CHARACTERIZING IQGAP1-C DOMAIN BINDING TO PHOSPHOINOSITIDES A Major Qualifying Project Report Submitted to the Faculty of WORCESTER POLYTECHNIC INSTITUTE In partial fulfillment of the requirements for the Degree of Bachelor of Science in Biochemistry Written by: Approved by: __________________ . Christopher Dallarosa Dr. Arne Gericke Date: April 2018 Table of Contents Acknowledgments........................................................................................................................... 3 Abstract ........................................................................................................................................... 4 Introduction ..................................................................................................................................... 5 Background ..................................................................................................................................... 6 History ......................................................................................................................................... 6 Scaffolding Proteins .................................................................................................................... 6 IQGAP Protein Family ................................................................................................................ 8 IQGAPs in Yeast Cells ............................................................................................................ 8 Mammalian IQGAPs .............................................................................................................. 9 IQGAP1 ....................................................................................................................................... 9 Domains ................................................................................................................................... 9 Expression ............................................................................................................................. 10 Location within the Cell ........................................................................................................ 10 Cellular Function ................................................................................................................... 11 Physiological Function .......................................................................................................... 13 IQGAP Functions Relevant to Cancer ................................................................................... 15 IQGAP1 and Phosphoinositide 3-Kinase (PI3K)/Akt Signaling .............................................. 18 Scientific Techniques .................................................................................................................... 20 Fluorescence Correlation Spectroscopy (FCS) ......................................................................... 20 Fluorescence Resonance Energy Transfer (FRET) ................................................................... 20 Circular Dichroism .................................................................................................................... 21 Proposed Research and Aims........................................................................................................ 22 Aims .......................................................................................................................................... 22 Aim 1- Purify full length IQGAP1 protein as well as IQGAP1 domains ............................. 22 Aim 2- Characterize the binding between IQGAP1 and PIP2 and PIP3 using LUVs .......... 22 Research Outline ....................................................................................................................... 22 Aim 1- Purify full length IQGAP1 protein as well as IQGAP1 domains ............................. 22 Aim 2- Characterize the binding between IQGAP1 and PIP2 and PIP3 using LUVs .......... 24 Methodology ................................................................................................................................. 25 Protein Expression..................................................................................................................... 25 Protein Purification ................................................................................................................... 25 1 Batch Method ........................................................................................................................ 25 Purity Assessment, Quantification of Protein Concentration, and Storage ............................... 26 Model Membrane Binding Studies ........................................................................................... 27 Spectrometer Settings ............................................................................................................ 27 Sample Preparation ................................................................................................................ 27 Large Unilamellar Vesicles (LUVs) Preparation .................................................................. 27 Lipid Titrations ...................................................................................................................... 27 Circular Dichroism .................................................................................................................... 27 Fluorescence Correlation Spectroscopy (FCS) ......................................................................... 27 Sample Preparation ................................................................................................................ 27 Fluorescent Labeling ............................................................................................................. 28 Large Unilamellar Vesicles (LUVs) Preparation .................................................................. 28 Results and Discussion ................................................................................................................. 29 Immuno-colocalization.............................................................................................................. 29 Protein Purification ................................................................................................................... 30 Batch Method ........................................................................................................................ 30 Fast Protein Liquid Chromatography .................................................................................... 32 Circular Dichroism .................................................................................................................... 33 Fluorescence Correlation Spectroscopy (FCS) ......................................................................... 33 Model Membrane Binding Study .............................................................................................. 35 Conclusions and Future Directions ............................................................................................... 40 References ..................................................................................................................................... 41 Appendix ....................................................................................................................................... 43 Protein Purification ................................................................................................................... 43 Fast Protein Liquid Chromatography .................................................................................... 43 Characterization ........................................................................................................................ 46 Fluorescence Correlation Spectroscopy ................................................................................ 46 Model Membrane Binding Study .......................................................................................... 47 2 Acknowledgments I would like the thank Dr. Arne Gericke for allowing to work in his lab for this project. His advice and the tools he provided were very helpful in completing this project. I would like to give a special thanks to Dr. V Siddartha Yerramilli. Throughout the entirety of the project he was there to help me with any problems that I had encountered, He taught me the skills and techniques to complete this project. I would also like to thank Alonzo Ross for providing assistance, and support with the project. I would also like to thank the Scarlata Lab for any assistance that they may have provided. 3 Abstract IQ-motif-containing GTPase-activating protein 1 (IQGAP1), also known as p195, is a ubiquitously expressed protein that is a member of the IQGAP family of scaffolding proteins. Due to the numerous binding partners IQGAP1 interacts with, this protein has been found to regulate a diverse range of cellular processes including: cell migration, cell proliferation, cytoskeletal dynamics, and intracellular signaling. Previous studies indicated that IQGAP1 binds to phosphoinositides, but the exact nature of the binding is not well characterized. Interactions between IQGAP1 and phosphoinositides can be characterized by in-vitro studies using purified proteins and phosphoinositide containing model membranes. Because IQGAP1 is a large multi- domain protein (190kDa), purifying it presented various challenges, including high protein degradation.