Department of Physics, Chemistry and Biology Master's Thesis Characterization of Protein Surface Interactions: Collagen and Osteocalcin Patrik Johansson 2013-06-17 LITH-IFM-A-EX--13/2780--SE Linköping University Department of Physics, Chemistry and Biology 581 83 Linköping Department of Physics, Chemistry and Biology Characterization of Protein Surface Interaction: Collagen and Osteocalcin Patrik Johansson Thesis work done at NESAC/BIO, University of Washington, Seattle. 2013-06-17 Supervisor Prof. Patrick Koelsch Examiner Prof. Kajsa Uvdal Linköping University Department of Physics, Chemistry and Biology 581 83 Linköping 2 Avdelning, institution Datum Division, Department Date Physics 2013-06-17 Department of Physics, Chemistry and Biology Linköping University Språk Rapporttyp ISBN Language Report category Svenska/Swedish Licentiatavhandling ISRN: LITH-IFM-A-EX--13/2780--SE Engelska/English Examensarbete _________________________________________________________________ C-uppsats D-uppsats Serietitel och serienummer ISSN ________________ Övrig rapport Title of series, numbering ______________________________ _____________ URL för elektronisk version Titel Title Characterization of Protein Surface Characterization: Collagen and Osteocalcin Författare Author Patrik Johansson Sammanfattning Abstract This work investigates how the proteins collagen type I and human osteocalcin interact with various surfaces. A pH- series of collagen adsorbed onto methyl terminated self-assembled monolayers has been made and the results indicate that less tropocollagen is found on the surfaces at pH below 6.0 and that biofilms made of larger fibrils with a more ordered 3D-structure are formed at pH 6.0 and above. This work also shows that it is possible to divide the amide I region of a vibrational Sum Frequency Generation (v-SFG) spectra into three peaks. These peaks can be correlated to the three amino acid residues glycine, proline and hydroxyproline, which have a high abundance in collagen. Analysis of different polarization combinations probing chiral and achiral contributions demonstrates that glycine has a higher contribution over proline and hydroxyproline in achiral responses, whereas hydroxyproline has similar or higher contribution than glycine in chiral responses, in which little to no signals from proline are detectable. v-SFG data for carboxylated and uncarboxylated osteocalcin respectively reveal that carboxylated osteocalcin has α-helices in the structure when Ca2+ ions are present in the solution, while the uncarboxylated version does not. Orientations for osteocalcin adsorbed onto hydrophobic, positively charged and negatively charged surfaces were determined by dividing peak areas of fragments from leucine, cysteine and carboxyglutamic acids from the positive Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) spectra. Also, it was exercised to find the v-SFG signal with a femtosecond SFG system by utilizing the non-resonant background of a gold substrate and then delay the visible laser beam to only get signals from the vibrating molecules. Self-assembled monolayers (SAMs) prepared from dodecanethiols were used to demonstrate this principle, but the approach is valid also for other molecular systems. Nyckelord Keyword collagen fibrils, osteocalcin, ToF-SIMS, SFG, chiral, XPS, AFM, surface analysis. 3 Abstract This work investigates how the proteins collagen type I and human osteocalcin interact with various surfaces. A pH-series of collagen adsorbed onto methyl terminated self-assembled monolayers has been made and the results indicate that less tropocollagen is found on the surfaces at pH below 6.0 and that biofilms made of larger fibrils with a more ordered 3D- structure are formed at pH 6.0 and above. This work also shows that it is possible to divide the amide I region of a vibrational Sum Frequency Generation (v-SFG) spectra into three peaks. These peaks can be correlated to the three amino acid residues glycine, proline and hydroxyproline, which have a high abundance in collagen. Analysis of different polarization combinations probing chiral and achiral contributions demonstrates that glycine has a higher contribution over proline and hydroxyproline in achiral responses, whereas hydroxyproline has similar or higher contribution than glycine in chiral responses, in which little to no signals from proline are detectable. v-SFG data for carboxylated and uncarboxylated osteocalcin respectively reveal that carboxylated osteocalcin has α-helices in the structure when Ca2+ ions are present in the solution, while the uncarboxylated version does not. Orientations for osteocalcin adsorbed onto hydrophobic, positively charged and negatively charged surfaces were determined by dividing peak areas of fragments from leucine, cysteine and carboxyglutamic acids from the positive Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) spectra. Also, it was exercised to find the v-SFG signal with a femtosecond SFG system by utilizing the non-resonant background of a gold substrate and then delay the visible laser beam to only get signals from the vibrating molecules. Self-assembled monolayers (SAMs) prepared from dodecanethiols were used to demonstrate this principle, but the approach is valid also for other molecular systems. Abbreviations: AFM - atomic force microscopy SHG - second harmonic generation OC - osteocalcin ToF-SIMS - time-of-flight secondary ion mass PBS - phosphate buffered saline spectrometry PCA - principal component analysis uOC - uncarboxylated osteocalcin pI - isoelectric point v-SFG - vibrational sum frequency generation SAM - self-assembled monolayer XPS - x-ray photoelectron spectroscopy SFG - sum frequency generation 4 Acknowledgements It was a very interesting experience for me to travel abroad to Seattle, WA, USA, and do my diploma work at NESAC/BIO. It has given me unique experiences and a lot of personal development. A lot of work was put into this project and I want to express my deepest gratitude to some people and organizations that have been of great importance. I would like to thank Professor Patrick Koelsch for great supervision and for always being available for discussions regarding obtained results or cultural experiences; professor David G. Castner for accepting me as a member in his group at NESAC/BIO; Dr. Dan Graham for all the help regarding the ToF-SIMS data and for developing the NESAC/BIO Toolbox used in this study and NIH grant EB-002027 for supporting that toolbox. I also want to thank Dr. Gerry Hammer for the acquisition of XPS data and for his help with the analysis of that data; Rami Foster for collecting the AFM images of the collagen samples; Blake Bluestein and Michael Robinson for their help acquiring the ToF-SIMS data. Sparbanksstiftelsen Alfas Internationella Stipendiefond för Linköpings Universitet, Bröderna Molanders Stiftelse and Anna Whitlock's Stiftelse provided scholarships that made it possible economically for me to undertake this adventure. Family and friends provided a lot of support, which has been an important source of motivation. Lastly, I want to thank my opponent, Gustav Emilsson, and my examiner, professor Kajsa Uvdal, for their invaluable input and support throughout all work. Linköping in May 2013 Patrik Johansson 5 Table of Contents 1 Introduction ........................................................................................................................ 7 1.1 Aims ........................................................................................................................................... 7 2 Theory ................................................................................................................................. 7 2.1 Collagen ..................................................................................................................................... 7 2.1.1 The Structure of Collagen ................................................................................................... 7 2.1.2 The Structure of Collagen Fibrils ........................................................................................ 8 2.2 Osteocalcin ................................................................................................................................ 9 2.2.1 The Role and Structure of Osteocalcin ............................................................................... 9 2.3 Techniques .............................................................................................................................. 10 2.3.1 Self-Assembled Monolayers for Surface Modification .................................................... 10 2.3.2 X-Ray Photoelectron Spectroscopy .................................................................................. 11 2.3.3 Time-of-Flight Secondary Ion Mass Spectrometry ........................................................... 12 2.3.4 Atomic Force Microscopy ................................................................................................. 13 2.3.5 Vibrational Sum-Frequency Generation Spectroscopy ..................................................... 14 3 Methods ............................................................................................................................. 17 3.1 Strategies ................................................................................................................................. 17 3.1.1 Collagen ...........................................................................................................................