Circular dichroism (CD) spectroscopy of the assembly and disassembly of SNAREs: The proteins involved in membrane fusion in cells - PubMed (original) (raw)

Circular dichroism (CD) spectroscopy of the assembly and disassembly of SNAREs: The proteins involved in membrane fusion in cells

Jeremy D Cook et al. Chem Phys Lett. 2008.

Abstract

In this study, we report for the first time that both t-SNAREs and v-SNARE and their complexes in buffered suspension, exhibit defined peaks at CD signals of 208 and 222 nm wavelengths, consistent with a higher degree of helical secondary structure. Surprisingly, when incorporated in lipid membrane, both SNAREs and their complexes exhibit reduced folding. In presence of NSF-ATP, the SNARE complex disassembles, as reflected from the CD signals demonstrating elimination of α-helices within the structure.

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Figures

Fig. 1

Circular dichroism data reflecting structural changes to SNAREs, both in suspension and in association with membrane. Structural changes, following the assembly and disassembly of the t-/v-SNARE complex is further shown. (A) CD spectra of purified full-length SNARE proteins in suspension and (B) in membrane-associated; their assembly and (NSF–ATP)-induced disassembly is demonstrated. (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 10 μM. In each experiment, 30 scans were averaged per sample for enhanced signal to noise, and data were acquired on duplicate independent samples to ensure reproducibility.

Fig. 2

AFM micrographs of t-/v-SNARE complex formed in buffered suspension (A, B) and when membrane-associated t-SNAREs and v-SNARE interact (C, D). (A) and (C) are low resolution images; (B) and (D) are high resolution images of the t-/v-SNARE complexes formed.

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