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Analysis of interactions between SNARE proteins using imaging ellipsometer coupled with microfluidic array.

Qi C, Zhang H, Liu L, Yang R, Yang Y, Kang T, Hao W, Jin G, Jiang T - Sci Rep (2014)

Bottom Line: The SNAREs were immobilized on the silicon wafer and then analyzed in a pairwise mode with microfluidic array, leading us to discover the interactions between Ykt6p and Sso2p, Sec22p and Sso2p.Moreover, by using the real-time function of the imaging ellipsometer, we were able to obtain their association constants (K(A)) of about 10(4) M(-1).We argue that the use of imaging ellipsometer coupled with microfluidic device will deepen our understanding of the molecular mechanisms underlying membrane fusion process.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Biophysics, Chinese Academy of Sciences, #15, Datun Rd., Beijing, 100101, China [2] Institute of Equipment Technology, Chinese Academy of Inspection and Quarantine, #3, Gaobeidian North Rd., Beijing, 100123, China [3].

ABSTRACT
The soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins are small and abundant membrane-bound proteins, whose specific interactions mediate membrane fusion during cell fusion or cellular trafficking. In this study, we report the use of a label-free method, called imaging ellipsometer to analyze the interactions among three SNAREs, namely Sec22p, Ykt6p and Sso2p. The SNAREs were immobilized on the silicon wafer and then analyzed in a pairwise mode with microfluidic array, leading us to discover the interactions between Ykt6p and Sso2p, Sec22p and Sso2p. Moreover, by using the real-time function of the imaging ellipsometer, we were able to obtain their association constants (K(A)) of about 10(4) M(-1). We argue that the use of imaging ellipsometer coupled with microfluidic device will deepen our understanding of the molecular mechanisms underlying membrane fusion process.

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SDS-PAGE gel of recombinant cytosolic domain of SNARE proteins stained with Coomassie Brilliant Blue.
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f1: SDS-PAGE gel of recombinant cytosolic domain of SNARE proteins stained with Coomassie Brilliant Blue.

Mentions: The cytosolic regions (without the transmembrane domain) of Sso2p, Sec22p and Ykt6p were expressed in E. Coli and purified to high quality (Fig. 1). To detect their interactions, we designed the microfluidic array as illustrated in Fig. 2a and Fig. 2b. According to the design, the interactions among three SNAREs were detected in 12 parallel areas (Fig. 2c). The application of target proteins will form a layer on a chip, and the interacting partners will form another layer on the chip (Fig. 2d). The light reflection intensity represented by grayscale value indicates the thickness of the SNARE protein film. Thus the increase of the grayscale value after the addition of potential interacting proteins could indicate the interaction strength. Fig. 3 shows the changes in grayscale values for bindings between Sso2p, Sec22p and Ykt6p. The corresponding P-values were calculated with single factor analysis of variance in Microsoft Office Excel24. As observed in Fig. 3, the protein A-IgG pair, the positive control on the microfluidic chip, demonstrated significant change in grayscale value in both directions (immobilizing protein A then adding IgG (P-value = 0.0002); immobilizing IgG then adding protein A (P-value = 0.0295)). Notably, for the Sso2p-Sec22p pair, we also observed significant change in grayscale value in both directions (immobilizing Sso2p then adding Sec22p (P-value = 0.0273); immobilizing Sec22p then adding Sso2p (P-value = 0.1227)). In addition, the Sso2p-Ykt6p pair could be regarded as positive interaction as there observed a significant change in grayscale when Ykt6p was added to the immobilized Sso2p (P-value = 0.0084). In static measurement, rinsing may cause remarkable false negatives especially for weak interactions. But false positives will not occur in high frequency. In this regard, the static ellipsometry measurement is reliable for weak interaction detection and it can be used as a rapid and convenient screening method before kinetic analysis.


Analysis of interactions between SNARE proteins using imaging ellipsometer coupled with microfluidic array.

Qi C, Zhang H, Liu L, Yang R, Yang Y, Kang T, Hao W, Jin G, Jiang T - Sci Rep (2014)

SDS-PAGE gel of recombinant cytosolic domain of SNARE proteins stained with Coomassie Brilliant Blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061542&req=5

f1: SDS-PAGE gel of recombinant cytosolic domain of SNARE proteins stained with Coomassie Brilliant Blue.
Mentions: The cytosolic regions (without the transmembrane domain) of Sso2p, Sec22p and Ykt6p were expressed in E. Coli and purified to high quality (Fig. 1). To detect their interactions, we designed the microfluidic array as illustrated in Fig. 2a and Fig. 2b. According to the design, the interactions among three SNAREs were detected in 12 parallel areas (Fig. 2c). The application of target proteins will form a layer on a chip, and the interacting partners will form another layer on the chip (Fig. 2d). The light reflection intensity represented by grayscale value indicates the thickness of the SNARE protein film. Thus the increase of the grayscale value after the addition of potential interacting proteins could indicate the interaction strength. Fig. 3 shows the changes in grayscale values for bindings between Sso2p, Sec22p and Ykt6p. The corresponding P-values were calculated with single factor analysis of variance in Microsoft Office Excel24. As observed in Fig. 3, the protein A-IgG pair, the positive control on the microfluidic chip, demonstrated significant change in grayscale value in both directions (immobilizing protein A then adding IgG (P-value = 0.0002); immobilizing IgG then adding protein A (P-value = 0.0295)). Notably, for the Sso2p-Sec22p pair, we also observed significant change in grayscale value in both directions (immobilizing Sso2p then adding Sec22p (P-value = 0.0273); immobilizing Sec22p then adding Sso2p (P-value = 0.1227)). In addition, the Sso2p-Ykt6p pair could be regarded as positive interaction as there observed a significant change in grayscale when Ykt6p was added to the immobilized Sso2p (P-value = 0.0084). In static measurement, rinsing may cause remarkable false negatives especially for weak interactions. But false positives will not occur in high frequency. In this regard, the static ellipsometry measurement is reliable for weak interaction detection and it can be used as a rapid and convenient screening method before kinetic analysis.

Bottom Line: The SNAREs were immobilized on the silicon wafer and then analyzed in a pairwise mode with microfluidic array, leading us to discover the interactions between Ykt6p and Sso2p, Sec22p and Sso2p.Moreover, by using the real-time function of the imaging ellipsometer, we were able to obtain their association constants (K(A)) of about 10(4) M(-1).We argue that the use of imaging ellipsometer coupled with microfluidic device will deepen our understanding of the molecular mechanisms underlying membrane fusion process.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Biophysics, Chinese Academy of Sciences, #15, Datun Rd., Beijing, 100101, China [2] Institute of Equipment Technology, Chinese Academy of Inspection and Quarantine, #3, Gaobeidian North Rd., Beijing, 100123, China [3].

ABSTRACT
The soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins are small and abundant membrane-bound proteins, whose specific interactions mediate membrane fusion during cell fusion or cellular trafficking. In this study, we report the use of a label-free method, called imaging ellipsometer to analyze the interactions among three SNAREs, namely Sec22p, Ykt6p and Sso2p. The SNAREs were immobilized on the silicon wafer and then analyzed in a pairwise mode with microfluidic array, leading us to discover the interactions between Ykt6p and Sso2p, Sec22p and Sso2p. Moreover, by using the real-time function of the imaging ellipsometer, we were able to obtain their association constants (K(A)) of about 10(4) M(-1). We argue that the use of imaging ellipsometer coupled with microfluidic device will deepen our understanding of the molecular mechanisms underlying membrane fusion process.

Show MeSH