From protein self assembly to crystallization

FROM PROTEIN SELF-ASSEMBLY TO CRYSTALLIZATION JIA YANWEI NATIONAL UNIVERSITY OF SINGAPORE 2005 FROM PROTEIN SELF-ASSEMBLY TO CRYSTALLIZATION JIA YANWEI (M Sc Hunan University, China) A THESIS SUBMITTED FOR THE DEGREE OF PHILOSOPHY DEPARTMENT OF PHYSICS NATIONAL UNIVERSITY OF SINGAPORE 2005 ACKNOWLEDGEMENTS First and foremost, I want to sincerely thank my supervisor, Professor XiangYang Liu, without whom this work would not have been possible His clear view of science and kind hearted nature made our lab an exceptionally pleasant, creative and exciting place to work It is truly my pleasure to have been part of it I thank for his valuable guidance and continuous encouragement throughout my research I also want to second show my sincere appreciation to Professor Janaky Narayanan for her invaluable advice and keen interest in this work She contributed enormously towards my learning biophysics and inspired me in many ways to develop new ideas and experimental techniques I take this opportunity to express my gratitude to Dr Christina Strom who has contributed significantly to this thesis and my PhD study I sincerely thank her for helping me throughout the period of my research by providing advice and support for editing the papers and in patent application I would also like to express my sincere gratitude to Dr Claire Lesieur Chungham for providing advice and practical instruction in biology I deeply appreciate the enlightening discussion with her in various allied fields I am going to miss the arguments and challenges between us that helped me learn a lot I also gratefully acknowledge the help and support of all my lab mates, past and present, who have spent countless hours of insightful discussion I am pleased to thank all of them, Keqin, Huaidong, Du Ning, Rongyao, Jingliang, Junying, i Dawei, Tianhui, Huiping, Liu Yu, Junfeng and Yanhua Special thanks are due to Mr Teo Hoon Hwee and Mr Chung Chee Cheong Eric for their support and help throughout my research work, as well as many other close friends who could not fit in the available space Furthermore, I would take this opportunity to thank my husband, Mr Zhou Yicong, who has provided constant support to me during the years of my research His love and encouragement kept my spirit high through the toughest part of this work I might not have completed this work without him Also I want to thank my beloved parents and brother for their love, encouragement and support they have given me during my years of study Finally, I thank the National University of Singapore for providing the scholarship during my study in NUS ii TABLE OF CONTENTS ACKNOWLEDGEMENTS SUMMARY i vii LIST OF TABLES x LIST OF FIGURES xi LIST OF SYMBOLS xvii CHAPTER INTRODUCTION 1.1 Crystals in Our Daily Life 1.2 Why is Protein Crystallization Important 1.2.1 Structural Biology and Drug Design 1.2.2 Bioseparation 1.2.3 Controlled Drug Delivery 1.3 Challenges in Research of Protein Crystallization 1.3.1 A Multiparametric Process 1.3.2 Purity 1.3.3 Solubility and Supersaturation 1.3.4 Nucleation, Growth and Cessation of Growth 1.3.5 Packing 1.4 Some Milestones in Research of Protein Crystallization 10 10 1.4.1 Nonionic Surfactant as Protein Crystallizing Agent 12 1.4.2 Prediction of Protein Crystallization 15 1.4.3 Kinetics of Protein Nucleation and Growth 17 1.5 Problems 19 iii 1.6 Objectives 20 1.7 Scope 20 CHAPTER MATERIALS AND METHODS 2.1 Materials 22 22 2.1.1 Proteins 22 2.1.2 Surfactant 22 2.1.3 Salts and Buffers 22 2.2 Techniques 23 2.2.1 Protein Crystallization 23 2.2.2 Static Light Scattering 27 2.2.3 Dynamic Light Scattering 32 2.2.4 Refractive Index Increment 37 2.2.5 Surface Tension 40 2.2.6 Fluorescence Spectroscopy 45 2.2.7 Cloud Point 51 CHAPTER SOLUBLE PROTEIN CRYSTALLIZATION WITH NONIONIC SURFACTANT 55 3.1 Introduction 55 3.2 Crystallization 56 3.3 Protein Interactions 59 3.3.1 Refractive Index Increment 59 3.3.2 Static Light Scattering 60 3.3.3 Dynamic Light Scattering 63 3.4 Origin of the Change in Protein Interactions 66 iv 3.4.1 Surface Tension Measurements 66 3.4.2 Fluorescence Measurements 69 3.4.3 Cloud point measurements 72 3.4.4 Depletion Force 75 3.5 Mechanism 77 3.6 Conclusions 79 CHAPTER SELF-ASSEMBLY OF PROTEIN IN CORRELATION TO PROTEIN CRYSTALLIZATION 80 4.1 Introduction 80 4.2 Amphiphilic Nature of Proteins 83 4.2.1 Origin of Surface Activity of Proteins 83 4.2.2 Surface Adsorption of Proteins 83 4.3 Critical Assembly Concentration (CAC) of Lysozyme 84 4.3.1 Protein Concentration Dependence 84 4.3.2 Salt Concentration Dependence 86 4.3.3 Correlation of CAC to Solubility 87 4.4 Two-dimensional Protein Assembly to Three-dimensional Crystallization 91 4.4.1 Limited and Infinite Aggregation/Assembly 91 4.4.2 Correlation between Protein 2D assembly and Crystallization 91 4.5 Conclusions 93 CHAPTER INTERFACIAL KINETICS OF PROTEIN CRYSTALLIZATION 5.1 Introduction 96 96 v 5.2 Protein 3D Crystallization 97 5.2.1 Thermodynamics Driving Force 97 5.2.2 Nucleation Barrier 98 5.2.3 Interfacial Kinetics 103 5.2.4 Kinetic Crystallization Coefficient 106 5.3 Protein 2D Assembly 109 5.3.1 Surface Assembly Process 110 5.3.2 Kinetics of Protein 2D Assembly 110 5.4 From Protein 2D Assembly to 3D Crystallization 113 5.4.1 Lysozyme 2D Assembly Kinetics 113 5.4.2 Kinetic Crystallization Window 118 5.4.3 Validation of the Kinetic Crystallization Window 120 5.5 Conclusions CHAPTER CONCLUSIONS 123 125 6.1 Conclusions 125 6.2 Recommendations for further study 129 REFERENCES 131 APPENDIX 145 List of Publications 145 vi SUMMARY Protein crystallization has attracted much attention due to its wide application in drug delivery and determination of protein structure However, it is difficult to crystallize protein since precise determination of crystallization conditions is often a time consuming task The purpose of this thesis is to understand the mechanism of protein crystallization and develop a prediction criterion for protein crystallization based on the kinetics of protein crystallization This thesis consists of three parts which are mainly based on the following publications International patent filed: X Y Liu and Y W Jia, Method for Prediction de novo Biomacromolecule Crystallization Conditions and for Crystallization of the same, File reference No PCT/SG2005/000051, Filing date: 21 February 2005 Papers: Y W Jia and X Y Liu, Self-assembly of Protein at Aqueous Solution Surface in Correlation to Protein Crystallization, Appl Phys Lett 86(2), 023903, 2005 Y W Jia and X Y Liu, Prediction of Protein Crystallization Based on Interfacial and Diffusion Kinetics, Appl Phys Lett 87(10), 103902, 2005 Y W Jia, J Narayanan, X Y Liu and Y Liu, Investigation of the Mechanism of Crystallization of Soluble Protein in the Presence of Nonionic Surfactant, Biophys J 89, 4245-4251, 2005 vii Y W Jia and X Y Liu, From Surface Self-assembly to Crystallization: Prediction of Protein Crystallization Conditions, J Phys Chem B, 110(13), 6949-6955, 2006 George et al have proposed a prediction criterion for protein crystallization, i.e., a “crystallization window”, based on protein molecular interactions, which is characterized by the second virial coefficient In this thesis, the mechanism of protein crystallization in the presence of nonionic surfactant is investigated with reference to protein molecular interactions From the protein crystallization results, it was found that interactions was repulsive in noncrystallization solution conditions, whereas intermolecular interaction was attractive and fell in the “crystallization window” in solution conditions that yield crystal The origin of the change in interaction was attributed to the adsorption of nonionic surfactant monomers on the hydrophobic parts of protein molecules and depletion force at high surfactant concentration Although the second virial coefficient is valid to predict protein crystallization in some cases, it fails in a lot of cases as well because it neglects the kinetics of protein crystallization, which is an essential part in protein crystallization A new prediction criterion for protein crystallization conditions was established based on the kinetics of protein crystallization studied via the two-dimensional assembly of protein at the aqueous solution surface Two-dimensional assembly of protein at the surface of aqueous solution followed the same behavior as amphiphilic molecules The critical assembly concentration (CAC) appearing in the protein solutions was found to coincide with the equilibrium concentration of protein crystal under given conditions This indicates viii Rreferences 1996 Concentration dependence of the film pressure of human serum albumin at the water/decane interface Colloids and Surfaces B: Biointerfaces 6:279-289 Chernov, A A 1984 Modern Crystallography III - Crystal Growth Berlin: Springer Verlag Chiew, Y C., D Kuehner and H W Blanch et al, Molecular Thermodynamics for Salt-induced Protein Precipitation, AICHE J 41 (9), 2150-2159 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