How does antifreeze protein stop ice nucleation

HOW DOES ANTIFREEZE PROTEIN STOP ICE NUCLEATION? DU NING NATIONAL UNIVERSITY OF SINGAPORE 2004 HOW DOES ANTIFREEZE PROTEIN STOP ICE NUCLEATION? DU NING (B. SCI., JiLin Univ., China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSICS NATIONAL UNIVERSITY OF SINGAPORE 2004 ACKNOWLEDGEMENTS First and foremost, I want to sincerely thank my supervisors, Professor Xiang-Yang Liu and Professor Janaky Narayanan, without whom this work would not have been possible. Thank you so much Professor Liu, your 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 all. Thank you so much for your valuable guidance and continuous encouragement throughout my research. Professor Narayanan, thank you so much for providing me with excellent advice and enthusiastic supervision that have helped me extend my knowledge in many fields. Your enormous contributions towards helping me learn biophysics and developing many of the ideas and experiments inspired me in many ways. I take this opportunity to express my gratitude to Professor Christina Strom who has contributed more than she can imagine to this thesis. I owe much to her for helping me throughout the period of my research by providing advice, support and editing the papers and this thesis. I would also like to express my sincere gratitude to Dr. Claire Lesieur for giving me a lot of advice and practical instruction in biology. I am also very grateful to Professor Zongchao Jia and Professor Hew Choy Leong for kindly providing us with the precious antifreeze proteins and scientifically productive discussions that form the basis for a large chunk of this thesis. I also gratefully acknowledge the help and support of all my fellow lab mates, past and present, who have spent countless hours of insightful discussion. I am pleased to i thank all of you, Yanwei, Keqin, Huaidong, Huiping, Zhang Jing, Prashant, Dawei, Junying, Junxue, Jingliang, Perry, Junfeng, Liu Yu, Rongyao, Yanhua, Caide, Tianhui and Zhou Kun. Special thanks are due to Mr. Teo and Eric for their support and help throughout my research work, as well as many other close friends that could not fit in the available space. I would like to thank those closest to me, whose presence helped me to complete my graduate work and made me feel at home, I extend them my deepest appreciation. My mother has been an inspiration throughout my life. She has always supported my dreams and aspirations. I would like to thank her for all that she means to me, and all she has done for me. Also I want to thank my beloved father and sister for their love, encouragement and support they have given me during my years of schooling. Finally, I would like to say thank you to my husband, whose constant support and encouragement got me through the toughest part of this work. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS SUMMARY i vi LIST OF TABLES viii LIST OF FIGURES ix NOMENCLATURE xv CHAPTER 1. INTRODUCTION 1.1 Freezing and Antifreeze Processes 1 1.1.1. Freezing in Nature 1.1.2 Antifreeze Action in Biological Systems 1.2 Antifreeze Proteins 1.2.1. Diversity in Antifreeze Proteins 1.2.2 Applications of Antifreeze Proteins 1.3 Overview on Antifreeze Mechanism of Antifreeze Proteins 1.3.1 General Features of Antifreeze Proteins 1.3.2 13 13 The key challenges in antifreeze mechanism by antifreeze proteins 17 1.4 Objectives 20 1.5 Scope and Summary 21 iii CHAPTER 2. EXPERIMENTAL TECHNIQUES 22 2.1 Micro-sized Ice Nucleation Technique 22 2.2 Applied Techniques 24 2.2.1 Dynamic Light Scattering 24 2.2.2 Tensiometer 28 2.2.3 Zetasizer 30 2.2.4 Fluorescence Spectrophotometer 33 CHAPTER 3. PRINCIPLES OF FREEZING AND ANTIFREEZE PROCESSES 3.1 Principles of Ice Nucleation 37 37 3.1.1 Structure of Ice 37 3.1.2 Ice Nucleation Kinetics 44 3.1.2.1 Thermodynamic Driving Force 44 3.1.2.2 Nucleation Barrier 45 3.1.2.3 Kinetics of Ice Nucleation: The Influence of Foreign Particles 52 3.2 Freeze Effect of Some Additives 72 3.2.1 Hydroxyapatite 72 3.2.2 Lysozyme 76 3.3 Antifreeze Effect of Antifreeze Proteins 79 3.3.1 Type III Fish Antifreeze Protein 79 3.3.2 Type I Fish Antifreeze Protein 84 3.3.3 Spruce Budworm Antifreeze Protein 87 3.4 Summary 89 iv CHAPTER 4. AGGREGATION OF ANTIFREEZE PROTEINS AND THE CORRELATION TO ANTIFREEZE KINETICS 90 4.1 Introduction 4.1.1. The role of hydrophobic groups of AFP III on protein-ice 90 90 interactions 4.1.2. Amphiphilic Nature of Proteins 4.2 Aggregation of AFP III Detected by Multiple Techniques 92 94 4.2.1 Surface Tension Measurement 94 4.2.2 Dynamic Light Scattering 96 4.3 Correlation of Aggregation to Antifreeze Kinetics 4.4 Summary 97 104 CHAPTER 5. THE ROLE OF ELECTROLYTE ON ANTIFREEZE ACTIVITY OF ANTIFREEZE PROTEINS 5.1 Introduction 106 106 5.1.1 Electrolyte Content in Fish Blood 106 5.1.2 The adsorption behavior of AFP III modified by electrolytes 108 5.2 The Effect of Ca(NO3)2 on the Interaction between AFP III Molecules and the Unfolding of AFP III 110 5.2.1 Surface Tension Measurement 110 5.2.2 Zeta Potential Measurement 112 5.3.3 Fluorescence Emission 113 5.3 The Role of Electrolyte on Antifreeze Activity of Antifreeze Proteins 115 5.4 Summary 121 CHAPTER 6. CONCLUSIONS 122 REFERENCES 126 v SUMMARY Antifreeze Proteins (AFPs), occurring in some polar animals and plants, are capable of inhibiting ice freezing at subzero temperatures. The application of AFPs can be found in medicine and industry where low temperature storage is required and ice crystallization is damaging. This includes improved protection of blood platelets and human organs at low temperature, increasing the effectiveness of the destruction of malignant tumors in cryosurgery, and improvement of the smooth texture of frozen foods. Previous studies on AFPs are mainly focused on the modification of the crystal morphology of ice and the inhibition of ice crystal growth in terms of the adsorption of antifreeze protein molecules on some specific surfaces of ice. In this study, we examine the effect of AFPs on the initial and key stage of ice crystallization, i.e. ice nucleation, which was neglected in most studies. The effect of AFP III on ice nucleation was examined based on a “micro-sized ice nucleation” technique. Techniques such as Dynamic Light Scattering, Size Exclusion Chromatography and Surface Tension Measurement etc., are also applied to examine the assembly and aggregation of AFPs in the bulk and at the surface of water. It follows from our experiments that at the microscale, the volume effect exerts a large influence on nucleation kinetics. Antifreeze proteins can inhibit the ice nucleation process by adsorbing onto both the surface of ice nuclei and dust particles, which leads to an increase of the ice nucleation barrier and the desolvation kink kinetics barrier, respectively. We also examined the impact of AFP aggregation and electrolyte on the antifreeze mechanism of AFPs. It was found that the antifreeze activity of AFPs can vi be enhanced either by their aggregation at higher concentration or by adding electrolyte into AFP solutions. The promotion in antifreeze activity is attributed to the optimal packing of AFP III molecules on the surface of the ice nuclei at or above the Critical Aggregation Concentration (CAC), and the screening effect of electrolyte on the surface charge of AFP molecules, respectively. This study enables us to obtain a comprehensive understanding on the antifreeze mechanism of AFPs for the first time. It will hopefully shed light on the understanding of antifreeze phenomenon by AFPs and the identification of effective antifreeze agents. vii LIST OF TABLES Table 1.1 Diversity of fish AF(G)Ps. Table 3.1 Lattice parameters of Ice Ih. 43 Table 3.2 Crystallographic parameters for ice Ih. 43 Table 3.3 The freezing temperature (for a droplet of constant volume) is dependent on the number and size of dust particles. 58 Table 3.4 The measured transition temperature (∆T)mid and the radius of the local curvature of foreign particles for different systems. 64 Table 3.5 Comparison of HAP and ice crystal structure. 73 Table 3.6 The effect of HAP crystals on the interfacial effect parameter and kink kinetic energy barrier for the nucleation of ice. 74 Table 3.7 The effect of lysozyme on the interfacial effect parameter and kink kinetic energy barrier for the nucleation of ice. 78 Table 3.8 The effect of AFP III on the interfacial effect parameter and kink kinetic energy barrier for the nucleation of ice. 81 Table 3.9 The effect of AFP I on the interfacial effect parameter and kink kinetic energy barrier for the nucleation of ice. 86 Table 3.10 The effect of CfAFP-501 on the interfacial effect parameter and kink kinetic energy barrier for the nucleation of ice. 87 Table 4.1 The size of AFP III molecules measured by Dynamic Light Scattering (DLS). 96 Table 5.1 Data on blood serum of three Antarctic fishes. Number of samples is shown in parentheses. 107 Table 5.2 The measured actual freezing temperature for different systems. 117 Table 5.3 The effect of AFP III on the interfacial effect parameter and kink kinetic energy barrier for the nucleation of ice. 118 viii Chapter The Role of Electrolyte on Antifreeze Activity of Antifreeze Proteins their compact packing when more protein is accommodated. Subsequently it decreased when the AFP III molecules reached their optimum packing at a Ca(NO3)2 concentration of 10mM. -19.6 DI water AFP III 2+ AFP III + 10 mM Ca 2+ AFP III + 100 mM Ca -20.0 -20.4 ln(τV) -20.8 -21.2 -21.6 -6 1.4x10 -6 1.5x10 -6 1.5x10 -6 1.5x10 -6 1.6x10 1/(T∆Τ ) Figure 5.6 The effect of AFP III and Ca(NO3)2 on the ice nucleation kinetics and the corresponding shift in the ln(τV) ~1/(T∆T 2) plot Table 5.3. The effect of AFP III on the interfacial effect parameter and kink kinetic energy barrier for the nucleation of ice Curve f m ∆(∆G≠kink/kT) add DI Water ≠ (20 nm filter) 0.168 0.48 - AFP III (3mg/ml) 0.250 0.35 ↑ 17.7 AFP III (3mg/ml) +10 mM Ca(NO3)2 0.283 0.30 ↑ 22.8 AFP III (3mg/ml) +100 mM Ca(NO3)2 0.266 0.32 ↑ 20.9 ≠ DI water: Deionized water 118 Chapter The Role of Electrolyte on Antifreeze Activity of Antifreeze Proteins (a) (b) Figure 5.7 Illustration of the adsorption of the AFP III molecules on the interface of ice nucleator (IN) and liquid and suppression of water molecules approaching the IN surface (a) without electrolyte (b) with electrolyte On the other hand, as shown in Table 5.3, the addition of 10 mM of Ca(NO3)2 to the AFP III solution raised ∆G≠’kink, whereas on the other hand, ∆G≠’kink decreased upon a further Ca(NO3)2 addition. This result is consistant with the trend of f, which is indicative of the interfacial arrangement between the ice crystalline phase and the foreign particles. When there are only AFP III molecules inside the solution, it is known that AFP III molecule will inhibit ice nucleation by adsorbing at the ice/fluid 119 Chapter The Role of Electrolyte on Antifreeze Activity of Antifreeze Proteins interface to block the incorporation of water molecules into the kink site. As discussed above, in analogy with the ice \ INs interface, by forming a more compact packing mode at the ice\ fluid interface at a Ca(NO3)2 solution of 10 mM, the AFP III molecules can block the approach of the water molecules onto ice surface more effectively. Accordingly, ∆(∆G≠kink) increased. As more Ca(NO3)2 was added into the AFP III solution, the AFP III molecules may have unfolded causing ∆(∆G≠kink) to decrease. (a) (b) Figure 5.8 Illustration of adsorption of AFP III molecules on the interface between ice and liquid and prevention of water molecules from approaching the ice surface (a) without electrolyte (b) with electrolyte 120 Chapter The Role of Electrolyte on Antifreeze Activity of Antifreeze Proteins 5.4 Summary In summary, we have quantified here the enhanced antifreeze activity of AFP III during ice nucleation by adding Ca(NO3)2 . This effect may be caused by the modified interfacial behavior at the ice\foreign particle interface and the ice/fluid interface, induced by the modified interactions among AFP III molecules upon adding Ca(NO3)2. This work could provide a model for the study of the specific role of an electrolyte in ice-binding activity. This new understanding of the electrolyte effect on the enhanced antifreeze activity of the AFP III suggests another way for increasing the antifreeze efficiency of the antifreeze proteins and provides us with new insight into the antifreeze mechanism of antifreeze proteins. 121 Chapter Conclusions CHAPTER CONCLUSIONS In this thesis, controlled ice nucleation in a micro-sized water droplet was examined using a micro-droplet suspension method. This is a well-controlled method to observe and measure ice nucleation. Because the water present in ecological and biological systems is mostly of the form of micro-sized water droplets, this method may be applied to study ice nucleation in these systems. By using this experimental method, we can greatly reduce the influence of the container and the foreign dust particles on ice nucleation. Another feature of our experimental study which was previously overlooked is taking into account directly the influence of the volume on the nucleation kinetics. The experimental data obtained by our method have provided results for the quantitative interpretation of ice nucleation. It was found that genuine homogeneous nucleation of ice never occurs even in ultraclean micro-sized water droplets because of the pre-existence of minute foreign particles. More surprisingly, at sufficiently low supercoolings, foreign nano-particles exert no effect on the nucleation barrier of ice; it is as if they had physically “vanished.” This effect, called the “zero-sized” effect of foreign particles (or nucleators), leads to the entry into the so-called inverse homogeneous-like nucleation domain, in which nucleation is effectively suppressed. This will then cause a sharp 122 Chapter Conclusions increase in the nucleation barrier at low supercoolings, which prevents the occurrence of nucleation in this so-called inverse homogeneous-like nucleation regime. The freezing temperature of water corresponds to the transition temperature from the inverse homogeneous-like nucleation regime to foreign particle-mediated heterogeneous nucleation. The freezing temperature of water is mainly determined by (i) the surface roughness of nucleators at large supercoolings, (ii) the interaction and structural match between nucleating ice and the substrate, and (iii) the size of the effective surface of nucleators at low supercoolings. Thus freezing promotion requires ice nucleators to be flat and large and have strong interactions and an optimal structural match with the ice nuclei, whereas antifreeze should be focused on the enhancement of the surface roughness of pre-existing foreign particles rather than other factors. Our experiments showed that the temperature of -40°C, commonly regarded as the temperature of homogeneous nucleation-mediated freezing, is actually the transition temperature from the inverse homogeneous-like nucleation regime to the foreign particle-mediated heterogeneous nucleation in ultra-clean water. Taking advantage of the inverse homogeneous-like nucleation, the interfacial tensions between water and ice in very pure water and antifreeze aqueous solutions were measured at a very high precision for the first time. Based on this “micro-sized ice nucleation method”, the inhibition effect of three types of antifreeze proteins-antifreeze protein type I (AFP I), type III (AFP III) and Spruce Budworm antifreeze protein on ice crystallization were examined quantitatively. It was found for the first time that all these antifreeze proteins can inhibit the ice nucleation process by adsorbing onto both the surfaces of the ice nuclei and the dust 123 Chapter Conclusions particles. The adsorption of AFPs on foreign particles will disturb the structural match between the nucleating ice and the dust particles, whereas the adsorption on the surface of the growing ice will inhibit the integration of water molecules into the ice lattice. These two effects can be identified from the increase of the ice nucleation barrier and the desolvation kinetics barrier. In addition, we suggest that the amphiphilic property may be a common characteristic of AFP I, III and CfAFP-501 and it could be one of the reasons why those proteins have a unique antifreeze function. This new understanding of the antifreeze mechanism of the AFP has never been examined before and is expected to provide us with guidelines in identifying new antifreeze proteins. Apart from this, it was found that AFP III molecules will aggregate in aqueous solutions at CAC due to their amphiphilic nature. This is not surprising considering their high hydrophobicity, as judged from the amino acid compositions. It is further suggested that this high hydrophobicity is a general feature of all four types of fish antifreeze protein (Sonnichsen et al. 1995). Since hydrophobicity is a dominant parameter among the various factors that may affect the surface activity of the protein (Magdassi et al. 1996), this may imply that aggregation in aqueous solution is a common characteristic of all fish antifreeze proteins. It is found that the ice nucleation kinetics can be modified at CAC by two effects. First, the aggregates appearing in solution may work as ice nucleators. Thus it is suggested that the aggregation should be suppressed for AFP to inhibit ice nucleation. The second effect, which is the dominant effect, is that more AFP III molecules will adsorb onto the surface of the ice 124 Chapter Conclusions nuclei and reach their optimal packing to inhibit ice nucleation at or above CAC. In this way, AFP III exhibits an enhanced inhibition effect on ice nucleation at CAC. In addition, we notice that the electrostatic interactions between protein molecules can be modified by adding an electrolyte. So we monitored the antifreeze effect of AFP III on ice nucleation by adding Ca(NO3)2, and found that it may enhance the antifreeze activity of AFP III. This effect may be caused by the modified adsorption behavior of AFP III at the ice/foreign particle interface and the ice/fluid interface. Upon adsorbing Ca2+ and NO3- ions, AFP III can form a more compact packing mode at these interfaces due to the screened electrostatic repulsions between them. This work could provide a model for the study of the specific role of the electrolyte in the ice-binding activity. This new understanding of the electrolyte role to enhance the antifreeze activity of the AFP III suggests another way for increasing the antifreeze efficiency of the antifreeze proteins and provides us with a comprehensive understanding of the antifreeze mechanism of antifreeze proteins. 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Springer-Verlag Berlin Heidelberg 1999. pp.326-327, 321 Zeldovich, J. B. (1943) On the theory of new phase formation: cavitation. Acta Physicochim. URSS 18, 1-22 Zettlemoyer, A.C., Tcheurekdjian, N., and Chessick, J.J. (1961) Surface properties of silver iodide. Nature 192, 653 Zettlemoyer, A. C. in Nucleation (Dekker, New York, 1969) 133 [...]... ice surface (a) without electrolyte (b) with electrolyte 120 xiv NOMENCLATURE AFGPs Antifreeze Glycoproteins AFP I Type I Fish Antifreeze Protein AFP III Type III Fish Antifreeze Protein CAC Critical Aggregation Concentration CfAFP Choristoneura fumiferana Antifreeze Protein DI water Deionized water DLS Dynamic Light Scattering HAP Hydroxyapatite IN Ice Nucleator Sbw AFP Spruce Budworm Antifreeze Protein. .. Figure 1.6, the ice crystal growth is inhibited because antifreeze proteins adhere to the ice surface, thus restricting ice growth on the exposed ice surface between adjacent antifreeze molecules The surfaces of these exposed area segments are more convex, such would not be the case in the absence of the antifreeze proteins The convex surface has a higher free energy than ordinary ice, and ice growth is... to explain the thermal hysteresis of antifreeze proteins is the adsorption-inhibition model In this model, it is believed that antifreeze proteins 14 Chapter 1 Introduction depress freezing point by adsorbing onto the ice surface thus inhibiting ice crystal growth (Raymond et al 1977; Knight et al 1991) Figure 1.6 Illustration of how antifreeze proteins inhibit ice crystal growth according to the adsorption-inhibition... onto the ice crystal surfaces and thereby inhibiting their growth Although some reports show the modification of the ice morphology caused by AFPs (Antson et al 2001), no study has thus far been carried out to show how AFPs inhibit ice crystallization, in particular ice nucleation 17 Chapter 1 Introduction In most cases, the formation of a new crystalline phase from the ambient phase proceeds via nucleation. .. process of ice crystallization from supercooled water In this process, water should undergo the stage of ice nucleation, followed by the growth of ice (Mutaftschiev 1993) This means that, in the case of ice crystallization, nucleation is the initial and one of the most important steps toward creating ice Actually, whether or not freezing takes place is determined to a large extent by ice nucleation. .. previous studies of the AFPs shows that they are mainly focused on the modification of the crystal morphology of ice and the inhibition of ice crystal growth in terms of the adsorption of antifreeze protein molecules on specific surfaces of ice (Antson et al 2001; Raymond et al 1977; Knight 2001) It is believed (Chao et al 1994; Yeh et al 1996; Raymond et al 1989) that antifreeze proteins lower the freezing... increased concentrations of antifreeze proteins lead to smaller ice crystals But the use of antifreeze proteins at high concentrations (about 20mg/ml) was associated with the formation of very destructive ice spicules, which caused the frozen samples to be structurally more damaged than the control samples Hence, in order to improve the quality of the frozen meat, antifreeze proteins have to be used at... of antifreeze proteins on ice is suggested to be based on a structural match between certain residues on the proteins and lattice sites on the ice surface As shown in Figure 1.7(a), the repeated sequences of AFP I follow a regular spacing between hydrophilic residues on one side of the helix The hydroxyl groups from the hydrophilic amino acids match the positions of certain oxygen atoms in the ice. .. type I AFP solution 16 Figure 1.8 SbwAFP model showing surface complementarity with the prism and basal planes of ice (a) A side view of the protein aligned above an ice prism plane Circles depict the lattice positions of water oxygen atoms in ice (b) A view perpendicular to that of a This view down the c-axis illustrates the match of adjacent loops to the ice surface As oxygen atoms have the same interatomic... certain oxygen atoms in the ice lattice, 15 Chapter 1 Introduction thus allowing hydrogen bonds to form between the antifreeze proteins and ice surface This binding causes the ice crystal to change to a bipyramid morphology (Fig 1.7(b)) Figure 1.7 (a) Binding of type I AFP to ice Type I AFP, represented by the green helix with projecting threonyl side chains adsorb on the ice surface, leads to shaping of . HOW DOES ANTIFREEZE PROTEIN STOP ICE NUCLEATION? DU NING NATIONAL UNIVERSITY OF SINGAPORE 2004 HOW DOES ANTIFREEZE PROTEIN STOP ICE NUCLEATION? . Applications of Antifreeze Proteins 9 1.3 Overview on Antifreeze Mechanism of Antifreeze Proteins 13 1.3.1 General Features of Antifreeze Proteins 13 1.3.2 The ke y challen g es in antifreeze. Type III Fish Antifreeze Protein 79 3.3.2 Type I Fish Antifreeze Protein 84 3.3.3 Spruce Budworm Antifreeze Protein 87 3.4 Summary 89 iv CHAPTER 4. AGGREGATION OF ANTIFREEZE PROTEINS AND