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. 2010 Apr 28;132(16):5596-7.
doi: 10.1021/ja101574d.

Membrane lipids influence protein complex assembly-disassembly

Leah Shin  1 , Won Jin Cho, Jeremy D Cook, Timothy L Stemmler, Bhanu P Jena
Affiliations

Membrane lipids influence protein complex assembly-disassembly

Leah Shin et al. J Am Chem Soc. .

Abstract

Approximately 11% smaller t-/v-SNARE ring complexes are generated using 50 nm cholesterol-associated vesicles as opposed to vesicles containing L-alpha-lysophosphatidylcholine (LPC), as observed using atomic force microscopy. Circular dichroism spectroscopy demonstrated that in the presence of LPC as opposed to cholesterol, N-ethylmaleimide-sensitive factor + adenosine triphosphate induces disassembly of beta-sheet structures but not the alpha-helical contents within the t-/v-SNARE complex.

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Figures

Figure 1
Figure 1
Representative AFM micrographs of approximately 50 nm in diameter liposomes and the t-/v-SNARE ring complexes formed when such clolesterol or LPC containing t-SNARE and v-SNARE proteoliposomes meet. Note the 50–53 nm in diameter cholesterol-containing liposomes (A–C). Similar size LPC-containing vesicles were prepared, and observed using the AFM (data not shown). Note the 6.89±0.61 nm t-/v-SNARE ring complexes formed when approximately 50 nm in diameter t-SNARE-cholesterol-liposomes interact with 50 nm v-SNARE-cholesterol-vesicles (D, F). Similarly, 7.746±0.646 nm t-/v-SNARE ring complexes are formed when cholesterol is replaced by LPC (E, F). (*p< 0.001).
Figure 2
Figure 2
Circular dichroism data reflecting structural changes to SNAREs associated with liposomes containing cholesterol (A) and LPC (B). Structural changes, following the assembly and (NSF-ATP)-induced disassembly of the t-/v-SNARE complex is further shown. (i) v-SNARE; (ii) t-SNAREs; (iii) t-/v-SNARE complex; (iv) t-/v-SNARE + NSF; and (v) t-/v-SNARE + NSF + 2.5 mM ATP, is shown. CD spectra were recorded at 25 °C in 5 mM sodium phosphate buffer (pH 7.5), at a protein concentration of 25 µM. In each experiment, scans were averaged per sample for enhanced signal to noise, and data were acquired on duplicate independent samples to ensure reproducibility. Note the decrease in α-helicity in the cholesterol groups as opposed to the LPC group following exposure of the t-/v-SNARE complex to NSF-ATP (Table 1).
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