The Interfacial Behavior of Bombyx Mori Silk Fibroin
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University of Massachusetts Amherst ScholarWorks@UMass Amherst Doctoral Dissertations 1896 - February 2014 1-1-1998 The interfacial behavior of Bombyx mori silk fibroin. Regina, Valluzzi University of Massachusetts Amherst Follow this and additional works at: https://scholarworks.umass.edu/dissertations_1 Recommended Citation Valluzzi, Regina,, "The interfacial behavior of Bombyx mori silk fibroin." (1998). Doctoral Dissertations 1896 - February 2014. 981. https://scholarworks.umass.edu/dissertations_1/981 This Open Access Dissertation is brought to you for free and open access by ScholarWorks@UMass Amherst. It has been accepted for inclusion in Doctoral Dissertations 1896 - February 2014 by an authorized administrator of ScholarWorks@UMass Amherst. For more information, please contact [email protected]. THE INTERFACIAL BEHAVIOR OF BOMBYXMORI SILK FIBROIN A Dissertation Presented by REGINA VALLUZZI Submitted to the Graduate School of the University of Massachusetts Amherst in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY September 1998 Department of Polymer Science and Engineering © Copyright by Regina Valluzzi 1998 All Rights Reserved THE INTERFACIAL BEHA\qOR OF BOMBYXMORI SILK FffiROIN A Dissertation Presented by REGINA VALLUZZI Approved as to style and content by; Samuel P. Gido, Chair Shaw-Ling Hsu, Mem David A. Tirrell, Member Rjclwd J. Farris, Department Head Polymer Science and Engineering ACKNOWLEDGEMENTS In the course of my studies in Polymer Science at the university of Massachusetts, I have had the privilege of working within some fine scientists, and have benefited fi-om their insights. My advisor, S. P. Gido has, in my opinion, a genuine appreciation for the spirit of scientific inquiry, and his persistence in the face of controversial results and sometimes daunting experimental problems has been exemplary. I would like to acknowledge my committee members S. L Hsu, D. A. Tirrell and thank them for their time, their input, and their attention to detail. Several fiiends and colleagues have made the UMass experience better than it would otherwise have been. I would like to thank Vema Lo, Georgia Dris, and Shi-Juang He for their good cheer and for sharing the trials and tribulations of entry into the research lifestyle. Drs. Scott Barton and Roger Porter have been sources of unwavering support and often necessary perspective. Lastly I would like to acknowledge my family for their patience in tolerating my absences while I pursued my degree. iv ABSTRACT THE INTERFACIAL BEHAVIOR OF BOMBYXMORI SILK HEROIN SEPTEMBER 1998 REGINA VALLUZZI, B.S. MASSACHUSETTS INSTITUTE OF TECHNOLOGY Ph. D., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Professor Samuel Patrick Gido A new crystal structure has been observed for Bombyx mori silk fibroin at air-water interfaces. This structure, silk III, incorporates a left-handed three-fold polyglycine II conformation and an approximately hexagonal lattice. Detailed crystaJlo graphic studies using electron diffraction data have been used to characterize the silk III crystal structure. There is a hexapeptide repetitive sequence found in the crystaUizable portions of silk fibroin. When this sequence, Gly-Ala-Gly-Ala-Gly-Ser, is in a threefold helical conformation, a row of alternating glycine and serine residues parallel to the helical axis results. One third of the helix thus becomes slightly hydrophilic, whereas the other two thirds consist of glycine and hydrophobic alanine residues. The data indicate that the helices are arranged so that the serine residues pack preferentially in serine-rich sheets in the (110) planes of the crystal. The result is a monoclinic crystal structure, where the basal plane angle y is 1 16° rather than the 120° expected for perfect hexagonal packing, due to the distortion in nearest neighbor interhelical packing distances that results when the serine residues have a preferred packing. The separation of hydrophobic and hydrophilic residues in a threefold helical conformation of fibroin suggests that the air- V water interface may stabilize the threefold conformation because this conformation allows the fibroin to behave as a surfactant at the interface. The sheet-like arrangement of serine residues deduced for the monoclinic silk III crystallites also supports a role for surfactancy in stabilizing the silk III structure at the air-water interface. If the crystallizable portions of fibroin are behaving as a surfactant, then the three-fold helical silk III structure should be oriented with the axes of the three-fold fibroin helices in the plane of the interface. This orientation is observed in uncompressed fibroin films which were picked up onto TEM grids. In LB films compressed to 16.7 mN/meter on the trough prior to being picked up onto TEM grids a uniaxial orientation is observed for silk III, with the helical axes perpendicular to the plane of the sample film. A surface compression of 34 mN/meter results in fihns containing silk II crystallites with the same uniaxial orientation, placing the helical axes perpendicular to the plane of the film. In addition to air-water interface experiments several experiments were carried out using aqueous-organic interfaces. A hydrated crystal structure incorporating a left-handed 6/2 helix which is still roughly three-fold, is observed at the water-hexane and water- chloroform interfaces. Large lamellar crystallites possessing a hexagonal habit were observed at both of these interfaces. A banded cholesteric mesophase which in some regions crystallizes into the same silk III hydrate structure as the lamellae was also observed and characterized. vi TABLE OF CONTENTS Page ACKNOWLEDGEMEP4TS iv ABSTRACT v LIST OF TABLES x LIST OF FIGURES xi Chapter L BACKGROUND 1 1.1 Silks 4 1. LI Silk Biology 5 1.2 Greater Implications 18 1,2.1 Fibrous Proteins 18 L2.2 Proteins at Interfaces 19 1.2,3 Globular Proteins 23 24 1.3 Objectives 25 2. SDLK ra, A NOVEL CRYSTAL STRUCTURE 25 2.1 Summary 2.2 Introduction 2.3 Experimental 2.3. 1 Sample Preparation 2.3.2 Characterization 2.3.3 Olfaction Data Analysis 2.3.4 Analysis of Possible contaminants 2.3.5 Results and Discussion 63 2.4 Conclusions THE FIBROIN : REFINEMENTS TO 3. THE INTERFACIAL STRUCTURE OF SILK MODEL 64 Vll 3.1 Summary 3.2 Introduction 3.3 Experimental 5.3.7 Sample Preparation i. 3. Characterization 2 ^g 3.4 Results and Discussion 73 3.4. 1 Polycrystalline data 77 3.4.2 Refinements to the Basic Hexagonal Silk Crystal Structure 83 3.4.3 Tilting Experiments with Uniaxially Oriented Samples 91 3.5 Conclusions 95 4. ORIENTATION EFFECTS IN SILK FIBROIN LANGMUIR-BLODGETT FILMS : EVIDENCE OF SURFACTANCY 96 4.1 Summary 96 4.2 Introduction 97 4.3 Experimental 100 4.4 Results and Discussion 101 4.5 Conclusions 120 5. A HYDRATED STRUCTURE OF SILK HI OCCURS AT AQUEOUS-ORGANIC INTERFACES ^ 122 5.1 Summary 122 5.2 Introduction 123 5.3 Experimental 130 5.4 Results and Discussion 132 5.5 Conclusions 152 6. SERICIN AND FIBROIN 154 6.1 Summary 154 6.2 Introduction 155 6.3 Preliminary Data 162 163 6.3. 1 The Unit Cell for /3-sheet sericin 176 6.4 Conclusions and Suggested Experiments 178 7. A COMPARISON OF HBROIN DVTERFACLVL AND BULK STRUCTURES '^^ 8. CONCLUSIONS 9. SUGGESTED FUTURE WORK 9.1 Microfibrils and Mesophase Formation 9.2 Other Water- Organic Interfaces viii APPENDICES A. ATOMIC COORDINATES FOR THE SILK ID CRYSTAL STRUCTURES 206 A. 1 Trigonal model (superlattice) 207 A.2 Monocl in ic model 216 A. 3 Hydrated orthorhombic model 249 A.4 Polyserine model for crystalline sericin 259 B. TRIGONAL VERSUS ORTHORHOMBIC LATTICE 270 C. SAMPLE COMPOSITION 271 REFERENCES 274 LIST OF TABLES Table Page 1 . Comparison of Diffraction data to Simulations from the trigonal silk III model 57 2. Angles between six- fold diffraction spots 71 3. Trigonal versus Monoclinic Unit Cell 72 4. Variation in Torsional Angles 94 5. Relative intensity changes with different orientations 102 6. Silk III Hydrate Model Data 143 X LIST OF FIGURES Figure Page 1. Asymmetric (3-sheet packing 2 2. The crankshaft conformation proposed for silk I 7 3. The silk II P-sheet conformation 8 4. The polyglycine II structure 15 5. Silk II 27 6. Brightfield TEM morphology image of a typical film region 28 7. Darkfield high angle annular STEM image of a typical LB film morphology 29 8. Polycrystalline Silk III diffraction 30 9. In the threefold conformation the glycine residues in the hexapeptide fibroin crystallizable repeat (Gly-Ala-Gly-Ala-Gly-Ser) alternate with the larger residues 31 10. There are different possible lateral arrangements of residues in the 003 planes in a silk III crystal 1 1 . Silk III lamellar crystallites consisting of 2 12. Superlattice structure, or "super cell", for the silk III model, 51 primitive unit cell repeats in each crystallographic direction 52 13. Simulated diffraction fi-om the trigonal model result fi-om uniaxially 14. Schematic diagrams of the diffraction rings that III cast films in the electron oriented silk III LB films and unoriented silk diffraction geometry 15. Tilted single crystal diffraction for the silk III lamellae 59 16. Different zone axes are sometimes observed xi 17. Hexapeptide fibroin sequence in a threefold conformation 66 1 8. Six fold diffi-action patterns fi-om LB films 74 19. Single crystal densitometer scan 75 20. Slow scan image of a silk LB film diffraction pattern showing the closely spaced component rings in the inner (4.5 A) reflection 80 21 . Densitometer scan of a typical diffraction pattern fi-om a uniaxially oriented LB film 81 22. Polycrystalline diffraction rings fi-om the chloroform interface 82 23. The combination of the six residue repeating sequence po\y(Gly-Ala-Gly-Ala- Gly-Ser) (representing the crystallizable component of silk fibroin) with a threefold helical conformation, results in a column of alternating glycine and serine residues parallel to the helical axis 84 24.