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. 2005 Sep;14(9):2246-57.
doi: 10.1110/ps.051489405.

Aggregation of granulocyte-colony stimulating factor in vitro involves a conformationally altered monomeric state

Stephen W Raso  1 , Jeff Abel, Jesse M Barnes, Kevin M Maloney, Gary Pipes, Michael J Treuheit, Jonathan King, David N Brems
Affiliations

Aggregation of granulocyte-colony stimulating factor in vitro involves a conformationally altered monomeric state

Stephen W Raso et al. Protein Sci. 2005 Sep.

Abstract

Aggregation of partially folded intermediates populated during protein folding processes has been described for many proteins. Likewise, partially unfolded chains, generated by perturbation of numerous proteins by heat or chemical denaturants, have also been shown to aggregate readily. However, the process of protein aggregation from native-state conditions is less well understood. Granulocyte-colony stimulating factor (G-CSF), a member of the four-helix bundle class of cytokines, is a therapeutically relevant protein involved in stimulating the growth and maturation of phagocytotic white blood cells. Under native-like conditions (37 degrees C [pH 7.0]), G-CSF shows a significant propensity to aggregate. Our data suggest that under these conditions, native G-CSF exists in equilibrium with an altered conformation, which is highly aggregation prone. This species is enriched in 1-2 M GdmCl, as determined by tryptophan fluorescence and increased aggregation kinetics. In particular, specific changes in Trp58 fluorescence report a local rearrangement in the large loop region between helices A and B. However, circular dichroism, reactivity toward cyanylation, and ANS binding demonstrate that this conformational change is subtle, having no substantial disruption of secondary and tertiary structure, reactivity of the free sulfhydryl at Cys17 or exposure of buried hydrophobic regions. There is no indication that this altered conformation is important to biological activity, making it an attractive target for rational protein stabilization.

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Figures

Figure 1.
Figure 1.
Crystal structure of recombinant human G-CSF. Shown are ribbon diagrams of G-CSF. The diagram on the left provides a view orthogonal to the long axis of the protein. On the right, the diagram is rotated 90° to give a view down the axis of the 4-helix bundle. The two native disulfides (36–42 and 64–74) and tryptophans 58 and 118 are shown as stick representations. Cysteine 17, the only free thiol in the protein, is shown as a space-filling model. The disulfide bond 64–74 was removed from the image on the right for clarity.
Figure 2.
Figure 2.
Concentration dependence of G-CSF aggregation kinetics in vitro. The loss of soluble G-CSF at 1 mg/mL (▪), 5 mg/mL (○), and 20 mg/mL (•) was measured as a function of time incubated in 0.1 M MOPS (pH 7.0) at 37°C. Lines through the data points do not represent a curve fit but are present to guide the eye.
Figure 3.
Figure 3.
A hyperfluorescent state of G-CSF is populated during equilibrium denaturation. The GdmCl-induced unfolding of G-CSF was monitored using tryptophan fluorescence, integrated intensity from 300–400 nm (□) and CD at 222 nm (•). The solid line through the CD data represents a curve fit to a two-state folding model. Denaturation was carried out in 0.1 M MOPS (pH 7.0) at 25°C. The protein concentration was 0.05 mg/mL. The data points represent the results from duplicate samples, and error bars showing the standard deviation were smaller than the data point symbols.
Figure 4.
Figure 4.
Biophysical characterization of the hyperfluorescent state of G-CSF. (A) The fluorescence emission spectra of G-CSF in 0 M(solid line), 1.5M(dotted), and 5M(dashed) GdmCl. The excitation wavelength was 280 nm and the protein concentration was 0.05 mg/mL. (B) The far-UV CD spectra of G-CSF in 0M(□) and 1.5M(•) GdmCl. A protein concentration of 0.5 mg/mL was used. (C) Size-exclusion chromatography shows that G-CSF remains monomeric under native conditions (top) and in 1.5 M GdmCl (bottom). The arrow indicates where a dimer would be expected to elute, based on column calibration standards. (D) ANS binding to G-CSF in 0, 1, 2, and 5 M GdmCl. The difference spectra of 25 mM ANS in the presence and absence of 0.1 mg/mL G-CSF are shown; the excitation wavelength was 360 nm. All of the experiments in A–D were performed in 0.1 M MOPS (pH 7.0) at 25°C.
Figure 5.
Figure 5.
Aggregation propensity of G-CSF in various concentrations of GdmCl. (A) Aggregation kinetics of 2 mg/mL G-CSF in several concentrations of GdmCl (shown), 0.1 M MOPS (pH 7.0) at 37°C. Lines through the data points are provided to guide the eye. (B) An overlay of the fraction of soluble protein remaining after 18 h under the conditions described for A (solid line) with a denaturation curve of GCSF, where tryptophan fluorescence was used to monitor protein conformation (•). Conditions for the denaturation curve were 0.05 mg/mL protein, 0.1 M MOPS (pH 7.0) at 25°C.
Figure 6.
Figure 6.
Equilibrium denaturation of W58Q and W118F G-CSF in GdmCl. (A) W58Q denaturation curve, (B) W118F denaturation curve, and (C) wild-type denaturation curve (•) and the normalized sum of the two mutant curves (□). All denaturation experiments were performed using 0.05 mg/mL in 0.1 M MOPS (pH 7.0) at 25°C.
Figure 7.
Figure 7.
Near-UV CD spectra of W58Q, W118F, and wild-type GCSF. Near-UV CD spectra of G-CSF in 0 M (solid lines) and 1.5 M (dashed lines) GdmCl. (A) W58Q, (B) W118F, (C) wild type. All spectra were collected at a protein concentration of 1.0 mg/mL in 0.1 M MOPS (pH 7.0) at 25°C.
Figure 8.
Figure 8.
Cysteine 17 and G-CSF aggregation. (A) Wild-type G-CSF (▪) and C17A (○) were subjected to the same aggregation assay depicted in Figure 2 ▶. All experimental conditions were the same, except that the protein concentration was 2.5 mg/mL. (B) The aggregated G-CSF from the 5 mg/mL experiment shown in Figure 2 ▶ was dissolved in SDS-containing buffer and analyzed by SEC. Protein peaks were detected by UV absorbance at 214 nm.
Scheme 1
Scheme 1
Scheme 2
Scheme 2
Scheme 3
Scheme 3
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