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. 2006 Jan 1;90(1):298-309.
doi: 10.1529/biophysj.105.070227. Epub 2005 Oct 7.

Structural characterization of apomyoglobin self-associated species in aqueous buffer and urea solution

Charles Chow  1 , Nese Kurt, Regina M Murphy, Silvia Cavagnero
Affiliations

Structural characterization of apomyoglobin self-associated species in aqueous buffer and urea solution

Charles Chow et al. Biophys J. .

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.

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Figures

FIGURE 1
FIGURE 1
ApoMb dynamic light scattering under different experimental conditions. (A) Time courses after protein solubilization in (▾) 10 mM sodium acetate buffer, 0.0 M urea, pH 6.0 (38 μM); (○) 10 mM sodium acetate buffer, 6.0 M urea, pH 6.0 (31 μM); (□) 10 mM sodium acetate buffer, 8.0 M urea, pH 6.0 (21 μM); (×) 10 mM sodium acetate buffer 0.0 M urea, pH 6.0, monomer peak collected during size-exclusion chromatography run (21 μM); (•)10 mM sodium acetate buffer, 6.0 M urea, pH 2.3 (157 μM). (B) ApoMb folding time course from 6.0 M urea and 100 mM sodium phosphate at pH 2.3. Refolding was initiated by 10-fold dilution into 100 mM sodium phosphate buffer at pH 6.4 (final pH 6.0). Final protein and urea concentrations were 12 μM and 0.6 M, respectively. Autocorrelation functions were analyzed by the cumulants method. Experimental dead times are not included in the plots.
FIGURE 2
FIGURE 2
(A) Zimm and (B) Kratky plots for apoMb-containing solution under different experimental conditions, denoted by symbols equivalent to those used in Fig. 1. Due to contributions from dust in the 0.0 M urea sample collected from SE chromatography, formula image and Rg were determined from linear fits to the data at large q2. Since the other samples contained large aggregates, the limiting slope as q2 approaches zero was used to estimate formula image and Rg.
FIGURE 3
FIGURE 3
Transmission electron micrograms of apoMb aggregates formed upon refolding the protein from a 6.0 M urea solution at pH 2.5 into 100 mM sodium phosphate buffer at final pH 6.0 (12 μM apoMb concentration), at room temperature. Data were collected after 17 h from refolding initiation.
FIGURE 4
FIGURE 4
Size exclusion chromatography analysis of apoMb under different experimental conditions: (A) 10 mM sodium acetate buffer 0.0 M urea, pH 6.0 (38 μM); (B) 10 mM sodium acetate buffer, 6.0 M urea, pH 6.0 (31 μM); (C) 10 mM sodium acetate buffer, 8.0 M urea, pH 6.0 (21 μM); (D) 10 mM sodium acetate, 6.0 M urea, pH 2.3 (157 μM); (E) Refolded from 100 mM sodium phosphate buffer, 6.0 M urea, pH 2.3, to final pH 6.0 (12 μM). The predicted elution volumes for monomer (m) and the void volume (v) in each panel are based on calibrations with protein standards performed under each of the experimental conditions A–E.
FIGURE 5
FIGURE 5
Far-UV circular dichroism spectra of apoMb samples under different experimental conditions: (▾) 10 mM sodium acetate buffer, 0.0 M urea, pH 6.0, 38 μM apoMb; (○) 10 mM sodium acetate buffer, 6.0 M urea, pH 6.0, 31 μM apoMb; (◊) 10 mM sodium acetate buffer, 8.0 M urea, pH 6.0, 21 μM apoMb; (•) 10 mM sodium acetate, 6.0 M urea, pH 2.3, 157 μM apoMb; (Δ) refolded from 100 mM sodium phosphate buffer, 6.0 M urea, pH 2.3, to final pH 6.0, 12 μM apoMb.
FIGURE 6
FIGURE 6
NMR analysis of apoMb refolding starting from an unfolded state in 6.0 M urea at pH 2.5. Refolding was triggered by a 1:10 dilution in buffer as described in the Materials and Methods section, under identical conditions to those of Fig. 1 B except that the final protein concentration was 50 μM. (A) Two representative 1H, 15N HSQC 1-dimensional (no 15N evolution) spectra taken during the time course of refolding. Data acquisition was initiated at the times indicated on the top left of the panels. (B) 2D 1H, 15N HSQC spectrum for apoMb refolding. Data acquisition was started 12 h after refolding initiation. This time is close to the end point for the aggregation time course of Fig. 1 B.
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