urea solutions and low pH values), whereas aggregates are predominant under milder unfolding conditions (highly concentrated urea solutions pH 6.0) [52]. …”

Quantitative analysis of aggregation in dilute solutions of effectively rigid biomacromolecules via the combination of oscillatory flow birefringence and viscoelasticity measurements: example study of aggregation of bovine fibrinogen in aqueous glycerol, and detection of a large aggregate formed on addition of guanidine hydrochloride

“…Note that the origin of this negative birefringence does not correspond to that of the negative birefringence reported by Haschemeyer and Tinoco [16] and by Haschemeyer [17]; the negative birefringence seen by them in the pH region of 4 to 5 was a result of a transverse permanent dipole moment being formed caused by the titration of two a-amino groups. Here the negative birefringence is the result of a negative intrinsic optical polarizability, which seems to us to suggest that extensive lateral aggregation has taken place, possibly involving of the order of 40 to 50 fibrinogen bmonomers.Q Extensive aggregation has very recently been seen in some protein solutions to which another denaturing agent, urea, has been added; these species were detected in light scattering experiments of Chow et al [18] here at Wisconsin. (We also attempted to reduce aggregation in our fibrinogen solutions by the addition of urea.…”