Interaction of horse heart and thermus thermophilus type c cytochromes with phospholipid vesicles and hydrophobic surfaces

Abstract

The binding of horse heart cytochrome c (cyt-c) and Thermus thermophilus cytochrome c(552) (cyt-c(552)) to dioleoyl phosphatidylglycerol (DOPG) vesicles was investigated using Fourier transform infrared (FTIR) spectroscopy and turbidity measurements. FTIR spectra revealed that the tertiary structures of both cytochromes became more open when bound to DOPG vesicles, but this was more pronounced for cyt-c. Their secondary structures were unchanged. Turbidity measurements showed important differences in their behavior bound to the negatively charged DOPG vesicles. Both cytochromes caused the liposomes to aggregate and flocculate, but the ways they did so differed. For cyt-c, more than a monolayer was adsorbed onto the liposome surface prior to aggregation due to charge neutralization, whereas cyt c(552) caused aggregation at a protein/lipid ratio well below that required for charge neutralization. Therefore, although cyt-c may cause liposomes to aggregate by electrostatic interaction, cyt-c(552) does not act in this way. FTIR-attenuated total reflection spectroscopy (FTIR-ATR) revealed that cyt-c lost much of its secondary structure when bound to the hydrophobic surface of octadecyltrichlorosilane, whereas cyt-c(552) folds its domains into a beta-structure. This hydrophobic effect may be the key to the difference between the behaviors of the two cytochromes when bound to DOPG vesicles.

FIGURE 1

FTIR spectra of cyt-c₅₅₂ in deuterated buffer (pD 8). (A) Experimental spectrum obtained after subtracting the buffer contribution. (B) Spectrum showing individual band components obtained from second-derivative analysis. (C) Experimental spectrum including the band components.

FIGURE 2

Optical density was recorded at 650 nm, while adding cyt-c to 25, 50, 100, 150, and 200 μM of DOPG in 5 mM HEPES,1 mM EDTA buffer, pH 7.6 (A). Detail of one curve to show the breakpoints (B).

FIGURE 3

Optical density was recorded at 650 nm, while adding cyt-c₅₅₂ to 25, 50, 100, 150, and 200 μM of DOPG in 10 mM Tris-HCl, pH 7.6 (A). Detail of one curve to show the breakpoints (B).

FIGURE 4

Linear relationship between the total protein concentration and the DOPG concentration at each breakpoint. (A) cyt-c; (B) cyt-c₅₅₂.

FIGURE 5

(A) Percentage of cyt-c amide I' groups involved in secondary-structure elements: white, cyt-c (4 mg/mL) in deuterated buffer; light gray, cyt-c bound to DOPG vesicles. (B) white, cyt-c₅₅₂ (4 mg/ml) in deuterated buffer; light gray, cyt-c₅₅₂ bound to DOPG vesicles.

FIGURE 6

Percentage of cyt-c₅₅₂ amide I' group involved in secondary-structure elements. (A) White, cyt-c (4 mg/ml) in deuterated buffer; dark gray, cyt-c (100 μg/ml) adsorbed onto OTS surface. (B) White, cyt-c₅₅₂ (4 mg/ml) in deuterated buffer; dark gray, cyt-c₅₅₂ (100 μg/ml) adsorbed onto an OTS surface.

FIGURE 7

Space-filling plot of the structures of cyt-c (A) and cyt-c₅₅₂ (B). Lysine and arginine residues are shown in red and hydrophobic residues in green. Right: View of the front side including the heme site (yellow); left: view of the back side of the heme. The figures were prepared with the program RASMOL.