4.5 Article

Measuring distances in supported bilayers by fluorescence interference-contrast microscopy: Polymer supports and SNARE proteins

Journal

BIOPHYSICAL JOURNAL
Volume 84, Issue 1, Pages 408-418

Publisher

BIOPHYSICAL SOCIETY
DOI: 10.1016/S0006-3495(03)74861-9

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Funding

  1. NIAID NIH HHS [R01 AI030557, R37 AI030557, AI30557] Funding Source: Medline
  2. NIGMS NIH HHS [P01 GM072694] Funding Source: Medline
  3. NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [R01AI030557, R37AI030557] Funding Source: NIH RePORTER
  4. NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [P01GM072694] Funding Source: NIH RePORTER

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Fluorescence interference-contrast (FLIC) microscopy is a powerful new technique to measure vertical: distances from reflective surfaces. A pattern of varying intensity is created by constructive and destructive interference of the incoming and reflected light at the surface of an oxidized silicon chip. Different levels of this pattern are probed by manufacturing silicon chips with terraces of oxide layers of different heights. Fluorescence collected from membranes that are deposited on these terraces is then used to measure the distance of the fluorescent probes from the silicon oxide surface. Here, we applied the method to measure the distance between supported lipid bilayers and the surface of oxidized silicon chips. For plain fluid phosphatidylcholine bilayers, this distance was 1.7 +/- 1.0 nm. The cleft distance was increased to 3.9 +/- 0.9 nm in bilayers that were supported on a 3400-Da polyethylene glycol cushion. This distance is close to the Flory distance (4.8 nm) that would be expected for a grafted random coil of this polymer. In a second application, the distance of a membrane-bound protein from the membrane surface was measured. The integral membrane protein syntaxin1A/SNAP25 (t-SNARE) was reconstituted into tethered polymer-supported bilayers. A soluble form of the green fluorescent protein/vesicle-associated membrane protein (GFP-VAMP) was bound to the reconstituted t-SNAREs. The distance of the GFP from the membrane surface was 16.5 +/- 2.8 nm, indicating an upright orientation of the rod-shaped t-SNARE/v-SNARE complex from the membrane surface.

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