1. Non-classical Export of Signal Peptide-less Proteins Studied via Vibrational Spectroscopy and Liposome Destabilization Techniques Andrew Doyle Functional Genomics Ph.D. Program and Department of Chemical and Biological Engineering 62 nd OSU International Symposium on Molecular Spectroscopy (06/21/07)

2. Classical Protein Translocation to the ER Lumen – Directed by Signal Peptide

3. Background Non–classical export of signal peptide-less proteins such as FGF-1 is not well understood. FGF-1 is involved in disorders such as restenosis, Alzheimer’s disease and cancer. Multi-protein complexes of FGF-1 (8.5-10 nm) traverse lipid bilayers by unknown mechanisms. Transport greatest in membranes comprising acidic phospholipids.

4. Proposed Non-classical Release Mechanism

5. Background Three-dimensional representation of the β-barrel structures of human FGF-1 and human mIL-1α. β- sheet domains are indicated in yellow and are depicted as rotating counter clockwise around the open centers of the structures. The structures were obtained from the Protein Data Bank of the NCBI (http://www.rcsb.org/pdb/).

6. In the presence of acidic phospholipids FGF-1 takes a partially unfolded molten globule conformation. It is known that FGF-1 binds to acidic phospholipids through domains in the C-terminus. Experiments were performed monitoring the dose dependent leakage of fluorescent dyes encapsulated within liposomes comprising different pL when exposed to WT FGF-1 and forms mutated in the C-terminus. Protein secretion and localization were also studied. Liposome Permealization Experiments and Localization Studies

7. Point Mutations in p40 Syt1 and FGF-1 Decrease Liposome Permealization

8. Attenuation of Protein Secretion in Mutants FGF-1

9. Intracellular Distribution of wt FGF1 and FGF1 Mutants K114,115A 37°C 42°C K126,127A FGF-HA wt

10. Silicon Gold ODT DpG 60 °C PBS Rinse PBS FGF-1 Addition FGF-1 in PBS Solution ? PBS Rinse PBS ? Experimental Design

11. Vesicle Fusion PBS Rinse 1 nM FGF-1 PBS Rinse Reversible Membrane Deformation

12. The spectrum after vesicle fusion indicates the formation of a highly ordered bilayer. The adsorption of the very small concentration of 1 nM FGF-1 induces conformational disorder in the HBM as is observed by the strengthening of methylene resonance modes and the slight loss of methyl resonance modes. A brief wash to remove a portion of the protein restores a large degree of order in the HBM. These observations show that the conformational disorder induced by FGF-1 is at least partially reversible. This data is the first recorded observing the in situ protein induced deformation of a biological membrane by SFS. Conclusions

13. Acknowledgements University of Maine –Dr. David J. Neivandt –Dr. Joerg Fick –Sarah Sterling Maine Medical Center Research Institute –Dr. Igor Prudovsky –Dr. Irene Graziani –Dr. Raffaella Soldi University of Heidelberg –Dr. Michael Grunze –Dr. Michael Himmelhaus Seeking Post-doc!!!!!!!

14. Interdisciplinary Ph.D. in Functional Genomics Interdisciplinary, inter-institutional Ph.D. program $30,000/year stipend funded by an NSF-IGERT training grant World-class faculty from the biological, computational and physical sciences Twinning: students work under two mentors from two different disciplines Information: www.umaine.edu/genomics