A Study on the Hyperactive Antifreeze Proteins from the Insect Tenebrio Molitor

A Study on the Hyperactive Antifreeze Proteins

from the Insect Tenebrio molitor

A thesis presented to

the faculty of the College of Arts and Sciences of Ohio University

In partial fulfillment

of the requirements for the degree

Master of Science

Young Eun Choi

November 2007

This thesis titled

A Study on the Hyperactive Antifreeze Proteins from the Insect Tenebrio molitor

YOUNG EUN CHOI

has been approved for

the Department of Physics and Astronomy

and the College of Arts and Sciences by

Ido Braslavsky

Assistant Professor of Physics and Astronomy

Benjamin M. Ogles

Dean, College of Arts and Sciences

Abstract CHOI, YOUNG EUN, M.S., November 2007, Physics and Astronomy Title: A study on the hyperactive antifreeze protein from the insect Tenebrio molitor (51 pp.) Director of Thesis: Ido Braslavsky Antifreeze proteins (AFPs) are class of proteins that protect organisms from damages caused by freezing, either by preventing freezing or minimizing frost damages. AFPs effectively lower the temperature at which water freezes. They are classified by the depression of the freezing temperature compared to the melting temperature, i.e. Thermal Hysteresis activity (TH): moderately active AFPs and hyperactive AFPs. It is still unknown what makes some AFPs hyperactive compared to the much less active classes of AFPs. Previous studies showed that fusion proteins of fish type III AFP bind independently to ice. This conclusion was derived from experiments with bulky proteins that were fused to this moderately AFP. One possible explanation for the increased activity of the hyperactive AFPs is that they might function cooperatively. To investigate this, the hyperactive AFP from the mealworm, Tenebrio molitor (TmAFP), was linked to bulky proteins. In this thesis, these fusion proteins were assayed by a nanoliter osmometer, a device that has been designed to measure TH of AFPs. The results indicate that the addition of large molecules to the TmAFP does not induce any loss of thermal hysteresis activity; these fusion proteins were rather more active than free TmAFP at almost all concentrations. Further, ice crystal morphologies obtained by the fusion proteins were the same as the ones of free TmAFP. Therefore, it is concluded that TmAFPs independently bind to ice and their enhanced thermal hysteresis activity does not result from cooperativity.

Approved: ______Ido Braslavsky Assistant Professor of Physics and Astronomy

Acknowledgements

I would like to acknowledge and thank my advisor, Dr. Ido Braslavsky, for the excellent guidance and the considerable help with numerous discussions. His support with patience and generosity encouraged me. For our collaboration, I am grateful to Dr. Peter Davies from Queen's university and to Dr. Deborah Fass and Maya Bar from the Weizmann institute of science, for providing proteins that I used for my experiments and for a lot of useful comments about the proteins. I would also like to thank Dr. Natalya Pertaya and Yeliz Celik for their assistant and suggestion while I was measuring the activity of AFPs. Finally, I thank my mother for her support and endless love. Without the support of my family, I could not finish this work. Thanks to all my friends for encouragement throughout my studies.

Table of Contents

Abstract...... 3

Acknowledgments...... 4

List of Figures...... 7

1. Introduction and literature review...... 8

1.1 What are antifreeze proteins?...... 9

1.2 Ice structure...... 10

1.3 The general mechanisms of antifreeze activity...... 11

1.3.1 Colligative phenomena ...... 12

1.3.2 Adsorption-inhibition: Gibbs-Thomson (Kelvin) Model...... 12

1.3.3 Nucleation inhibition ...... 17

1.4 Molecular structures of antifreeze proteins...... 18

1.5 Hyperactive antifreeze proteins ...... 22

1.6 Previous studies of type III AFP with attached bulky protein...... 23

1.7 Aim of the thesis ...... 26

2. Sample preparation and experimental methods ...... 27

2.1 Materials ...... 27

2.1.1 Tenebrio Molitor AFP (TmAFP)...... 27

2.1.2 MBP-His6-TEV-TmAFP...... 27

2.1.3 His6-eGFP-TmAFP ...... 29

2.2 Experimental Equipment ...... 30

2.3 Experimental procedures and measurements...... 32

2.3.1 Protein purification by adsorption to ice...... 32 6

2.3.2 Procedure and measurement of protein concentration...... 35

2.3.3 Procedure and Measurement of the Thermal Hysteresis activity ...... 36

3. Results...... 38

3.1 Ice crystal growth and morphology as a function of the TmAFPconcentration...... 38

3.2 Thermal hysteresis activity of TmAFP-fusion proteins...... 40

4. Conclusion ...... 43

5. Discussion and future work ...... 45

References...... 47

List of Figures

Figure 1: Ice structure ...... 11

Figure 2: Illustration of the thermal hysteresis...... 16

Figure 3: Adsorption-Inhibition of ice crystal growth...... 16

Figure 4: A schematic representation of the curvature of the ice developed between adjacent AFP molecules...... 17

Figure 5: The structures of AFPs...... 20

Figure 6: The structure of TmAFP...... 21

Figure 7: Ice crystal morphologies of insect AFP and fish AFP ...... 23

Figure 8: Thermal Hysteresis of fish type III AFP and its fusion proteins...... 25

Figure 9: Ice crystals formed in the presence and absence of AFP-fusion proteins and their constituents ...... 26

Figure 10: A schematic representation of MBP-His6-TEV-TmTHP (TmAFP)...... 29

Figure 11: A schematic representation of His6-eGFP-TmTHP (TmAFP) ...... 29

Figure 12: The experimental apparatus...... 31

Figure 13: Screen shot of the Labview interface for controlling the temperature of the ice crystals...... 32

Figure 14: The cold finger apparatus...... 34

Figure 15: Thermal hysteresis activity (°C) vs. concentration (μM) for TmAFP ...... 39

Figure 16: Growing direction of fish AFP and insect AFP ...... 39

Figure 17: Ice crystal morphologies ...... 41

Figure 18: The thermal hysteresis activity of TmAFP and TmAFP-fusions...... 42

Figure 19: Schematic representation of cooperativity and non-cooperativity ...... 44

1. Introduction and literature review

A number of different types of proteins have evolved in fish [1], insects [2], plants [3,4], and other organisms [5-7] to interfere with ice crystal growth. The proteins that protect the organisms by preventing or reducing the damage caused by freezing are called “antifreeze proteins” (AFPs), and also known as “thermal hysteresis proteins” (THPs), “ice structuring proteins” (ISPs) or “ice binding proteins” (IBPs). AFPs have played an important role opening up a fascinating field of research; in agriculture, AFPs can protect crops from frost damage, preserve food, and improve the quality of the frozen food. They are also beneficial in cryosurgery and in cryopreservation of tissues, cells, and organs [8,9]. However, the interaction between ice crystal and protein in the AFPs solution and mechanism of ice binding leading to growth inhibition are not well understood yet. Fish have several types of AFPs. Most of them have only a moderate ability to depress ice nucleation down to -1.5 °C while several insects have AFPs that are more active and can depress ice growth down to – 6 °C. The latter are called hyperactive AFPs. Previous studies with AFP from a fish (type III) showed that they function independently [10]. This conclusion was obtained by measuring the thermal hysteresis activity of fusion molecules that were built from two molecules – an AFP and a bulky non-AFP molecule. They showed that these proteins are active even though they have a big additional molecule. Thus, the fact that the additional molecules did not disturb their activities indicates that these AFPs operate independently. It would be interesting to know if the hyperactive AFPs function independently as well. In this thesis, a study of the physical interactions of ice and the hyperactive AFPs from the yellow mealworm beetle Tenebrio molitor (TmAFP) will be presented. This hyperactive protein has the potential to serve as a platform for future applications. TmAFP fusion proteins were constructed by our collaborators. These included the construction of TmAFP with two different bulky molecules [11]. In the first chapter of this thesis, I will explain the background and basic knowledge about the AFPs in general and how the AFPs bind to ice surfaces. I will present the hyperactive AFPs, including what they are, what the morphology of ice under

influence by AFPs is, and how TmAFP binds to the ice surface. Then I will detail the previous studies that have been done with fish type III AFP and its fusion proteins: Non- AFP bulky molecules. In the second chapter, I will describe the experimental setup, sample preparations and materials. In the third chapter, I will describe the results of experiments I did with the TmAFP fusion proteins. The last, I will discuss these results and conclude.

1.1 What are antifreeze proteins?

It has been more than thirty years since AFPs were first discovered in Antarctic marine teleost fish where they function to lower the freezing point in the icy seawater (−1.9°C), which was colder than the typical freezing temperature of fish blood (−0.7°C) [1]. It became apparent that AFPs are common in the blood serum of teleost fish inhibiting icy seawater. AFPs could depress the freezing temperature of the fish blood several hundred times more efficiently than regular salts; just a few millimolar of AFP provides one degree of the temperature depression that is needed to protect the fish from freezing in the icy seawater. The difference between the melting temperature and the depressed freezing temperature is termed thermal hysteresis (TH). Although AFPs were first discovered in fish, the phenomenon of thermal hysteresis was initially seen in larvae of the beetle Tenebrio molitor [2]. Recent studies have shown that there are AFPs identified in many other insects [2], plants [3,4], fungi [5], and bacteria [6,7] as well as fish of both the northern hemisphere and southern hemisphere [12]. Antifreeze proteins bind to the surface of ice crystals to prevent their growth, and lower the non-equilibrium freezing temperature of aqueous solutions below the melting temperature without significantly changing the melting temperature itself as will be explained below [13]. In addition, AFPs inhibit ice nucleation by passivating small particles that might initiate nucleation [14] and prevent ice recrystallization [14,15] which is a devastating phenomenon in which large crystals grow larger on the expense of small crystals and cause damage during their growth.

A single crystal with the well-defined shape can be obtained in a super-cooled AFP solution (below 0°C). The shape of the ice crystal growing in the AFP solution is diverse depending on the specific activity of the AFP, which varies depending on the structure and sequence of the type of AFPs [16]. The DNA sequences of these AFPs show that they develop independently. This parallel development of many AFPs leads to their diversity [17,18] .

1.2 Ice structure

Ice has different types of crystal phases depending on the pressure and temperature[19]. The related structure of the AFPs around 0 °C is hexagonal ice crystal (ice Ih) Figure 1(A)). AFPs do not change this basic structure, but they do change the growth habits and morphology of the ice crystals. Ice Ih is an arrangement of water molecules with fixed positions for the oxygen atoms that are held together tetrahedrally by hydrogen bonds. The distance between two oxygens is 2.8 Å. Ice Ih has a Wurzite structure; this structure forms two hexagonal plates called basal planes with a distance of 4.5 Å between two neighbor oxygens that face out the plane, and prism planes that consist of six equivalent sides (Figure 1(B)). The direction normal to the basal plane is called the c-axis; the hexagonal plate of the crystal is defined by the three a-axes perpendicular to the c-axis. The cell size in the c-direction is 7.4 Å and in the a-direction is 4.5 Å. When ice crystal grows in the water, it is added to the prism plane rather than the basal plane and crystallites form disc-like sheet. However, in the presence of AFPs, ice growth differs significantly from that of free AFPs; bypyramidial crystallites and columnar spicules are formed instead of sheets. For instance, type III AFPs bind to the prism plane at steps, and form the hexagonal bypyramidal crystals [20]. The ice crystal grows towards an apex by preventing c-axis further growth and ice growth on the prism faces is inhibited (Figure 1(C)).

Figure 1: Ice structure. (A) The atomic structure of hexagonal Ice Ih. Figure (A) is taken from http://www.lsbu.ac.uk/water/ice1h.html (B) Hexagonal ice structure (Ice Ih) (C) A diagram of hexagonal bipyramidal structure formed by most fish AFPs. Figures and caption of (B,C) were modified from [13] with permission of the Cryobiology .

1.3 The general mechanisms of antifreeze activity

With the discovery of several AFPs, different mechanisms were proposed in order to explain the activity and interaction between AFPs and ice. The adsorption-inhibition mechanism published by Raymond, J.A. and A.L. DeVries (1977) [21] remains the

standard reference for understanding of the basic mechanism. It was explained that the protein prohibits the ice growth by binding to the ice surfaces without the need to completely block the access of water molecules to the ice surface. Rather it creates a new rough surface with low equilibrium temperature as explained below. It was believed that binding of AFPs to the ice surface involved in adsorption is due to the formation of hydrogen bonds that are the dominant force for the interaction with ice and protein. Later, it was shown that hydrophobic interaction and van der Waals interaction play a role as well. Indeed, the ice binding surfaces of AFPs are relatively hydrophobic with a regular spacing of amino acids that matches the spacing of the ice structure [22-25]. However, the activity of the AFP is not completely understood yet. Further, if nucleation did not occur, there would be no ice growth. AFP influence on nucleation as well which will be discussed in 1.3.3

1.3.1 Colligative phenomena

A colligative phenomenon is dependent on the molar concentration of solute molecule, regardless of its chemical property. The freezing temperature and the melting temperature can be lowered by the molar concentration of solution. For every molar of free ions, the melting temperature is depressed by 1.86 oC [15]. With the colligative phenomenon, there is no difference between the freezing temperature and the melting temperature [15]. On the contrary, the antifreeze activity is a non-colligative phenomenon in that there is TH and the depression of the freezing temperature depends on the physical properties of the AFPs.

1.3.2 Adsorption-Inhibition: The Gibbs-Thomson Model

The generally accepted mechanism for AFP activity is an adsorption-inhibition mechanism in which AFPs in the solution are allowed to bind to an ice surface, resulting in the inhibition of ice growth because of the Gibbs-Thomson effect [21,23,26,27]. The Gibbs-Thomson effect relates the curvature of an ice crystal to the local melting

temperature, which is lower than the equilibrium temperature of the bulk, causing the ice front to grow as convex shapes, and eventually to stop growing. The explanation in the thermal dynamic terms about the antifreeze mechanism is expanded from the review article by Yeh and Feeney [15]. When a small ice crystal is growing, the molecules that join that crystal increase its volume as well as its surface. These two factors contribute to the free energy change. Thus, ΔGG=Δ +Δ G (1) VS

where Δ Gv, is the volume free energy change, and ΔGs is the surface free energy change. Next, the relation G = Nμ is used, where N is the number of molecules in the solid and μ

dG dG dG is the chemical potential. μ = = V + S dN dN dN

For the solid phase, the chemical potential μ S due to the surface of an ice crystal is expressed as: dG γdA μ = S = (2) S dN dN

dGS γdA γdA dV dA This equation can be also expressed as: μ S = = = × = γ × Ω dN dN dV dN dV where γ is the surface tension (for water, γ= 0.030 J / m 2 [28]) and Ω is the volume of dA 2 one mole of water molecules. For a spherical ice with radius ρ, the curvature = dV ρ 4 2 whereV = πρ 3 , and A = 4πρ 2 . Thus, μγ= Ω . So, the total chemical potential of 3 s ρ the solid phase is: 2Ωγ μρ()=∞+ μ () (3) ice ice ρ where μice (ρ =∞ ) is the chemical potential for the bulk ice with a surface of infinite radius. For the melt phase, the free energy is GHTS= − [29], and the chemical ∂∂GH ∂ S potential is μ ==−T . The dependence of the chemical potential on the ∂∂NN ∂ N temperature can be expressed in the linear approximation:

∂∂μ S μμ()TT≈+−=+−− () ( TTT ) μ ()( )( TT ). (4) mmmm∂∂TN

At equilibrium melting temperature Tm between a bulk ice ( ρ = ∞ ) and water, Δ=Δ−Δ=GHTS0, the entropy loss per molecule during freezing, ΔS is equal the ΔH enthalpy change (latent heat of fusion per molecule: ΔH ): Thus Δ=S . Tm For simplicity, one can assume that the entropy of the ice is much smaller that of ∂S the water, ≈ΔS . So the total chemical potential of the melt phase is: ∂N

ΔH (Tm − T ) μsoln (T) = μsoln (Tm ) + (5) Tm where Tm − T is the difference between the equilibrium melting temperature and the freezing temperature of a bulk ice. When two phases are at equilibrium, the chemical potential of the solution is the same as the chemical potential of the ice phase:μice (ρμρ=∞ ,TT = m ) = soln ( =∞ , TT = m ) . A crystal with radius ρ will be in equilibrium with the solution at temperature that will satisfy the conditions:

μice(,)ρμρTT= soln (,) From equation (3) and (5), ΔH (T − T ) 2Ωγ m = (6) Tm ρ Thus, the lowering of the freezing temperature ( Δ T) due to the presence of the finite- sized ice crystals with radius ρ is given by: 2ΩγT ΔT = (T − T ) = m (7) m ρΔH Equation (7) describes that the critical supercooling of a spherical particle is directly proportional to the interfacial energy and inversely proportional to the radius of the ice crystal [15]. By using equation (7), the radius of curvature can be expected depending on the measurement of thermal hysteresis. For example, if one wants to calculate the radius of curvature when the thermal hysteresis is 2 °C, the constant value of water molecule can

15 be used : the latent heat of fusion per molecule ΔH = 334×18 J /mol [30], the surface tension γ =0.030 J / m 2 [28], and the volume of one mole of water molecule Ω =

18cm3 / mol= 18×10−6 m3 / mol . Equation (7) is changed to the formula for the radius of 2ΩγT curvature: ρ = m . So, we can expect the radius of the curvature to decrease 2 °C ΔTΔH of the non-equilibrium freezing temperature: 2ΩγT 2×× (18 10−63mmol / ) × (0.030 Jm / 2 ) × (273.15 K ) ρ ==m =25 nm ΔΔTH(2 K ) × (334 × 18 Jmol / ) Therefore, the maximal distance between proteins can be determined which is in the order of 50 nm. This typical length is bigger than the typical size of the proteins which is on the order of 5 nm, and suggests that full cover of the ice surface by the AFPs is not necessary for the protection from freezing. Indeed measurements of the AFP surface concentration by fluorescence microscopy indicate that the distance between the proteins is about 20 nm [31]. When AFP molecules bind to the ice surface, they inhibit the further growth of ice at the point of contact. Further ice growth between adjacent adsorbed AFPs in the surface of ice will develop a local curvature that increased as the ice is growing. This process makes harder that water adds to the ice surface, resulting in the depressed local- equilibrium temperature which is lowered below the melting temperature of the bulk. This difference between the depressed non-equilibrium freezing temperature and the melting temperature is called thermal hysteresis (TH). During the thermal hysteresis gap, the shape of ice crystals remains stable, and at the depressed non-equilibrium freezing temperature, it starts to grow (Figure 2). This progress of adsorption-inhibition is illustrated in Figure 3 [27] and Figure 4. It has been observed that AFPs shape the seed ice crystals during TH measurements. Such ice crystals have the characteristic morphology of the facet caused by the inhibition of the growth of the adsorbed surfaces to which the AFPs bind.

Figure 2: Illustration of the thermal hysteresis. The ice crystal morphology of fish type III AFP is used. Within the thermal hysteresis gap, the crystal remains stable. It grows explosively when it reaches the non-equilibrium temperature. Figure courtesy of Dr. Natalya Pertaya.

Figure 3: Adsorption-Inhibition of ice crystal growth. (A) AFPs (open circles) in solution contact with the ice surface (hatched line) at 0°C. (B) AFPs bind to the ice surface at a supercooling. The curvature between bound AFPs inhibits ice growth by the Gibbs-Thomson effect. Figure and caption were modified from [32] with permission of the Biophysical Journal.

Figure 4: A schematic representation of the curvature of the ice developed between adjacent AFP molecules. It shows that as the ice is growing between the AFP molecules (red circles), its local curvature is increased; the local radius of the ice crystal becomes smaller. Thu, this configuration is stable at temperature lower than the bulk melting temperature.

1.3.3 Nucleation Inhibition

Ice crystallization is a freezing process in supercooled water. In this process, water should undergo the stage of ice nucleation, followed by the growth of ice. Depending on whether or not freezing takes place, ice nucleation can be determined. Antifreeze proteins prevent freezing, either by the inhibition of nucleation or by the inhibition of crystal growth when a crystal is present in the solution. Inhibition of nucleation can not be monitored directly as the nucleation seeds are too small to be observed by light microscopy, but the change in the kinetics of the nucleation reveals their activity. The inhibition of nucleation by antifreeze proteins is probably by passivating nucleation seeds [14,15,33].

1.4 Molecular structures of antifreeze proteins

AFPs are diverse in their structure. They can be classified to antifreeze glycoproteins (AFGPs), four different types of fish antifreeze proteins (types I, II, III, IV AFPs), and four distinct types of insects antifreeze proteins (the spruce budworm, the pyrochroid beetle, the yellow mealworm, the snow flea AFPs). In addition, there are plant, fungi and bacteria AFPs. The different structures of the proteins have different ice binding affinities to few crystal directions, resulting in the different morphology of crystals that grow in solution of the AFPs. In order to examine the detailed mechanism about what is the function of the AFPs, it is needed to understand the characterization of the molecular structure of AFPs. Even though it is one common characteristic that they are stable at or near 0°C, they show considerable variations in their secondary structure. The first AFPs detailed below is fish AFPs including AFGPs, called also moderately active AFPs because the thermal hysteresis is in range from 0.7 ° to 1.5°. Next, the insect AFPs are discussed. They are called hyperactive AFPs due to their higher thermal hysteresis activity (above 1.5°).

AFGPs were found in the fish (Pathogenia borchgrevinki and Dissostichus mawsoni). It was discovered that AFGPs from their blood serum had glycotripeptide backbone with three repeated tripeptide units (Alanine-Alanine-Threonine) and a disaccharide residue where two sugar groups are linked [12,34,35]. Eight distinct fractions of AFGPs have been isolated, ranging in molecular mass from 2.7 kDa to 32 kDa, and they have different number of tripeptide repeats [36]. AFGPs exist in a solution as extended left handed three-fold helices and do not have much α-helical content [37]. In this conformation, the disaccharide groups are positioned on one side of molecule where they present stable, hydrophilic, and hydrogen bonding side, capable of interacting with ice surface, while the hydrophobic Alanine groups are in the primary position on the opposite side [38]. However, the three-dimentional structure of AFGPs is not determined yet.

Type I AFPs are found in Winter flounders (Pleuronectes americanus), Yellowtail flounders (Limanda ferruginea), Shorthorn sculpins (Myoxocephalus

19 scorpius), and Grubby sculpins (Myoxocephalus aenaeus). These AFPs are rich in alanine like AFGPs, and have long single α-helix for their secondary structure (Figure 5). There are four repeating (Thr/Asp or Thr/Asn) ice-binding motifs to form a binding surface [32]. Type II AFPs are found in Sea raven (Hemitryptrrus), Smelt (Osmerus mordax) and Atlantic herring (Clupeah harentus harengus) and are the largest of the non- glycoprotein fish AFPs. The most significant feature of these AFPs is that they are globular, cysteine rich molecules (8.3 mol % for the sea raven and up to 9.1 mol % for the Atlantic herring). In contrast to type I AFPs, type II AFPs have limited α-helix content (Figure 5). Type II can be divided in calcium dependent (herring and smelt) and calcium independent (sea raven) [39]. Type III AFPs are found in both Northern and Antarctic Eelpout (Macrozoarces anerucanus). They are globular protein and their structures are not dominated by any particular amino acids, neither alanine nor cysteine residues. They have unusual fold, consisting of short β-strands with a single turn of α-helix [20,34,40,41] (Figure 5). Type IV AFPs are discovered in the Longhorn sculpin (Myoxocephalus octodecimspinosis). They might have an 22 amino acid repeats, but there is no proposed three dimensional structure [42-44]. Even though the thermal hysteresis activity of AFPs was first studied in the mealworm Tenebrio molitor over 40 years ago, only four insect AFPs have been characterized. They are relatively small from 8.4 kDa to 9.0kDa. TmAFP is a small (8.4KDa), highly disulfide-bonded, right-handed β-helix protein, consisting of seven repeated 12-amino acid loops (TCTxSxxCxxAx where x is any amino acid) with high threonine and cysteine contents [45]. Each repeat forms one loop of beta-helix and is internally disulfide-bonded, and the 12-amino acid loops are almost identical. Seventeen threonine residues are placed in the protein, and among them, nine threonine residues form the ice-binding face. The spacing of the hydroxyl group is a nearly perfect match to the prism plane [46].The two threonines are in each repeat, which forms 4.64Å by 7.44Å (Figure 6). Note that these spacing are similar to the spacing of the ice crystal. The AFP from fire beetle(Dendroides Canadensis) is very close to TmAFP in that it is rich in threonine and cysteine, and has almost identical 12-13 amino acid repeats [40,47,48].

Figure 5: The structures of AFPs. Four types of fish AFPs and two types of insect AFPs with α-helix in red, β-sheet in yellow, and backbone in gray. PDB accession codes are given in parentheses. (A) Fish type I AFP from winter flounder (1J5B) (B) Fish type II AFP from sea raven (2AFP) (C) Fish type III AFP from North-Atlantic ocean pout (1KDE). (D) Insect AFP from Tenebrio molitor (1L1I) (E) Insect AFP from spruce budworm (PDB: 1Z2F). All diagrams were generated by using Rasmol from Protein Data Bank (PDB).

The AFP from the Spruce budworm (Choristoneura fumiferana), called sbwAFP, is similar with two insect AFPs mentioned before, but with some modifications. It has high serine and threonine content, and left-handed β-helix structure (Figure 5) with 15- amino acid loops [23]. It has the four TxT (where x is any amino acid) motifs located on one face of the protein that match the ice lattice on both prism and basal plane. Despite structural differences between sbwAFP and TmAFP, their ice binding sites have similar arrays of threonine residues on one side of prism plane. In addition, all three insect AFPs contain the TxT motif, so this sequence might be the ice-binding motif. In sbwAFP, it has been identified that TxT is the ice-binding site by mutagenesis [23].

(A) (B)

Figure 6: The structure of TmAFP. (A) Side view of the TmAFP β -helix with the β - sheets (TCT sequences) indicated by green arrows and the disulphide bonds in yellow. Threonine side chains on the β -sheet surface are shown with oxygen atoms in red. (B) End-on view of the β -helix. Figure and caption were taken from [46] with permission of the Nature.

1.5 Hyperactive antifreeze proteins

Although it was recognized early that AFPs from insects are effective, it has been recently reported that some AFPs from bacteria [6], primitive insects [49], and fish [50] are also considerably more active at depressing the freezing temperature than most fish AFPs. The more active AFPs, usually above 1.5°C of TH, are called “hyperactive AFPs”. Ice crystals formed in the structurally distinct and novel AFPs remain stable to lower the freezing temperature, and grow in a direction perpendicular to the c-axis (along the a- axis) when cooled down to the freezing temperature; hyperactive AFPs explode perpendicular to the c-axis, whereas fish AFPs burst the parallel to the c-axis [13] (Figure 16). In addition, the difference in TH at a millimolar concentration between insect and fish AFPs is approximately two orders of magnitude; it is 10-100 times more active. For instance, non-equilibrium freezing temperature for a 20µM spruce budworm AFP (sbwAFP) is observed at -1.08°C, while a 400µM of winter flounder type I AFP depress the freezing temperature by -0.27 °C (Figure 7) [23]. Thus, sbwAFP depresses the freezing temperature about four times at one-twentieth concentration of the type I fish AFP. There is the difference of ice morphology shown in the Figure 7. In the presence of the sbwAFP, the hexagonal ice crystal is obtained that bursts perpendicular to the c-axis, and the bipyramidal ice crystal that burst out of the tips along the c-axis is produced by type I AFP. Our collaborator Prof. Peter Davies in department of Biochemistry at Queen's university suggests that the ability of hyperactive AFP to block growth in the c-axis is the reason for their hyperactivity [13].

(A)

(B)

Figure 7: Ice crystal morphologies of insect AFP and fish AFP. (A) 20 µm spruce budworm antifreeze protein (sbwAFP). (B) 400 µm winter flounder type I AFP. There was no observable change in the ice crystals as the temperature was lowered until the non-equilibrium freezing point was exceeded at -1.08 °C for sbwAFP, and at -0.27 °C for type I AFP. Figure and caption were taken from [23] with permission of the Nature.

1.6 Previous studies of type III AFP with attached bulky proteins

It has been proposed that the cooperative interaction between AFPs is needed for the complete inhibition of ice crystal growth, and the all ice surfaces were completely covered by cooperative intermolecular interaction between AFPs [51] (Figure 19 (A)). To test this hypothesis, DeLuca et al. [10] constructed fusion proteins with globular type III AFP (7 kDa) that are larger in diameter than free type III AFP: the thioredoxin (TRX, 12kDa) and the maltose-binding protein (MBP, 42 kDa) were added to type III AFP by constructing thioredoxin-type III-His-tag fusion gene (TRX-AFP) and maltose-binding protein-type III- His-tag fusion gene (MBP-AFP). 'His' stands for the common use of six histidine amino acids, and is added to proteins for purification purpose. In this article, a wide range of concentrations from μM to mM was measured for thermal hysteresis activity of AFPs. At almost all concentrations, type III fusion proteins were at least as

24 active as the free type III AFPs. Especially, at the low concentration, two fusion proteins were more effective than free type III AFPs; two fusions were up to two or three times more active in the concentration 0-0.5 mM, and MBP-AFP was more active than TRX- AFP in this range (Figure 8). This article also described that MBP and TRX by their own did not show thermal hysteresis activity, and they did not have an influence on the ice crystal morphology (Figure 9). This paper suggests that the type III AFPs fusion proteins make more active rather than eliminate the thermal hysteresis activity because the ice surface is covered by the each molecule and it is more difficult for the ice to grow over fusion protein with large domains. The enhanced activity of the AFP-fusion proteins can be explained by the Gibbs-Thomson effect. Thus, this paper showed that there is no cooperativity for type III AFP.

Figure 8: Thermal Hysteresis of fish type III AFP and its fusion proteins. (a) The thermal hysteresis activities (°C) of MBP: AFP ( ) and TRX: AFP ( ) as a function of concentration are compared to that of fish type III AFP ( ). Results are the mean ± standard deviation (SD) of triplicate determinations. (b) Expansion of a sub-mM AFP concentration range. Figure and caption were modified from [10] with permission of the Biophysical Journal.

Figure 9: Ice crystals formed in the presence and absence of AFP-fusion proteins and their constituents. (a) in the absence of protein (b) in the presence of thioredoxin (10 mg/ml) in 100 mM NH4HCO3 (pH 7.9) (c) in the presence of maltose-binding protein (20 mg/ml) in 100 mM NH4HCO3 (pH 7.9). Photographs were taken at 0.02°C of supercooling. The right-hand panel shows ice crystals formed in the presence of 100 mM NH4HCO3 (pH 7.9) and mM concentrations of (d) type III AFP, (e) TRX: type IIIAFP, and (f) MBP: type IIIAFP at 0.2°C undercooling Figure and caption were taken from [10] with permission of the Biophysical Journal.

1.7 Aim of the thesis In the previous section, the non-cooperativity of type III AFP was discussed. It would be informative for the understanding of AFPs to check if this non-cooperativity is applicable to hyperactive AFPs as well. To check this, we collaborated with a group in the Weizmann Institute Science (WIS) that produced a Tenebrio molitor AFP (TmAFP) linked with bulky proteins [11]. In this thesis, these fusion proteins were assayed by a nanoliter osmometer; a device that has been designed to measure TH of AFPs. The results, which I will detail in the next section, indicate that the addition of large molecules to the TmAFP does not induce any loss of thermal hysteresis activity; these fusion proteins were rather more active than free TmAFP at almost all concentrations. Furthermore, ice crystal morphologies obtained by the fusion proteins were the same as the ones of free TmAFP. In the next section, I will describe the methods, which were used, the results of the experiments, and the conclusions of the thesis.

2. Sample preparation and Experimental Method

2.1 Materials

All TmAFP proteins were provided by the Group of Prof. Deborah Fass from the Department of Structural Biology at Weizmann Institute of Science (WIS) in Israel. Maya Bar, a PhD student of Dr. Fass group, constructed the fusion proteins (MBP-His6- TEV-TmAFP and His6-eGFP-TmAFP) [11].

2.1.1 Tenebrio Molitor AFP (TmAFP)

TmAFP extracted from the larvae of the yellow mealworm Tenebrio molitor, and its sequence had been determined [45]. TmAFP is highly disulfide bond protein, having twelve amino acid repeat sequences (TCTxSxxCxxAx). It has a cysteine of 18.8 % and a threonine content of 22.4 %, which is known as the ice-binding site. The molecular weight is 8.38 kDa calculated from amino acid sequences. The entire amino acid sequences are (from Expert Protein Analysis System (ExPasy) http://ca.expasy.org/uniprot/O16119): 10 20 30 40 50 60 QCTGGADCTS CTAACTGCGN CPNAVTCTNS QHCVKATTCT GSTDCNTAVT CTNSKDCFEA

70 80 QTCTDSTNCY KATACTNSTG CPGH

Cysteine residues are colored yellow and they are linked with disulfide bonds.

2.1.2 MBP-His6-TEV-TmAFP

The first fusion protein is the MBP- His6-TEV-TmAFP construct which was originally used to assist in the production of TmAFP [11]. The same bulky protein MBP (42kDa) that was used in the work on AFP III (7kD) was fused to the TmAFP (8.4 kDa). This fusion protein includes the His6 tag (HHHHHH) and the TEV protease cleavage site (ENLYFQG) between MBP and TmAFP (Figure 10).

The His6 tag, also known as polyhistidine tag, is an amino acid sequence that consists of six histidine residues and is used for purification of the proteins as it has high affinity to Nickel or Cobalt. It is usually added to the N-terminus (known as amino- terminus, NH-2-terminus) or C-terminus (known as carboxyl-terminus, or COOH- terminus) of the protein. The His6 tag is also designed with specific recognition site for cleavage by the TEV protease between MBP and TmAFP; the TEV protease is a site- specific protease that is found in the Tobacco Etch Virus (TEV), and it is a useful reagent for cleaving fusion proteins. Insertion of the cleavage site should not interfere with protein function. The total amino acids linked in the MBP-His6-TEV-TmAFP are 479, and the molecular weight is 51982Da calculated using the ProtParam tool of the ExPasy proteomics server [52]. The entire sequence is the following: The six-Histidine tag is colored blue and TEV is colored green.

10 20 30 40 50 60 MKIEEGKLVI WINGDKGYNG LAEVGKKFEK DTGIKVTVEH PDKLEEKFPQ VAATGDGPDI

70 80 90 100 110 120 IFWAHDRFGG YAQSGLLAEI TPDKAFQDKL YPFTWDAVRY NGKLIAYPIA VEALSLIYNK

130 140 150 160 170 180 DLLPNPPKTW EEIPALDKEL KAKGKSALMF NLQEPYFTWP LIAADGGYAF KYENGKYDIK

190 200 210 220 230 240 DVGVDNAGAK AGLTFLVDLI KNKHMNADTD YSIAEAAFNK GETAMTINGP WAWSNIDTSK

250 260 270 280 290 300 VNYGVTVLPT FKGQPSKPFV GVLSAGINAA SPNKELAKEF LENYLLTDEG LEAVNKDKPL

310 320 330 340 350 360 GAVALKSYEE ELAKDPRIAA TMENAQKGEI MPNIPQMSAF WYAVRTAVIN AASGRQTVDE

370 380 390 400 410 420 ALKDAQTNSS SHHHHHHDYD IPTTENLYFQG EFGSQCTGG ADCTSCTAAC TGCGNCPNAV

430 440 450 460 470 480 TCTNSQHCVK ATTCTGSTDC NTAVTCTNSK DCFEAQTCTD STNCYKATAC TNSTGCPGH

Figure 10: Schematic representation of MBP-His6-TEV-TmTHP (TmAFP) constructs. The TEV recognition site is represented by a caret. Figure was taken from [11] with permission of the Protein Expression and Purification.

2.1.3 His6-eGFP-TmAFP

The second fusion protein is the His6-eGFP-TmAFP that is a 27 kDa protein. The eGFP stands for Enhanced Green Fluorescence Protein that has fluorescence properties [53]. His6 is added at the N-terminal of eGFP and then TmAFP is linked (see Figure 11). The total amino acids linked are 346, and the molecular weight is 37729 Da that is also calculated based on the amino acid sequences [52]. The entire sequence is the following: The six–Histidine tag is colored blue and bold letter is the TmAFP

10 20 30 40 50 60 MGSSHHHHHH SSGLVPRGSH MVSKGEELFT GVVPILVELD GDVNGHKFSV SGEGEGDATY

70 80 90 100 110 120 GKLTLKFICT TGKLPVPWPT LVTTLTYGVQ CFSRYPDHMK QHDFFKSAMP EGYVQERTIF

130 140 150 160 170 180 FKDDGNYKTR AEVRFEGDTL VNRIELKGID FKEDGNILGH KLEYNYNSHN VYIMADKQKN

190 200 210 220 230 240 GIKVNFKIRH NIEDGSVQLA DHYQQSTPIG DGPVLLPDNH YLSTQSALSK DPNEKRDHMV

250 260 270 280 290 300 LLEFVTAAGI TLGMDELYKL GSQCTGGADC TSCTAACTGC GNCPNAVTCT NSQHCVKATT

310 320 330 340 350 360 CTGSTDCNTA VTCTNSKDCF EAQTCTDSTN CYKATACTNS TGCPGH

Figure 11: Schematic representation of His6-eGFP-TmTHP (TmAFP) constructs. Figure was taken from [11] with permission of the Protein Expression and Purification.

2.2 Experimental equipment

Experimental setup consists of a custom-built temperature controlled stage (Figure 12(A)). A thermistor in conjunction with two thermoelectric cooling elements is embedded in the cell (Figure 12(B)). A circular metal plate (~ 0.8cm) in the cell is used to suspend the protein solution into the immersion oil droplet (Figure12 (C, D)). The cell itself is cooled by circulation of cold water. Cold water is circulated to provide the heat sink for the thermoelectric cooling elements by the pump inside the cell and warm water is going out from the cell. Water circulation is controlled by a temperature controllable Water Cooled Bath/Circulator (Neslab RTE-111). Dry air from Drierite calcium sulfate (CaSO4) is blown to the cell to keep it free of moisture; drierite is a desiccant used to remove moisture, and when it starts to have loaded with humidity, it changes the color from blue to pink that indicates absorption of moisture. This custom-built arrangement can allow the precise control of the temperature of the solution. It can be changed in range from the room temperature to -40°C with a precision of ±0.01°C by temperature controller (Newport Temperature controller Model 3040, Irvine, CA). A time of 0.1s is required for the temperature change of 0.01°C. This system was controlled by a Labview interface (Figure 13) that was developed in Dr. Braslavsky's lab.

0.5 cm

Figure 12: The experimental apparatus. (A) Custom-built temperature controlled apparatus from left to right- temperature controller between monitor and VCR, microscope, drierite (CaSO4), air pump, and water pump. (B) A schematic drawing of the temperature controlled ( Modified from [31] with permission of the Biophysical Journal) (C) The cell with a circular metal plate. (D) The circular metal plate holds a small droplet of protein solution. Pictures were taken in the lab.

Figure 13: Screen shot of the Labview interface for controlling the temperature of an ice crystal. It shows the image of the crystal with the typical lemon shape of the TmAFP as it recorded during the experiment.

2.3 Experimental procedures and measurements

2.3.1 Protein purification by adsorption to ice

Protein purification is a process to isolate a single type of protein from a complex mixture. It is important for the characterization of the function, structure and interactions of the protein of interest because it produces a large quantity of purified proteins for use. Adding a tag to the fusion protein gives it a specific binding affinity. Usually the recombinant protein is the only protein in the mixture with this affinity, which aids in separation. The fusion protein used in my experiment has the histidine tag that has affinity to nickel or cobalt ions. Thus by immobilizing nickel or cobalt ions, an affinity

33 support that specifically binds to histidine-tagged proteins can be created. Furthermore, the AFPs themselves have a specific affinity to ice, thus they can be used for their own purification [54]. In the self-purification method of AFPs, ice is grown on a cold finger, as the AFPs selectively adsorb to the ice while other proteins and salts do not. The ice part contains a purified ice binding proteins while the liquid part contains a crude mixture. To purify two fusion proteins, the cold finger was constructed before measuring the thermal activity of these proteins. Before starting the cold finger, the sample needed to be diluted to less than 1 mg/ml protein concentration and less than 100mM salt in a total volume of 80-120 ml [54]. As shown in the Figure 14, the cold finger was connected to a temperature controllable Water Cooled Bath/Circulator (Neslab RTE-111) containing water/ ethylene glycol mixture. The fusion protein solution was placed in a150 ml beaker insulated with polystyrene foam. To mix the protein solution well while the ice was accumulated to the cold finger, the small magnetic stirrer was placed inside the beaker. The cold finger was inserted into the beaker, and then solution started to cool down. The temperature was gradually lowered in the step of 0.1°C every 30 minutes until the clear ice was formed around the bottom of the cold finger. The ice suspended into the cold finger should be clear because cloudy ice indicates that the cooling step was too quick [54]. The ice with cold finger was removed from the unfrozen liquid sample, and washed with distilled water. It was placed into the clean beaker until detached from the cold finger. After melting, it was used to measure the antifreeze protein activity.

(A)

(B)

Figure 14: (A) The cold finger apparatus. (B) General method for ice-binding protein extraction: A: seeding the cold finger with ice; B–D: growing the ice; E: the ice fraction containing the bound AFPs. Figure and caption were taken from [54] with permission of the Biochemical and biophysical research communications.

2.3.2 Procedure and Measurement of protein concentration

All protein assays were prepared in 20 mM ammonium bicarbonate (NH4HCO3) buffer and clarified by centrifugation. To measure the protein concentration, first, the extinction coefficient that indicates how much protein can be absorbed at a certain wavelength was calculated by the amino acid composition using the ProtParam ExPasy tool. The absorbance was determined by UV spectrophotometry (Clippinger #367, Dr. Tees’s lab). Before use of the UV spectrophotometry, the instrument was warmed up at least 10 minutes, and adjusted to detect the desired wavelength. Proteins in solution absorb ultraviolet light with absorbance maxima at 280 and 200 nm. The spectrophotometry was calibrated to zero absorbance with buffer solution only in the quartz cuvette that is called the blank; for MBP-TmAFP, 6 M of guanidine hydrochloride (GuHCl) was used in order to unfold the proteins for accurate measurement of absorption at 280 nm. The eGFP-TmAFP has strong absorption at 488 nm. In this case, 20 mM

NH4HCO3 of buffer is used for the blank. This buffer does not unfold the protein. After making the zero absorbance measurement, the small amount of the protein to be measured was added to the blank. The absorbance of the protein solution was measured and the protein concentration was calculated by using the Beer-Lambert law:

A=εbc (8) where A is absorbance (no units), ε is the extinction coefficient with units of M-1 cm-1, b is the path length of the cuvette (1cm is usually used), and c in the concentration of the protein solution with units of M.

1) MBP-TmAFP

Absorbance at 280 nm is 0.035, and 800 μl of 6 M of GuHCl for the blank and 6μl of the protein were used. The extinction coefficient is 70820 M-1 cm-1. Using these values in equation (8), the concentration of the MBP-TmAFP is found to be 66 ± 1.0 μM (= 3.45 mg/ml where the molecular weight is 51982.3 g/mol)

2) eGFP-TmAFP

800 μl of NH4HCO3 (20 mM) and 6 μl of protein were used to measure the absorbance. 0.017 of the absorbance was obtained. When 10 μl of protein was added, the absorbance was 0.027. The extinction coefficient is 55900 M-1 cm-1 at 488 nm. Again by using the equation (8), the concentration determined is 40 ±1.0 μM (=1.51mg/ml where the molecular weight is 37729.2 g/mol).

2.3.3 Procedures and Measurement of the Thermal Hysteresis activity

Before measuring the thermal hysteresis activity, it was necessary to clean the circular metal plate of any contamination completely. First, isopropanol was used to remove the oil and dirt. The circular metal plate was placed in a beaker with isopropanol and sonicated at 40 °C for between 10 and 15 minutes. After the sonication, it was cleaned again with isopropanol, ethanol, and acetone. Air was blown to dry and to clean the holes as a last step. Through these steps, the hole to contain the oil and AFP solution was cleaned enough for the experiments. When the cleaning was finished, a small droplet of oil was loaded on the back of the circular metal plate, and then placed in the cell (Figure 12 (C,D)). The temperature was controlled with the Labview interface (Figure 13) that was connected to the nanoliter osmometer temperature controller (Newport Model 3040). Sub-microliter volumes of AFP solution were introduced into one of the holes of the circular metal plate, by using the pipette needle with micro tip. These needles were prepared by pulling a diameter of 1 mm glass tubes in a pipette puller device. The circular metal plate has seven tiny holes (diameter 0.41 mm) (Figure 12 (D)). The droplet of AFP solution should not touch the edge of the hole. If it touched the edge of the hole, it interferes with the accurate measurement of thermal hysteresis activity. After loading the small droplet of AFP solution, the temperature was lowered until the droplet of protein completely freezes (called first freezing temperature) usually the droplet nucleated at -30°C. Then the

37 temperature was gradually increased to melt the ice until only one small crystal left in the droplet. After a single ice crystal was obtained, the temperature was changed in a controlled manner, with a resolution of 0.01°C, to find the melting point. At the melting temperature, the size of an ice crystal was decreased, and just below the melting temperature, an ice crystal retained in size and shape. To make sure that the crystal is stabilized and to allow the AFP to accumulate to the surface, it remained for 10 minutes and then the freezing point was determined by slowly cooling a drop of AFP solution that contains a small seed ice crystal at a rate of 0.01°C per 4s until the ice started to grow. After certain decrease of the temperature, the crystal cannot resist the temperature decrease anymore and it grew explosively to freeze the whole droplet. This growth occurred very rapid. All temperature controls were through the labview interface connected with a nanoliter osmometer. Observation and recording were through a microscope and video camera. The ice crystal morphology and its growth were monitored during the thermal hysteresis measurement. The experiment was observed through the microscope that has Ultra-Long-Working-Distance objectives of 40 x (numerical aperture (NA=0.5), WD=10mm) or 50 x (NA=0.55, WD=8.7 mm) and recorded with a SONY Hyper HAD camera. I used two systems to record the video; in one of the system, the video was recorded to a JVC VCR. In another system, the video signal was directly digitized by IMAQ card compressed by a DivX Codec 6.5.1 decoder in the labview program and saved directly into the computer hard drive. At later time, the movies were edited by DivX Author program.

All concentrations of the proteins in range from μM to mM were measured at least three times, and over a hundred of experiments were fulfilled to obtain the accurate and consistent measurement. In order to obtain the accurate thermal hysteresis, one protein drop was used maximum three times because in the high concentration, there were bubbles and salts left, and sometimes proteins lost their activities such as when the original shape shrank or the melting and freezing temperature were in the different range from previous measurements after several trials. For consistency, the size of the ice crystal measured was around 20μm.

3. Results

3.1 Ice crystal growth and morphology as a function of the TmAFP concentration

Ice crystal morphology was observed by microscope, and it was lemon-shaped that has the prominent tips connected by the c-axis. After observing the ice crystal whose size is around 20 μm, and waiting 10 minutes to make the ice crystal stabilized, the temperature was cooled down 0.01ºC every 4 seconds. When ice crystal was cooled below the melting temperature until it reached the non-equilibrium freezing temperature, the lemon-shaped ice crystal directly burst normal to c-axis (Figure17 (A)). This burst direction is different from the one of moderately active AFPs such as fish AFPs. The fish AFPs grow explosively along the c-axis (Figure 16). The thermal hysteresis of the most concentrated TmAFP at 1.5 mg/ml (170μM) was ~2.52°C with ± 0.43°C of standard deviation (SD). The relationship between thermal hysteresis activity and TmAFP concentration was shown in Figure 15. This graph shows similar trend with the one of the published paper obtained by Marshall et al. (2002) as the concentration increases [55]. At low concentration of 0.075 mg/ml (7μM) although the lemon shape was observed, no thermal hysteresis was detectable (less than 0.02°C). However, our result is lower than that measured by Marshall. The possible reason might be that the molar concentration of solution (20mM NH4HCO3) is different from that of previous study that used 100 mM NH4HCO3 for all assays. As mentioned in the introduction, thermal hysteresis can be affected by the molar concentration of the solution. The other reasons might be systematic difference in the procedures such as the size of the ice crystals and the rate of the cooling.

TmAFP 4.50

4.00

3.50

3.00

2.50

TH(°C) 2.00

1.50

1.00

0.50

0.00 0 50 100 150 200 Concentration(μM) TmAFP TmAFP_paper

Figure 15: Thermal hysteresis activity (°C) versus concentration (μM) for TmAFP. (♦) shows our result and the result of the published paper (Marshall et al. (2002)[55] with permission) is indicated by (■).Results are the mean ± SD (standard deviation).

20 µm

Figure 16: Growing direction of fish AFP and insect AFP. (A) The fish type III AFP. It grows directly along the c-axis. It was observed through the microscope with 50 x magnification. (B) The TmAFP (insect AFP). It burst normal to the c-axis. The 40x magnification of microscope was used. Photographs were captured by DivX author.

3.2 Thermal Hysteresis Activity of TmAFP-fusion proteins

The fusion proteins were assayed in 20 mM NH4HCO3 and purified by centrifugation for the thermal hysteresis measurements.

1) MBP-TmAFP

The MBP-TmAFP was soluble up to at 3.45 mg/ml (= 66.4 μM). First, this highest concentration was tried; however, it was not clear to observe the ice crystal and measure the thermal hysteresis activity because I observed that there were lots of bubbles during the TH measurements. This situation usually happened and observed at high concentration of the proteins. However, it has not been solved yet. This might be an intrinsic property of these AFP at high concentration. After diluting the original highest concentration, the thermal hysteresis was measured. The thermal hysteresis of MBP-TmAFP at 1.73 mg/ml (33.2μM) was approximately 3.15 ºC. This thermal hysteresis is almost two times more active than the TmAFP of the concentration around 30 μM. Especially, in the high concentration, there was large range of variation compared to other concentration measurements (Figure 18).because it is hard to obtain the clear well-defined lemon shape and the consistent size of the ice crystal. Through the thermal hysteresis measurements of the nine different concentrations in range from 33.2 μM to 1.11 μM, the lemon shape was obtained, and also the MBP- TmAFP directs the crystal to burst perpendicular to the c-axis (Figure 17(C)).

2) eGFP-TmAFP

The eGFP-TmAFP had limited solubility at 1.54 mg/ml (40.85μM) that our lab had. It had the thermal hysteresis reading of about 1.54 °C. The thermal hysteresis of 1.155 mg/ml (30.63 μM) diluted two-thirds from1.54 mg/ml (40.85μM) had 1.60 ° C. in these two highest concentration, there is no detectable difference of thermal hysteresis observed. This fusion protein also produced the lemon shape (Figure 17(B)). I could observe the consistent trend of the graph with the TmAFP and MBP-TmAFP, and below

41 the concentration 0.024 mg/ml, there was no distinct burst of ice crystal growth (thermal hysteresis is about 0.03°C). In the low concentrations, the two fusion proteins were more active than the free TmAFP; especially all ranges of the MBP-TmAFP concentration were up to two times more effective (Figure 18). MBP and eGFP were not only free from the thermal hysteresis activity, but also did not affect the ice crystal morphology.

Figure 17: Ice crystal morphologies. (A) TmAFP at concentration 18.8 μM. (B) eGFP- TmAFP at concentration 10μM. (C) MBP-TmAFP at concentration 1.33 μM. Pictures are taken by Divx program. All explosions happened normal to the c-axis. Figure (A) was observed through by 40x magnification of microscope, and the 50x magnification of microscope was used for (B, C). Photographs were captured by DivX author.

(A) TH vs.Concentration 5.00

4.50

4.00

3.50

3.00

2.50 TH (°C) TH 2.00

1.50

1.00

0.50

0.00 0 50 100 150 200 Concentration(μM) TmAFP MBP- TmA FP eGFP-TmAFP

(B) low concentration

2 TH (°C)

0 01020304050

-1 Concentration (μM)

MBP- TmA FP eGFP-TmAFP TmAFP

Figure 18: The thermal hysteresis activity of TmAFP and TmAFP-fusions. (A) The thermal hysteresis of MBP-TmAFP (■) and eGFP-TmAFP (▲) as a function of concentration are compared to that of the TmAFP (♦). Results are the mean ± SD. (B) Expansion of (a) in the low concentration range.

4. Conclusion

In this thesis, I confirmed that the ice crystal morphology induced by the hyperactive TmAFP was lemon-shaped, and had the noticeable tips connected by the c- axis. Especially, at the low concentration, it was clear to observe the lemon shape with tips. In TmAFP solutions, the ice crystal grows explosively perpendicular to the c-axis, whereas the ice crystal of the moderately active AFPs from fish burst parallel to the c- axis. A possible explanation is that the ice crystal of TmAFP binds to the surface of ice and prevents its growth in the c-axial direction at the basal plane. Even though the large companion domains were introduced by constructing the fusion proteins of the TmAFP (MBP-TmAFP and eGFP-TmAFP), the ice crystal morphology and the burst direction did not change at all. Without significantly affecting the melting temperature, two fusion proteins depressed more the non-equilibrium freezing temperature than the free TmAFP did. Thus, the fusion proteins make a more powerful thermal hysteresis activity; rather than eliminate or reduce the thermal hysteresis activity. The fusion proteins were more active than the free TmAFP at almost all concentrations tested. These results resemble the findings that were obtained from the fish type III AFP fusion proteins [10], and the explanation for this increase in activity is valid for the TmAFP as well. As the concentration of TmAFP is increased, the curvature of the surface needed to grow over between the bound AFPs is also increased. The ice crystal surface is more covered by the additional molecule of the fusion protein than the free TmAFP does, so it makes more difficult for the ice to grow over the surface. When the larger fusion proteins bind to ice surface, it is less susceptible for engulfment of the TmAFP, thereby lowering the non- equilibrium freezing temperature. Thus, the cooperativity is not required for hyperactive TmAFP to bind to the ice, and inhibit the further growth of the ice crystal as DeLuca et al concluded [10]. The possible reason is that if each molecule of AFPs binds to the ice surface with tight contacts to each other, when the large molecules are added, one might expect that these molecules will disturb the operation of the AFPs (Figure 19(A)). However, as shown in the results, the ice crystal morphology of the fusion proteins is the same as that of free TmAFP; the ice binding affinities (sites) are also the same. Thus, the

44 cooperative intermolecular interaction between AFPs is not required. The AFPs bind independently to the ice (Figure 19(B)).

Figure 19: Schematic representation of cooperativity and non-cooperativity. (A) Shows a situation in which AFPs bind to ice with tight contacts between themselves (cooperativity). If large molecules are added to the AFPs, these molecules will presumably disturb the operation of the AFPs. (B) If AFPs bind independently to ice surface (non-cooperativity), then when large molecules are added to the AFPs, these molecules will presumably not disturb the operation of the AFPs.

5. Discussion and future work

AFPs have an important role in avoiding freezing and in preventing freezing damages in organisms due to ice nucleation inhibition and ice recrystallization. They may be invaluable in cryosurgery and cryopreservation of cells, tissues, and organs [8,9]. They may protect membranes and metabolic activity. Furthermore, they may contribute to the agricultural industry in that AFPs prevent frost damage, preserve food, improve the quality of the frozen food, and extend the harvest season in cooler climates. Insect AFPs are more suitable for industrial application because they are considerably more active than the fish AFPs. The recombinant fusion approach may provide significant advantage; the lower concentration of TmAFP fusions can be employed to inhibition of ice recrystallization by using the same degree of thermal hysteresis. They can be used to provide high yield in an expression system or purification protocol [10]. The AFP fusion approach also may provide advantages in transgenic systems. This research showed that the eGFP-TmAFP have similar activity to TmAFP. As the eGFP is a fluorescence molecule, it opens the possibility to use this contrast with fluorescence microscopy and to learn about the TmAFP activity by direct observation of the fluorescence of molecules that bind to the ice. Another extension of this research could be to use the ability of fusion protein to drag bulky proteins into ice. This could be exploited as a purification method for big molecules. For example, several membrane proteins are hard to purify and it might be beneficial to purify them with the help of AFP proteins by dragging the fusion protein into the ice in a cold finger experiment. Furthermore, even bigger particles might be dragged into the ice. This might have advantages in some research. For example, if one can drag nanoparticles into the ice, it could be beneficial in research on heat generation of nanoparticle [56,57]. Thus, our lab and Prof. Richardson in chemistry plan to investigate that the AFPs can be used to examine the heat generation on nanoparticles embedded in an ice matrix. In this research, metallic nanoparticles are embedded in the ice and illuminated by laser. The heat generated by these nanoparticles melts the ice and the amount of heat that absorbed can be calculated. Without the AFP these nanoparticles tend

46 to be aggregated and to be concentrated in veins at the boundary between ice grains. Thus, AFPs could be used to drag nanoparticles into the ice and might prevent the aggregation of these nanoparticles. In this thesis, I check whether or not the hyperactive TmAFP needs the cooperative intermolecular interaction between AFPs to completely inhibit the further growth of ice crystal by using fusion proteins. As shown in the results, there was no change of the observed ice crystal morphology. Rather, the fusion proteins were more active in thermal hysteresis activity. This increased thermal hysteresis activity can be explained by the Gibbs-Thomson effect; the heavy molecules added to the TmAFP retard the growth of the ice crystal. Thus, I conclude that cooperativity is not required for hyperactive TmAFP which is in similar to the finding with moderately active fish type III AFP.

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