Personnel
David J. Neivandt
University of Maine, Orono
Department of Chemical and Biological Engineering
319 Jenness Hall
(207) 581-2288
dneivandt@umche.maine.edu
http://www.umche.maine.edu/
chb/faculty/dneivandt.htm
Lab Members:
Joerg Fick (Post-doc)
Andrew Doyle (Ph.D Student)
Gerard Gagnon (Ph.D. Student)
Sarah Sterling (M.Sc. Student)
Jonathan Tse (Undergraduate Student)
Dale Veilleux (Undergraduate Student)
Research:
Phospholipid membrane order and deformation studied by Sum Frequency Generation Vibrational Spectroscopy (SFS)
The past twelve months has seen the publication by my group in collaboration with those of Michael Grunze (IMB/Heidelberg) and Igor Prudovsky (IMB/MMCRI) of the first study of protein induced membrane deformation by sum frequency generation vibrational spectroscopy (SFS) (Doyle et al. Langmuir 2004, 20, 8961). This study was performed employing a model membrane consisting of a gold substrate supporting a monolayer of octadecanethiol upon which a single lipid leaflet was formed via vesicle fusion of 1,2 diacyl-sn-glycero-3-[phosphor-rac-(1-glycerol)], pG. Deformation of the membrane by the signal peptideless protein FGF-1 was examined and determined to be reversible upon removal of the protein. This work marks the beginning of a collaboration in which we aim to elucidate the non-classical transport mechanism of FGF-1, a major regulator of embryogenesis and a critical participant in angiogenesis, certain types of cancer, and restenosis.
Although the above mentioned study demonstrated the power of SFS to probe molecular level conformational order and the orientation of lipid species comprising a model membrane, it did so on a membrane system with limited physiological relevance. Clearly such studies would ideally be performed directly on live cells in-vitro, or in-vivo. The physical constraints on sampling live cell systems with large high power SFS laser spectrometers, however, are daunting. In addition, if such a spectrum were recorded then it would likely defy interpretation due to the vast number of lipid, protein and polysaccharide species sampled and hence contributing to the spectrum. In order for SF studies to yield biologically meaningful information it is therefore essential that membrane analogues are employed and that they are of true physiological relevance. Unfortunately the physiological relevance of the membrane systems employed in all SF studies to date is limited. Specifically, all work in the field has investigated model membranes formed either directly on solid substrates with the associated issues of effects on membrane fluidity, prevention of effective incorporation of transmembrane proteins and the impossibility of transmembrane transport, or at an interface other than the biologically relevant aqueous/aqueous interface such as the oil/solution interface, or at the air/water interface. My group, in collaboration with the Mason group, is consequently developing an alternative system that is more biologically relevant by building upon state-of-the-art knowledge concerning ‘cushioned’ model membranes and leveraging my expertise in substrate development for SF spectroscopy.
In order to address the issues of maintaining membrane fluidity whilst providing physical space for protein transport and transmembrane protein incorporation, researchers such as Ringsdorf, Sackmann, Israelachvili, Offenhausser, Tanaka, Knoll and Frank, have developed model membranes where the lipid bilayer is spatially decoupled from the solid substrate via a lipopolymer tether, or more commonly, by a hydrated polymer/hydrogel ‘cushion’. The thickness of the spacing layer is selected such that it is greater than the dimensions of the intracellular domains of the transmembrane protein. For large cell adhesion proteins for example the spacing should be greater than 10nm. Polymers such as polyethyleneimine, polyacrylamide, and polysaccharides including dextran cellulose and chitosan have all been employed to form hydrated polymer layers. A schematic representation of such a hydrogel supported membrane illustrating the effective incorporation of active transmembrane proteins is given in below.
Figure 1. Schematic representation of a) lipid membrane supported directly on a solid substrate illustrating the difficulty in incorporating active transmembrane proteins, b) lipid membrane supported on a hydrogel cushion which enables the effective incorporation of active transmembrane proteins.
The model membrane system under development will employ this ‘cushioned’ membrane approach (where the membrane is applied by Langmuir-Blodgett deposition) while placing it on a gold coated support. The presence of the gold layer will facilitate application of the full power of SFS through enabling the determination of both the absolute orientation and degree of conformational order of the lipid species. My group in collaboration with that of Dr. Paul Davies, University of Cambridge has pioneered the development of SF model systems in which the molecules of interest such as surfactants or polymers (or potentially lipid and protein analogues) are displaced from a gold surface via a spacing layer of a selected material. The motivation has been to enable the study of the surfactant or polymer layer at the spacing layer/air or spacing layer/solution interface where the material comprising the spacing layer is chosen by the user to be of interest. The presence of the gold layer under the spacing material is critical as it enables the determination of the absolute orientation of the surfactant or polymer species through examination of the phase of the resonances in the SF spectra (‘peaks’ or ‘dips’). In the model membrane under development the spacing layer is the hydrogel cushion.
A critical aspect of the model system is that while it does allow the determination of the orientation of the molecule at the upper surface of the spacing layer from the phase of the SF signal, to do so requires a calibration be performed to account for the fact that varying the thickness of the spacing layer also results in a change in phase of the SF signal. Generating the required calibrations has necessitated the development and experimental verification of detailed theoretical models. The models are essentially based on thin film interference effects, as illustrated for a dielectric spacing layer on gold in Figure 2.
Development work to date has focused on a detailed literature review, and preliminary experiments verifying that the polysaccharide chitosan may be spin coated onto carboxcylic acid functionalized gold substrates and that the resultant films are stable in buffer solutions. Future work will involve characterization of the film thickness and swelling, theoretical prediction of the optimal film thickness for SFS and fluorescence imaging, and Langmuir-Blodgett deposition of lipid films.
a)
b)
Figure 2. a) Schematic representation of the theoretical model developed for predicting the SF spectrum arising from a resonant species at the dielectric/air interface of a dielectric material of varying thickness on gold. b) The predicted variation in phase of a CH3 resonance (plotted as SF intensity) versus thickness of the dielectric material (d, in microns). Note that the interfacial species such as a lipid would reside at the dielectric/air interface in this system but has been omitted from the figure for clarity.
Recent Publications:
1. McGall, S.J. ; Davies, P.B. ; Neivandt, D.J. “Development of a Biologically Relevant Calcium Phosphate Substrate for Sum Frequency Generation (SFG) Vibrational Spectroscopy”, J. Phys. Chem. A 109, 8745 (2005)
2. Holman, J. ; Davies, P. B. ; Nishida, T. ; Ye, S. ; Neivandt, D. J. “Sum Frequency Generation from Langmuir Blodgett Multilayer Films on Metal and Dielectric Substrates” J. Phys. Chem. B 109, 18723 (2005) Feature and Cover Article
3. Lambert, A.G.; Davies, P.B.; Neivandt, D.J. “Implementing the Theory of Sum Frequency Generation Vibrational Spectroscopy: A Tutorial Review” Appl. Spect. Reviews 40, 103 (2005)
4. Poirier, J. S. ; Tripp, C. P. ; Neivandt, D.J. “Templated Surfactant Re-Adsorption on Polyelectrolyte Induced Depleted Surfactant Surfaces” Langmuir 21, 2876 (2005)
5. Doyle, A. W.; Fick, J.; Himmelhaus, M.; Eck, W.; Graziani, I.; Prudovsky, I.; Grunze, M.; Maciag, T.; Neivandt, D.J. “Protein Deformation of Lipid Hybrid Bilayer Membranes studied by Sum Frequency Generation Vibrational Spectroscopy (SFS)” Langmuir 20, 8961 (2004)
6. Holman, J.; Ye, S.; Neivandt, D.J.; Davies, P.B “Studying Nanoparticle-Induced Structural Changes Within Fatty Acid Multilayer Films Using Sum Frequency Generation Vibrational Spectroscopy” J.A.C.S. 126, 14322 (2004)
7. McGall, S.J. ; Davies, P.B. ; Neivandt, D.J. “Interference Effects in Sum Frequency Generation Vibrational Spectra of Thin Polymer Films: an Experimental and Theoretical Investigation ”, J. Phys. Chem. B. 108, 16030 (2004)
8. Holman, J. ; Neivandt, D.J. ; Davies, P.B. “Nanoscale Interference Effect in Sum Frequency Generation from Langmuir-Blodgett Fatty Acid Films on Hydrophobic Gold”, Chem. Phys. Letts. 386, 60 (2004)
9. Holman, J. ; Davies, P.B. ; Neivandt, D.J. “Sum Frequency Spectroscopy of Langmuir-Blodgett Fatty Acid Films on Hydrophobic Gold”, J. Phys. Chem. B. 108, 1396 (2004)
10. Casford, M.T.L. ; Davies, P.B. ; Neivandt, D.J. “A Study of the Co-Adsorption of an Anionic Surfactant and an Uncharged Polymer at the Aqueous Solution/Hydrophobic Interface by Sum Frequency Spectroscopy”, Langmuir 19, 7396 (2003)
11. McGall, S.J. ; Davies, P.B. ; Neivandt, D. J. “Sum Frequency Vibrational Spectroscopy of the Comb Copolymer Cetyl Dimethicone Copolyol”, J. Phys. Chem. B. 107, 4718 (2003)
12. Lambert, A.G. ; Neivandt, D.J. ; Briggs, A.M. ; Usadi, E.W. ; Davies, P.B. “Enhanced Sum Frequency Generation from a Monolayer Adsorbed on a Composite Dielectric/Metal Substrate”, J. Phys. Chem. B. 106, 10693 (2002)
13. Lambert, A.G. ; Neivandt, D.J. ; Briggs, A.M. ; Usadi, E.W. ; Davies, P.B. “Interference Effects in Sum Frequency Spectra from Monolayers on Composite Dielectric/Metal Substrates”, J. Phys. Chem. B., 106, 5461 (2002) Cover Article
14. Windsor, R. ; Neivandt, D.J. ; Davies, P.B. “Temperature and pH effects on the Co-Adsorption of Sodium Dodecyl Sulfate and Poly(ethylenimine)”, Langmuir, 18, 2199 (2002)
15. Windsor, R. ; Neivandt, D.J. ; Davies, P.B. “Adsorption of Sodium Dodecyl Sulfate in the presence of Poly(ethylenimine) and Sodium Chloride studied using Sum Frequency Vibrational Spectroscopy”, Langmuir, 17, 7306 (2001)
16. Kawai, T. ; Neivandt, D.J. ; Davies, P.B. “Sum Frequency Generation on Surfactant-Coated Gold Nanoparticles”, J.A.C.S., 122, 12031 (2000)
17. Lambert, A.G. ; Neivandt, D.J. ; McAloney, R.A. ; Davies, P.B. “A Protocol for the Reproducible Silanisation of Mica Validated by Sum Frequency Spectroscopy and Atomic Force Microscopy”, Langmuir, 16, 8377 (2000)
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