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Background Current membrane models have been formed:

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CH. CH. 2. 3. SF Intensity (arb). d (nm). Dominant Periodicity = 246 nm Minor Periodicities = 276 nm, 2.252 m m.

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slide1
CH

CH

2

3

SF Intensity (arb)

d (nm)

Dominant Periodicity = 246 nm

Minor Periodicities = 276 nm, 2.252 mm

Model Cellular Membrane for the Study of Non-Classical Protein Transport Sarah M. Sterling1,2, Lei Li3, Joerg Fick1, Michael D. Mason1,2,4, Igor Prudovsky2,4,5, David J. Neivandt1,2,41Department of Chemical and Biological Engineering, University of Maine, Orono, ME 044692Graduate School of Biomedical Sciences, University of Maine, Orono, ME 04469 3Department of Physics and Astronomy, University of Maine, Orono, ME 044694Institute for Molecular Biophysics, Orono, ME 044695Maine Medical Center Research Institute, Scarborough, ME 04074

Motivation:

Due to the correlation between membrane structure and function, a great deal of work is focused on determining the distribution, kinetics, and structure of lipid and protein membrane constituents, often as a result of their interactions. One such interaction is the non-classical export of signal peptide-less proteins (SPLs). This small subset of proteins does not utilize the Endoplasmic Reticulum (ER)-Golgi pathway of release, yet the proteins still play roles in cell survival, cell growth, and angiogenesis. Combining spectroscopic and microscopic techniques allows for a better understanding of this unknown mechanism. Development of a planar model membrane system replicating in a controlled manner, the cross-section of a biological membrane has been necessary in order to enable application of many of these techniques.

  • Background
  • Current membrane models have been formed:
  • Directly on solid substrates (on 1-2 nm water layer)
  • At the oil/solution interface
  • At the air/water interface
  • Disadvantages:
  • Prevent effective incorporation of transmembrane proteins with intracellular domains
  • Impossibility of transmembrane transport
  • Not the biologically relevant aqueous/aqueous interface
  • The model membrane system (above) provides an alternative physiologically relevant system, building upon knowledge concerning ‘cushioned’ model membranes.
  • Advantages:
  • Biologically relevant model membrane
  • Through use of the gold enables determination of both the orientational and conformational of constituent species by Sum Frequency Generation Vibrational Spectroscopy
  • Model Membrane Preparation
  • Chitosanhydrogel was prepared and spin-coated on functionalized gold substrates with hydrogel thickness in the range of 53-63 nm
  • Thickness variation per film is within 1 nm from center to edge and no significant change (desorption) was noted when placed in neutralizing buffer
  • Measurement of degree of swelling in 10 mM HEPES at pH 7.4 via ellipsometry has shown a swelling ratio of 2.3
  • Deposition of lipid bilayer on the hydrogel via the Langmuir-Blodgett/Langmuir-Schäfer method (LB/LS) has been successful
  • LB depositions have shown favorable transfer ratios with zwitterionic and acidic phospholipids, specifically DMPC and DMPG
  • Atomic force microscopy determined the rms roughness of an LB film on hydrogel to be 1.1 nm

Sum Frequency Generation (SFS)

A sum frequency signal is produced by overlapping two pulsed laser beams temporally and spatially at an interface. One beam is at a fixed visible frequency while the other is a tunable frequency infrared (IR) beam. Photons are emitted at the sum of the two frequencies. Detecting these photons as a function of the IR wavelength produces a vibrational spectrum of interfacial molecules from which the following information may be obtained:

Polar Orientationspectralpeaks or dips

Molecular Conformationresonances in the spectrum

Interference Effect in SFS Spectra

Due to the hydrogel spacing layer between the lipid bilayer and the gold surface, an optical interference effect occurs which requires calibration:

Schematic representation of the model membrane system comprising a lipid bilayer supported on a hydrogel spacing layer attached to a gold coated substrate.

CH

3

Schematic representation of LB/LS deposition.

CH

3

Currently, fluorescence correlation spectroscopy is being used to determine lipid membrane diffusion coefficients, verifying the phase transition temperature. Shown here are preliminary results.

Acknowledgements: The Institute for Molecular Biophysics, the Pulp and Paper Foundation of the University of Maine, the University of Maine Graduate School of Biomedical Sciences, and the National Science Foundation – Major Research Instrumentation CHE-0722759 grant for Financial Assistance

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