Thermal Stability of the Ribosomal Protein L30e from

Hyperthermophilic Archaeon Thermococcus celer by

Protein Engineering

LEUNG Tak Yuen

B.Sc. (Hon.), CUHK

A Thesis Submitted in Partial Fulfillment of the Requirement

For The Degree of Master of Philosophy in Biochemistry

July 2003 The Chinese University of Hong Kong

The Chinese University of Hong Kong holds the copyright of this thesis. Any person(s) intending to use a part or whole of the materials in the thesis in a proposed publication must seek copyright release from the Dean of the Graduate School /;/輕塑\ \ L； ：^ \

U^VE^ /, / ^O^LIBRARY SYSTEMX./ Table of Contents Acknowledgments { Abstract “ Abbreviations 衍 Abbreviations of amino acids iv Abbreviations of nucleotides iv Naming system for TRP mutants v

Chapter 1 I ntroduction 1 • 1 Hyperthermophile and hyperthermophilic proteins 1 1.2 Hyperthermophilic proteina are highly similar to their mesophilic 2 homologues 1.3 Hyperthermophilic proteins and free energy of stabilization 3 1.4 Mechanisms of protein stabilization 4 1.5 The difference in protein stability between mesophilic protein and 4 hyperthermophilic protein 1.6 Ribosomal protein L30e from T. celer can be used as a model 9 system to study thermostability 1.7 Protein engineering of TRP 10 1.8 Purpose of the present study 12

Chapter 2 Materials and Methods 2.1 Bacterial strains 13 2.2 Plasmids 13 2.3 Bacterial culture media and solutions 13 2.4 Antibiotic solutions 13 2.5 Restriction endonucleases and other enzymes 14 2.6 M9ZB medium 14 2.7 SDS-PAGE 14 2.8 Alkaline phosphatase buffer 15 2.9 DNA agarose gel 15 2.10 Gel loading buffer, DNA 16 2.11 Ethidium bromide (EtBr), 1 Omg/ml 16 2.12 Constructing mutant TRP genes 16 2.12.1 Polymerase Chain Reaction (PGR) 17 2.12.2 Gel electrophoresis 19 2.12.3 DNA purification from agarose gel 19 2.12.4 Construction of R39A, R39M, K46A, K46M, E47A, E50A, R54A, 19 R54M 2.12.5 Construction of double mutant R39A/E62A, R39M/E62A 20 2.13 Sub-cloning 21 2.13.1 Restriction digestion 22 2.13.2 Ligation vector with mutant TRP gene insert 22 2.13.3 Amplifying vector carrying mutant TRP gene insert 22 2.13.4 Mini-preparation of DNA 22 2.13.5 Preparations of competent cells 23 2.13.6 Transformation of Escherichia coli 24 2.13.7 Screening tests 25 2.14 Over expression of mutant TRP 26 2.14.1 Transformation 26 2.14.2 Expression 26 2.14.3 Cell harvesting 27 2.14.4 Expression checking 27 2.14.5 SDS-PAGE 27 2.14.6 Staining the acrylamide gel 28 2.15 Purification of mutant TRP protein 28 2.15.1 Cells lysis 28 2.15.2 Chromatography 29 2.15.3 Concentrating TRP as protein stock 31 2.16 Protein stability 32 2.16.1 Chemical stability 33 2.16.2 Thermal stability 34

Chapter 3 Results 3.1 Construction of mutant TRP genes 36 3.1.1 PGR mutagenesis 36 3.1.2 Sub-cloning of mutant TRP gene to express vector pET8c 37 3.2 Expression and purification of mutant TRP 38 3.3 Protein stability 39 3.3.1 Free energy of unfolding 39 3.3.2 Thermal stability 43

Chapter 4 Discussion 4.1 Effect of R39, K46, E62, E64 47 4.2 Double mutation at R39 and E62 50 4.3 Effect of R54 51 4.4 Effect of E47 and E50 53 4.5 Conclusion 54

References 57 Appendix 64 Acknowledgements

I would like to express my sincere gratitude to my supervisor Dr. K.B. Wong for his supervision and guidance in my M.Phil, study. I am very thankful for colleagues of M. M. W. 507 for their support. I also take this opportunity to express my appreciation to my family for their patience and understanding.

i Abstract:

In recent years, an increased interest in the origin of protein thermal stability has

gained attention both for its possible role in understanding the forces governing the

folding of a protein and for the design of new highly stable engineered biocatalysts.

Although many efforts have been made to isolate thermostable protein from

thermophilic organisms in the ho pe to unravel the s tmctural basis und erlying the

increased thermal stability of thermophilic protein, there are still no generalizations

in explaining the principles of stabilization. Here we used a ribosomal protein L30e

(TRP) from the hyperthermophilic archaeon Thermococcus celer as a model system to study thermostability of protein at high temperature. TRP resists thermal unfolding

at over 90�C O. n the other hand, its mesophilic counterpart, yeast ribosomal protein

L30e (YRP), which contains about 37% sequence identity with TRP, undergoes

irreversible unfolding at about 45°C. By comparing their protein sequences and crystal structures, TRP has more surface charged amino acid residues. In this study,

Arg39, Lys46, Glu47, Glu50, Arg54, Glu62 and Glu64 were under investigation. To

see if these charge-charge interactions contribute to the thermostability of TRP, site- directed m utagenesis has been c arried out. Ar ginine residue h as been mutated to

alanine and methionine while glutamic acid has been mutated to alanine in order to remove the extra charge-charge interaction. A total of twelve mutants 一 10 single mutants and 2 double mutants were constructed (R39A, R39M, K46A, K46M, E47A,

E50A, R54A, R54M, E62A, E64A, R39A/E62A and R39M/E62A). The conformational stability of the mutated proteins has been studied by circular

dichroism (CD). We found that charged residues R39, K46, R54 and E62 played an

important role in stabilizing TRP.

ii 摘要

近年來’蛋白質熱穩定性硏究愈趨成熟。這有助了解蛋白質的折疊機制和設計發展

熱穩定生物催化劑。雖然科硏人員已從眾多嗜熱生物中分離出不少熱穩定蛋白作爲

硏究對象，但仍未能得出一個簡單而明確的熱穩定機制。因此，我們選取了一種原

始的熱球菌Thermococcus ce/e/•的核糖體蛋白L30e (TRP)作爲硏究蛋白質熱穩定性

的模型。TRP能在9(rC以上不變性，而與它有37%同源性的嗜溫成員-酵母核糖

體蛋白L30e (YRP)則在45°C以上便被不可逆地變性。比較二者之間蛋白段序列和

結晶結構時發現’ TRP的表面具有較多的帶電殘基。爲證明這些帶電殘基之間的電

荷作用是否構成蛋白的熱穩定性，本硏究選取了 Arg39, Lys46, GluSO, Arg54, Glu62

與Glu64進行定點突變，其中，精氨酸突變成丙氨酸與甲硫氨酸，而谷氨酸則突變

成丙氨酸。這些突變去除額外的電荷作用。我們構建了十二個突變體，包括十個單

突變體(R39A, R39M, K46A, K46M, E47A, E50A, R54A, R54M，E62A, E64A)和兩個雙

突變體（R39A/E62A與R39M/E62A)。使用環二向色譜(circular dichrosim)硏究這些突

變體的構像穩定性時發現殘基R39, K46, R54與E62對於TRP的穩定性起重要作

用。

ii Abbreviations

AGu(H20) Free energy of unfolding GdmHCl Guanidine Hydrochloride IPTG Isopropyl-P-D- thiogalactopyranoside LB A Luria broth with ampicillin LBAC Luria broth with ampicillin and chloramphenicol Na2S04 Sodium Sulphate NaCl Sodium Chloride E.coli Escherichia coli NaOAc Sodium Acetate PB Phosphate Buffer PMSF Phenylmethylsulfonylfluoride Tm Melting temperature TRP T. celer ribosomal protein L30e YRP Yeast ribosomal protein L30e WT TRP wild type

iii Abbreviation of amino acids A Ala Alanine C Cys Cysteine D Asp Aspartate E Gill Glutamate F Phe Phenylalanine G Gly Glycine H His Histidine I lie Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagines P Pro Proline Q Gin Glutamine R Arg Arginine S Ser Serine T The Theronine V Val Valine W Trp Tryptophan Y Tyr Tyrosine

Abbreviation of nucleotides A Adenine C Cytosine G Guanine T Thymine U Uracil N Any nucleotide

iv Naming System for TRP mutant Twelve mutants were constructed in the present study. The following figure illustrates the naming system for the mutants.

R39A Mutation of arginine-39 to alanine R39M Mutation of arginine-39 to methionine K46A Mutation of lysine-46 to alanine K46M Mutation of lysine-46 to methionine E47A Mutation of glutamate-47 to alanine E50A Mutation of glutamate-50 to alanine R54A Mutation of arginine-54 to alanine R54M Mutation of arginine-54 to methionine E62A Mutation of glutamate-62 to alanine E64A Mutation of glutamate-64 to alanine R39A/E62A Double mutation of arginine-39 to alanine and glutamate-62 to alanine R39M/E62A Double mutation of arginine-39 to methionine and glutamate-62 to alanine

Wild-type amino acid R Mutated amino acid A stands for arginine stands for alanine

^/ R39A means the mutant which at X3 9 A身 ^

f position 39 is mutated to alanine

Position of amino acid

V Introduction

Chapter 1 Introduction

1.1 Hyperthermophile and Hyperthermophilic Proteins

In last 30 years, the number of study on hyperthermophile, organisms that

thrive in hot environments, has constantly increased. Current theory suggests

that hyperthermophiles were the first life-forms to have arisen on Earth (Stetter

1996.) Hyperthermophilic enzymes can therefore serve as model systems for

use by biochemists who are interested in understanding molecular mechanisms

for protein thermostability, understanding enzyme evolution, and the upper

temperature limit for enzyme function.

Proteins isolated from hypertheromophiles are intrinsically stable and active at

high temperatures. To understand how thermophilic proteins remain stable and

active at high temperatures is not only of great academic interest but also has

potential applications in biotechnology (Bruins et al 2001). For example,

thermostable enzymes are gaining wide industrial and biotechonological

interest due to the fact that their enzymes are better suited for harsh industrial

processes (Zeikus et al. 1995). One extremely valuable advantage of

conducting biotechnological processes at elevated temperatures is reducing the

risk of contamination by common mesophiles.

Hyperthermophilic proteins can also be used as models for the understanding

1 Introduction

of thermostability and thermo-activity, which is useful for protein engineering.

Engineering of thermostable industrial enzymes offers the benefit of increased

rate of chemical reactions at higher temperatures.

1.2 Hyperthermophilic Proteins Are Highly Similar to Their Meosphilic

Homologues

With the exception of phylo genetic variations, what differentiates

hyperthermophilic and mesophilic enzymes is only the temperature ranges in

which they are stable and active. Otherwise, hyperthermophilic and mesophilic

enzymes are highly similar: (i) the sequences of homologous

hyperthermophilic and mesophilic proteins are typically 40 to 85% similar

(Davies et al 1993; Auerbach et al. 1998; Chi et ciL 1999; Hopfner et ciL 1999.)；

and (ii) they have the same catalytic mechanism (Bauer et al. 1998; Vieille et al.

1995; Zwickl et al. 1990.).

2 Introduction

1.3 Hyperthermophilic Proteins and Free Energy of Stabilization

The free energy of stabilization (AGstab, where AG^b =風她—TASstab) of a

protein is the difference between the free energies of the folded and the

unfolded states of that protein. It directly measures the thermodynamic stability

of the folded protein. AH咖b (the stabilization enthalpy) and (the

stabilization entropy) are large numbers that vary almost linearly with

temperature in the temperature range of the activities of most enzymes. Also a

function of temperature, AGstab is usually small (Dill 1990; Jaenicke 1991.).

Most AGstab data are for small monomelic proteins (Privalov, et al. 1974)

because studies are hindered by the fact that the thermal denaturation of most

proteins is irreversible: complete denaturation is often almost immediately

followed by aggregation and precipitation.

As a consequence of the enthalpic and/or entropic stabilizations occurring in a

hyperthermophilic protein, the AGstab-versus-r curve of this protein will be

different from that of its mesophilic counterpart. Figure 1.1 illustrates the three

theoretical ways by which increased protein thermodynamic stability can be

achieved (Nojima et al. 1977): (a) the AGstab-versus-7 curve of a

hyperthermophilic protein can be shifted toward higher AG^b values, (b) it can

3 / X / \ / ..Z-， Z \\ ,/ / \ TeIIIperailire

,fZ / / \ (b) (a) M： (c)

Figure 1.1 Comparison of theoretical AG versus T curves for mesophilic and hyperthermophilic proteins. M, theoretical AG versus T curve for a mesophilic protein, (a), (b), and (c), theoretical AG versus T curves for a hyperthermophilic protein. In curve (a), the hyperthermophilic protein has the same temperature of maximal stability {Ts) as the mesophilic protein, and the AG versus 7 curve of the hyperthermophilic protein is shifted upward to higher AG values In curve (b), hyperthermophilic and mesophilic protein have same Ts values and the same AG values at Ts. The AG versus T curve of the hyperthermophilic protein is flatter. In curve (c), hyperthermophilic and mesophilic proteins have different Ts values but have the same AG at their respective Ts. The AG versus T curve of the hyperthermophilic protein is shifted toward higher temperatures. Introduction

be shifted toward higher temperatures, or (c) it can be flattened (due to smaller

difference in partial molar heat capacity between the protein's folded and

unfolded states [AC^]).

1.4 Mechanisms of Protein Stabilization

The hydrophobic effect is considered to be the major driving force of protein

folding (Dill 1990). Hydrophobicity drives the protein to a collapsed structure

from which the native structure is defined by the contribution of all types of

forces (e.g., hydrogen bonds, ion pairs, and Van der Waals interactions). Given

the central role of t he h ydrophobic e ffect in protein folding, it was easy to

assume that the hydrophobic effect is also the major force responsible for

protein stability. In f act, in the 1 ast 20 years, the sequencing, structure, and

mutagenesis information accumulated confirmed that hydrophobicity is, indeed,

a main force in protein stability.

1.5 The Difference in Protein Stability between Mesophilic Protein and

Hyperthermophilic Protein

Comparing the mesophilic and thermophilic proteins, two observations suggest

that both of them have a common basic stability afforded by the conserved

4 Introduction protein core: (i) hydrophobic interactions and core residues involved in secondary structures are better conserved than surface area features, and (ii) numerous stabilizing substitutions are found in solvent-exposed areas (as observed in mesophilic and hyperthermophilic protein structures comparisons and in protein directed-evolution experiments). The high level of similarity encountered in the core of mesophilic and hyperthermophilic protein homologues suggests that even mesophilic proteins are packed almost as efficiently as possible and that there is not much room left for stabilization inside the protein core. The role of packing in stabilizing thermophilic proteins at high temperatures were challenged by structural comparison between mesophilic and thermophilic proteins, which did not always reveal significant differences in their packing (Vogt and Argos 1997; Karshikoff and Ladenstein

1998). Stabilizing interactions in hyperthermophilic proteins are often found in the less conserved areas of the protein. Sequence-structure comparison of homologous proteins from thermophilic and mesophilic origin provides insights on the structural basis of thermostability of proteins. A number of factors, f or example, increased number o f hydrogen b onds and s alt-bridges, better packing of hydrophobic core, stabilization of secondary structural features achieve high thermostability.

5 Introduction

Sequence/Structure Comparisons

By comparing the sequences and/or structures of protein families from mesophilic and thermophilic organisms, proteins from thermophiles tend to contain more charged amino acid residues on the protein surfaces (Fukuchi and

Nishikawa 2001). Vogt and Argos (1997) observed that thermophilic proteins tend to have more hydrogen bonds and ion pairs after they have compared the structures of 16 protein families whose high-resolution structures are determined for both mesophilic and thermophilic counteiparts.

Ion Pairs

Ions pairs are usually present in small numbers in proteins and because they are not highly conserved, they are not a driving force in protein folding (Dill 1990).

Earlier work by Perutz (1978) has suggested, however, that electrostatic interactions represent a significant stabilizing force in folded proteins.

The role of charge-charge interactions is still controversial though it is prevalent that the surfaces of thermophilic protein have more charged residues.

Some reports demonstrated the importance of salt-bridges (Spek et al 1998;

Vetriani et al. 1998; Xiao and Honig 1999; Takano et al. 2000) while some reports showed that surface salt-bridges contribute very little to protein stabilities (Fersht and Serrano 1993; Matthews 1993; Strop and Mayo 2000.).

6 Introduction

However, recent studies have highlighted the importance of long-range

Coulombic interactions among charged residues on protein thermostability

(Grimsley et al 1999; Loladze et al. 1999; Perl et al 2000; Spector et al 2000;

Martin et al. 2001.). These studies concluded that salt bridges with favorable geometry are likely to be stabilizing.

Proline Residue

Mattews et al. (1987) proposed that proteins of known three-dimensional structure could be stabilized by decreasing their entropy of unfolding. In the unfolded state, glycine, without a (3-carbon, is the residue with the highest conformational entropy. Proline, which can adopt only a few configurations and restricts the configurations allowed for the preceding residue (Sriprapundh

2000), has the lowest conformational entropy. Thus, proline located at the loop regions may contribute to protein stability by entropically destabilizing the denatured state. A number of thermophilic and hyperthermophilic proteins also use this stabilization mechanism (Nakai 1999).

Hydrogen Bonds

Hydrogen bonds are typically defined by a distance of less than 3 人 between the H-donor and the H-acceptor and by donor-hydrogen-acceptor angle below

90�Sinc. e the identification of hydrogen bonds is highly dependent on the

7 Introduction distance cutoff and because a number of hyperthermophilic protein structures have not been refined to sufficiently high resolution, studying the role of H bonds in thermostability by structure analysis has not provided clear-cut answers.

Helix Dipole Stabilization

Helix dipoles can be stabilized by negatively charged residues near their

N-terminal end, as well as by positively charged residues near their C-terminal end. In general, though N and C capping compete with other stabilization and destabilization mechanisms (e.g., propensity for H bonds or ion pairs, and conformational strain), and the stabilization provided may be marginal.

Thermococcus celer and its ribosomal protein L30e

Thermococcus celer is a spherical hyperthermophilic euryarchaeote indigenous to anoxic thermal waters in various locations throughout the world. The spherical cells contain a tuft of polar flagella and are thus highly motile.

Thermococcus celer is an obligately anaerobic chemoorganotroph that grows on proteins and other complex organic mixtures with sulphur as electron acceptor at temperature 85°C.

Ribosomal protein L30e is a small and single-domain protein. It is a component o f the large subunit ofribosome and is highly conserved in the

8 Introduction

eukaryotic and archaeal genomes. To our knowledge, the only L30e structure

reported is that from yeast (Mao and White 1999; Mao and Williamson 1999).

In yeast, the L30e protein can bind to its own mRNA and inhibit its splicing

and translation (Vilardell and Warner 1997; Li et al. 1996; Eng and Warner

1991). Experiments have shown that ribosomal protein L30e isolated from T.

celer and yeast differ greatly in their conformational stability. T. celer L30e

was found to be extremely thermostable, having a melting temperature of 95�C

while the mesophilic L30e from yeast is only marginally stable, which

undergoes irreversible unfolding at

1.6 Ribosomal protein L30e from T. celer can be used as a model system to

study thermostability

Thermophilic proteins, like their mesophilic counterparts, are composed of the

same 20 common amino acid residues. The overall structures of thermophilic

proteins are often very similar to their mesophilic counterparts. A good model

system is required in order to find out how thermophilic proteins remain stable

at high temperature.

Since the ribosomal protein L30e is a small protein (100 residues) and isolated

from T. celer, a thermophile t hat g rows at an optimal t emperature a t SS'^C,

9 Introduction

thermophilic L30e can be used as a model system to study thermostability of

proteins.

By comparing the sequences, the ribosomal protein L30e from T. celer (TRP)

shares 37% identities to its mesophilic counterpart, the yeast L30e (YRP). TRP

resists unfolding at over 90� Cwhile YRP undergoes irreversible unfolding at

around 45°C (Mao and Williamson 1999). Prominently, TRP does not contain

any cysteine residue so there is no disulfide bond to stabilize its structure.

High resolution structures of the thermophilic L30e from T. celer were recently

solved by our research group (Wong et al 2001). By comparing the structures

of L30e (Figure 1.2) from mesophilic and thermophilic organisms, we can

design mutants of T. celer L30e to study its thermostability.

In addition, both TRP and YRP can be efficiently expressed and purified. Large

quantities (in mg level) of both proteins obtained will provide enough amount

of samples for analyses.

1.7 Protein Engineering of TRP

When comparing the sequence between TRP and YRP, the T. celer protein has

a dramatic increase in negatively charged residues (figure 1.3). There are 14

residues of T. celer L30e whose c orresponding positions in yeast L30e a re

10 Z] A^f^ � I

z ；喊基，/、；;y :

TRP YRP

Figure 1.2 The structure of TRP and YRP are nearly superimposable Ribosomal protein L30e is a small protein (100 residues).TRP share 37% sequences identities to its mesophilic counter part, YRP. However, TRP can resist unfolding at over 90� Cwhile YRPundergoes irreversible unfolding at around 45�C. 0 10 20 30 40 50

TRP VDFAFEL^AODTGK IVMGARKS 工 Q YAKMGGAKL 工工 VA 兰 NA^PQI K竺D 工 E

YRP APVKSQEILNQIA」&/IICSGKYTLGYKLTVKSLRQGKSKLIII_AANTPVLRKSELE

TRP YYA.5LSGIPVY 竺 FGC4TSVELGTLLGRPHTVSALAVVDPGES 芒工：LALGG^ YRP YY.AMLSKTKVYYFQGGNNELGTAVGKLFRVGVVSILEAGDSDILTTLA--- 60 ^ ^ ^

Figure 1.3 Sequencing comparison of TRP and YRP TRP has a dramatic increase in negatively charged residues than YRP. There are 14 residues of T.celer L30e whose corresponding positions in yeast L30e are either uncharged or oppositely charged. The extra charges in TRP may form more favorable charge-charge interactions in TRP that contribute to its thermostability. Introduction either uncharged or oppositely charged. At physiological pH, yeast L30e has a net charge of +8 while T. celer L30e is near-neutral, due to the extra acidic residues in the thermophilic protein. The extra charges may form more favorable charge-charge interactions in TRP that contribute to its thermostability and they are clustered in two regions: (1) helix 1 and helix-5 (2) between strand-2,3 and helix-3. It is likely that charge-charge interactions among these residues are evolved to improve the thermostability of TRP without affecting its biological functions.

Therefore, in order to study the role of charge-charge interactions among those

residues (E6, R8, K9, D12, K22, R39, R42, D44, E47, R54, E62, E64, R92)

(figure 1.4) on protein stability, site-directed mutagenesis on TRP can be used.

Single and double mutants can be made. Negatively charged residues, for

example glutamate and aspartate, can be mutated to alanine. Positively charged

residues, like arginine and lysine, can be mutated to alanine and methionine.

Methionine is chosen to m imic the hydrocarbon side c hain of arginine a nd

lysine. Site-directed mutagenesis will be carried out by polymerase chain

reaction. Thermodynamics parameters (AGu, Tm) of the mutants can be

measured as described for the wild-type protein. The changes in stability upon

11 Introduction

mutation should be calculated by:

AAG = AGu(mutant) -AGu(WT)

1.8 Purpose of the present study

Twelve mutants of TRP are constructed (R39A, R39M, E62A, E64A, E47A,

E50A, R54A, R54M, K46A, K46M，R39A/E62A, R39M/E62A) to examine

the determinant factors involved protein stability. The mutations mainly

involve the elimination on the charge residue of TRP and hence the

charge-charge interaction in TRP. The conformational stability of the mutated

TRP is determined by circular dichroism (CD).

12 L^ R39 ^"••^：：， Figure 1.4 The extra charge residues in TRP � TRP has 14 extra charged residues which are E50 緣;：广“ % / either uncharged or oppositely charged in YRP. 烂‘‘ ••‘ yjf m j , f^l In order to study the charge-charge interaction .. M^^jf^m of TRP, site-directed mutagenesis was \ \ . • employed.

R92 � Ie90 Materials and Methods

working concentration of 100 jig/ml.

A stock solution of chloramphenicol (Sigma Chemical Co.) was prepared at

lOOmg/ml with absolute ethanol (Sigman Chemical Co.). Aliquots were stored

at -20°C and used at the working concentration of 100 jug/ml.

2.5 Restriction endonucleases and other enzymes

Restriction endonuclease us ed in digesting plasmid D NA w ere supplied, with

their recommended reaction buffers, by Amersham International (UK) and New

England Biolabs, Inc. (USA).

T4 DNA ligase was purchased from Amersham (UK) with its 10 x ligation buffer

supplied.

2.6 M9ZB medium

The medium was prepared by adding Ig ammonium chloride, 3g potassium

phosphate, 6g sodiumphosphate, lOgbacto-tryptone and 5g sodium chloride

powder in 1 L distilled water. After autoclaving the medium, 1 ml magnesium

sulphate and 10 ml 20% glucose solution.

2.7 SDS-PAGE:

1 X SDS gel-loading buffer

It contained 50mM Tris.Cl (pH 6.8), lOOmM dithiothreitol, 2% SDS

(electrophoresis grade), and 0.1% bromophenol blue, 10% glycerol. 1 X SDS

14 Materials and Methods

gel-loading buffer lacking dithiothreitol can be stored at room temperature.

Dithiothreitol should then be added, just before the buffer is used, from a IM

stock.

Ammonium persulfate. 10% ( w/v)

This solution was prepared by adding 1 g ammonium persulfate powder in 10 ml

distilled water. It could be stored at room temperature.

Tris-glycine electrophoresis buffer

The buffer was prepared by 25 mM Tris, 250mM glycine (electrophoresis

grade)(pH 8.3), 0.1% SDS. A 5X stock can be made by dissolving 15. Ig of Tris

base and 94 g of glycine in 900ml of deionized H2O. Then, 50ml of a 10% (w/v)

stock solution of electrophoresis-grade SDS is added, and the volume is adjusted

to 1000ml with H2O.

2.8 Alkaline phosphatase buffer

100 mM NaCl, 5mM MgCb, 100 mM Tris.Cl (pH 9.5).

2.9 DNA agarose gel

TAE buffer, SOX

This buffer is prepared by adding 242g Tris base, 136. Ig sodium acetate, 19g

Na2EDTA.2H20 in 700 ml distilled water. Adjust pH to 7.2 with acetic acid and

add water to a final volume of 1 L.

15 Materials and Methods

2.10 Gel loading buffer, DNA

This buffer is prepared by a dding 4g sucrose, 0.025g bromophenol blue, and

0.025g xylene cyanol in 10 ml distilled water. Store at 4°C.

2.11 Ethidium bromide (EtBr), lOmg/ml

This solution is prepared by adding 0.2g ethidium bromide in 19.8ml distilled

water. It could be stored in room temperature and need to prevent from exposure

to light.

Methods:

2.12 Constructing Mutant TRP genes

Using wild type TRP gene as a template and a pair mismatch primer, mutants

of TRP were created by site-directed mutagenesis. The mismatch nucleotides

were flanked by 15 matched nucleotides at both sides. Full length mutated

TRP gene was created with two rounds overlapping polymerase chain

reactions (PGR). In the first round PGR, two gene fragments, one from 5'

common primer to mismatch and the other from 3’ common primer to

mismatch, were separately amplified. Then, the two fragments self-prime

each other and the ~300kb full length mutant gene was amplified. The

amplified gene segments after PCR were separated by gel electrophoresis

and then extracted by GeneClean kit (QIAquick Gel Extraction Kit (250)).

16 Endogenous primer \ £；， CI 3’ 5 3’ 3， Wt TRP 飞， 5'. 5' 3 Endogenous primei

round PGR

3 — — ___y\ 3' 5' 八 3， y — 3’ 5' — V “ ：：:accS gene fragment Mutated TRP gene fragmeni

^ iw

Two fragments self prime each other

^JJl^""' round PCR

八 s/ Site directed mutated TRP

Figure 2.1. Diagrammatic flow of the site directed mutagenesis. The mutated TRP gene was created by 2 rounds of PCR reactions Materials and Methods

2.12.1 Polymerase Chain Reactions fPCR)

The PGR mutagenesis steps are described in figure 2. land the reaction

mixture are summarized in table 2.1..

Volume 邮。 1 Ox Pfu buffer 5 dNTP (lOmM) Forward primer (0.1 nM)* Reverse primer (0.1 nM)* 1 Pfu polymerase 0,5jLil Template (wild type TRP gene) o.5jLil Total volume

Table 2.1 PGR reaction mix

The twelve mutants primer were used are listed below in table 2.2:

17 Materials and Methods

Table 2.2 Primer used in Site Directed Mutagenesis

Common Primer

Forward Primer - TRP-NF-Ncoi

5' CGC TTG CCA TGG TTG ATT TTG CTT TCG AA 3' Reverse Primer - TRP-CR-BamHI 5' TCG CGG ATC CTC ACT CTT TAG CGC CCA A 3'

Mutagenic Primer Forward Primer - TRP-R39A-F

5' CTG ATC ATC GTT GCC GCC AAC GCG CGC CCT GAT 3' Reverse Primer - TRP-R39A-R 5' ATC AGG GCG CGC GTT GGC GGC AAC GAT GAT CAG 3' Forward Primer - TRP-R39M-F 5' CTG ATC ATC GTT GC ATG AAC GCG CGC CCT GAT 3' Reverse Primer - TRP-R39M-R 5' ATC AGG GCG CGC GTT CAT GGC AAC GAT GAT CAG 3' Forward Primer - TRP-E62A-F 5’ GGT ATT CCA GTG TAT GCC TTC GAG GGC ACC TCC 3’ Reverse Primer - TRP-E62A-R 5' GGA GGT GCC CTC GAA GGC ATA CAC TGG AAT ACC 3_ Forward Primer - TRP-E64A-F 5’ CCA GTG TAT GAG TTC GCC GGC ACC TCC GTG GAG 3' Reverse Primer - TRP-E64A-R 5' CTC CAC GGA GGT GCC GGC GAA CTC ATA CAC TGG 3' Forward Primer - TRP-E47A-F 5' CGC CCT GAT ATT AAA GCC GAC ATC GAA TAG TAG 3' Reverse Primer - TRP-E47A-R 5' GTA GTA TTC GAT GTC GGC TTT AAT ATC AGG GCG 3' Forward Primer - TRP-E50A-F 5' ATT AAA GAA GAC ATC GCC TAG TAG GCA CGT TTG 3’ Reverse Primer - TRP-E50A-R 5' CAA ACG TGC GTA GTA GGC GAT GTC TTC TTT AAT 3' Forward Primer - TRP-R54A-F 5’ ATC GAA TAG TAG GCA GCC TTG AGC GGT ATT CCA 3' Reverse Primer - TRP-R54A-R 5' TGG AAT ACC GCT CAA GGC TGC GTA GTA TTC GAT 3'

18 Materials and Methods

Forward Primer - TRP-R54M-F 5' A TC GAA T AC T AC GCA A TG TTG AGC GGT A TT CCA 3' Reverse Primer - TRP-R54M-R 5' TGG AAT ACC GCT CAA CAT TGC GTA GTA TTC GAT 3' Forward Primer - TRP-K46A-F 5' AAC GCG CGC CCT GAT A TT GCC GAA GAC A TC GAA T AC T AC 3' Reverse Primer - TRP-K46A-R 5' GTA GTA TTC GAT GTC TTC GGC AAT ATC AGG GCG CGC GTT 3' Forward Primer - TRP-K46M-F 5' AAC GCG CGC CCT GAT ATT ATG GAA GAC ATC GAA TAC TAC 3' Reverse Primer - TRP-K46M-R 5' GTA GTA TTC GAT GTC TTC CAT AAT ATC AGG GCG CGC GTT 3'

2.12.2 Gel Electrophoresis

The agarose gel was prepared 1. 0 to 2.0 %( w/v) dissolving in 1x TAE buffer

with O.05~ l/ml ethidium bromide. The DNA samples were mixed with 6x

loading buffer making up a 1x sample with final volume ~ 12~1. The 1x

samples were loaded into the wells and were electrophoresized in a constant

90V gel tank with Ix TAE as conducting buffer. DNA separated on the

agarose gel with sizes was visualized under ultra violet light and were

photographed in the GelDoc (B & L Gel Doc System, Imago).

2.12.3 DNA Purification from Agarose Gel

DNA purification was carried out as described in the company manuals

using commercial kit. (QIAquick Gel Extraction Kit)

2.12.4 Construction ofR39A, R39M, K46A, K46M, E47A, E50A, R54A, R54M

19 Materials and Methods

Oligo R39A-F, R39M-F, K46A-F, K46M-F, E47A-F, E50A-F, R54A-F,

R54M-F were used as the forward primer in PCR mutagenesis. Reverse

primer TRP-CR-BamHI was used. In addition, oligo R39A-R, R39M-R,

K46A-R, K46M-R, E47A-R, E50A-R, R54A-R, R54M-R were used as the

reverse primer while TRP-NF-Ncol was used as the forward primer to

generate the second gene fragment in the PCR mutagenesis. One

microgram of pET8c with TRP wild type gene cloned was used as the

template. The amplified gene products were used as the template in the

PCR and TRP-NF-NcoI, TRP-CR-BamHI were used as the forward and

backward primer. The mutant TRP genes R39A, R39M, K46A, K46M,

E47A, E50A, R54A, R54M could be amplified.

2.12.5 Construction of double mutant R39A/E62A. R39M/E62A

Oligo R39A-F and R39M-F were us ed as the forward primer in PCR

mutagenesis and TRP-CR-BamHI was used as the reverse primer. In

addition, oligo R39A-R and R39-R were used as the reverse primer in

PCR mutagenesis and TRP-NF-NcoI was used as the forward primer. One

microgram of pET8c with E62A mutant TRP gene cloned was used as the

template. The two amplified gene products were used as the template in the

2nd mutagenesis. TRP-NF-NcoI and TRP-CR-BamHI were used as the

20 Materials and Methods

forward and backward primer and to generate the double mutants

R39A/E62A, R39M/E62A.

2.13 Sub-Cloning

2.13.1 Restriction Digestion

The mutant gene was inserted into the cloning vector pET8c. Both the insert

and the vector were cut by restriction enzyme BamHI and Ncol for sticky

ends at 37°C for 3 hours. The reaction mixture was summarized as below:

Insert Plasmid

Distilled water — n^]

10XNEB2 buffer 2|a] 2jal

lOX BSA 2^1 2^1

DNA 14|al 31^1

Ncol l|a] (lOU) l^il (lOU)

BamHI l|al (lOU) l^] (lOU)

21 Materials and Methods

2.13.2. Li gating vector with mutant TRP gene insert

The digested vector and insert were ligated by T4 DNA ligase at 14�C

overnight (-16 hours). The reaction mixture of ligation was shown below:

Reagent Volume l\x\

lOX ligase buffer 2.0

Insert 12.5

Vector 5.0

T4 DNA ligase 0.5

2.13.3 Amplifying vector carrying mutant TRP gene insert

The ligated vector was transformed into DH5a E. coli competent cell. The

transformed cells were spread on LB plate with ampicillin. Single colony

was picked and inoculated into 5ml LB with 5|LI1 ampicillin. The culture is

grown at 37� Cfor 16 hours. The plasmids were harvested with CONCERT

TM Rapid Plasmid Miniprep System 250 reactions by GibcoBRL Life

Technologies.

2.13.4 Mini-preparation of DNA

The method was based on the CONCERT™ Rapid Plasimd Miniprep

System by GibcoBRL Life Technologies. Within this method, all the

centrifugation steps were performed at maximum speed (>10,000g) in a

22 Materials and Methods

table-top microcentrifuge. The overnight culture of plasmid-containmg cells

was collected by centrifugation for 30 sec. All the supernatant were removed

by aspirating or pipetting. The cell pellet was resuspened by 200jul Cell

Resuspension Solution. The Cell Lysis Solution (250)il) was added and

mixed gently by inverting about 10 times. Afterwards, 250^1 of the

Neutralization Solution were added and gently mixed by inverting. The cell

debris was removed by centrifugation for 10 min. The cleared lysate was

transferred into a Spin Column. The matrix was centrifuged for 1mm. Wash

Buffer (750jul) was added and the mixture was centrifuged for Imin. The

filter was washed again with 250|li1 Wash Solution. Finally the plasmid DNA

was eluted by lOOjul sterile water (with 65�C an) d stored at -20� Cuntil use.

2.13.5 Preparation of competent cells

The techniques for transformation of E.coli were according to Hanahan

(1985). E.coli strain DH5 a was streaked from frozen stock or stab culture

on to LB plate. After overnight incubation at 37� Ca, single colony was

transferred under sterile conditions to 5ml of LB medium. The culture was

shaken in a 37°C water bath overnight and 1% inoculate 200ml of fresh LB

in a 1 L flask until the cell culture reached O.D. 600 = 0.3-0.4. The cells

were chilled at 4°C for at least 15 minutes, then pelleted at 5,000 rpm for 10

23 Materials and Methods

minutes in 4°C Hitachi centrifuge.

The medium was drained thoroughly before resuspending the cells in 15ml

ice-cold Ca/glycerol buffer (10% 0.6M CaCk, 2% 0.5M Pipes pH7, 15%

pure Glycerol). Pelleted at 5000 rpm for 5 minutes in 4� CHitachi centrifuge.

Repeated the procedure twice and keep the cells in Ca/glycerol buffer for 30

minutes. Finally resuspended the pellet in 4-5 ml ice-cold Ca/Glycerol buffer.

Aliquots the competent cells into 1.5 ml microcentrifuge tubes and the

aliquots were rapidly frozen in liquid nitrogen before storage (preferably, for

not longer than 6 months) in a —70� Cfreezer.

2.13.6 Transformation to Escherichia coli

The competent cells DH5a (or BL21 (DE3) pLysS) were thawed in hand.

0.5-1.0|jJ plasmid was added to the competent cells. The cells were cooled

on ice for 30 minutes, heat shocked at 42°C without shaking for two minutes

and then cold shock for 10 minutes. The recombinant plasmid was

introduced into the expression competent cells. Four hundred ml LB medium

was added and the cells were incubated at 37� Cfor 45 minutes with 250 rpm

in order to express the ampicillin (for BL21 (DE3) pLysS, and

choramphenicol) resistance marker on the plasmid.

24

� Materials and Methods

For plasmid amplification, the competent cells were centrifuged down at

13000 rpm for 2 minutes and resuspensed with 100 jul LB medium. Then 100

cells were spread on the pre-warmed LBA plate, which exerted

ampicuillin selection, and were incubated at 37� Cfor 16 hours.

For expression, the competent cells were diluted with 10 folded LB medium

and spread on the pre-warmed LBAC plate, which exerted ampicillin and

choramphenicol selection, and were incubated at 37� Cfor 24 hours.

2.13.7 Screening tests

The plasmid amplified and extracted from miniprep system was tested for

successful mutant gene insertion.

Restriction digestion test

The plasmid extracted was digested by restriction enzymes BamHI and Ncol

in digestion mixture illustrated in table 4. The digested mixtures were

electrophoresized a nd bands with the size of t he insert (�300bp) and the

vector were seen on the agarose gel under ultra violet light.

PGR test

The plasmid extracted was the template for PGR. The reaction mixture was

loaded as illustrated in table 1. The PGR products were electrophoresized

25 Materials and Methods

and a very bright band of the mutant gene's size (�300bp) can be seen.

Sequencing

To ensure the correct mutant gene sequence, lOjul of the plasmids carrying

the mutant gene were sequenced by Tech Dragon Ltd.

2.14 Over expression of mutant TRP

2.14.1 Transformation

Expression vector pET8C with the mutant TRP gene insert was transformed

into E. coli expression host, BL21 (DE3) pLysS.

2.14.2 Expression

Ten colonies on the LB AC plate were picked and inoculated to 10ml of

M9ZB bacterial culture medium with 100 jug/ml ampicillin and 100 jug/ml

chloramphenicol for selection.

The starter culture was incubated at 37°C with 280rmp for 5 hours until the

culture medium was opaque. Starter culture was then inoculated into IL of

M9ZB with lOOjug/ml of chloramphenicol, lOOjug/ml of ampicillin as 1%

inoculation at 37°C with 280rmp. When the cell density reached OD600 =

0.6-1.0, where the cells reached their mid log phase.

isopropylthio-P-galactoside (IPTG) at final concentration of 0.2 mM was

added to the bacterial culture to induce TRP protein expression. One ml of

26 Materials and Methods

100 |ig/ml ampicillin and 1 ml of 100 jug/ml chloramphenicol were added

upon induction to supplement the possible exhaustion of antibiotics.

2.14.3 Cell Harvesting

The cells were harvested by centrifiigation at 6,400rpm at 4� Cfor 12

minutes. The pellet was collected for further purification and the supernatant

was discarded. The cells were stored at -80°C if they were not immediately

purified.

2.14.4 Expression Checking

One m 1 of competent cell samples were c ollected before and after IPTG

induction in order to check for the expression of mutant protein in competent

cells culture.

2.14.5 SDS-Polyacrvlamide Gel Electrophoresis fSDS-PAGE)

SDS-Polyacrylamide gels were run with the Mini-Protean III electrophoresis

cell (BioRad Laboratories, Inc.). The running SDS-PAGE gel comprised of

3/4 separating gel in length. To ensure a horizontal boundary, isopropanol

was added temporary to ensure a firm boundary. After the separating layer

was polymerased, the gel was topped with stacking gel with wells for sample

loading. The samples were mixed with 2x loading buffer and carefully

loaded to the gel. The SDS-PAGE gel was run at 35mA until the dye front

27 Materials and Methods

just reached the bottom of the gel.

2.14.6 Staining the acrvlamide gel

The acrylamide gel was removed and stained by Coommassie blue stain. (30

o/o (v/v) ethanol, 10 % (v/v) acetic acid and 0.15 % (w/v) Coommassie

Brilliant Blue R-250) at 70� Cfor 30 minutes. Then it was destained with

destaining solution containing 25% ethanol and 8% acetic acid with a piece

of paper towel at room temperature overnight.

2.15 Purification of Mutant TRP Protein

Mutant TRP proteins were purified by chromatography — by ion-exchange

column, affinity column and finally size exclusion column.

2.15.1 Cells Lysis

The pelleted cells were resuspended with 30 ml of 20 mM sodium acetate

buffer (NaOAc), pH 5.4. 150jul of 100 mM phenylmethyl sulfonyl fluoride

(PMSF), a protease inhibitor, was added to prevent the protease release after

cell lysis degrading TRP. The cells were lysed by sonication (Vibra Cell™,

Sonics & Materials Inc.), 4s pulse, 80% output on ice for 10 minutes. 30jul of

(3-mercaptoethanol (1 jul/ml) was added to the lysate to denature the mutant

proteins. The crude lysate was centrifuged at 15,000rpm, 4� Cfor 30 minutes

to pellet the cell debris. Both the supernatant and the pellet was sampled and

28 Materials and Methods

analyzed m the SDS-PAGE gel. The mutant protein was found to be present

in the supernatant and the supernatant was collected for chromatography.

2.15.2 Chromatography

The chromatography separated TRP from the other by its three properties —

positive charge at the buffer's pH (pH 5.4), affinity with nucleotide as its

ribosomal protein and the size. The columns were drove with AKTA prime

from Amersham Pharmacia Biotech.

2.15.2.1 Ion exchange chromatography

Five ml Hi-Trap™ SP Sepharose column (Amersham Pharmacia Biotech.) is

a cation exchanger, to which TRP could bind when buffer pH was 5.4. The

column was equilibrated with 20mM NaOAc, pH 5.4 at constant flow rate of

6ml/min. The protein was eluted by a linear sodium chloride gradient, from

0-lM NaCl, in 20mM NaOAc, pH 5.4 buffer over 300ml. Mutant TRP

protein were collected around 0.3M NaCl. The fractions with high OD280

were analyzed in SDS-PAGE gel. Fractions with high concentration of TRP

mutant and low concentration of impurities were pooled together for the

affinity column.

2.15.2.2 Affinity Chromatography

Five ml Hi-Trap^^ Heparin column (Amersham Pharmacia Biotech.) is an

29 Materials and Methods

affinity column binding nucleic acid binding protein. The column was

equilibrated with 20mM NaOAc, 0.2M NaCl, pH 5.4 at constant flow rate of

6ml/min. Then the sample was filtered with 0.2|Lim filter and loaded to the

column. To avoid non-specific ionic interaction, the binding buffer has

�0.15 Mionic strength. Therefore the elution NaCl gradient was setup from

0.2 to l.OM NaCl over 240ml. The mutant TRP was eluted at about 0.45 M

NaCl. The fractions were analyzed and those with high concentration were

collected and c oncentrated to >5ml Centriprep (Millipore) at 4� Cfor gel

filtration column.

2.15.2.3 Size Exclusion Chromatography

Superdex 75 gel filtration column was used to provide a stationary phase to

separate protein according to their size and shape, working as a molecular

sieve. The fractions containing TRP after running heparin column was

pooled and concentrated >5ml for sharp elution. The gel filtration column

was equilibrated with 20 mM NaOAc, 0.2 M Na2S04, pH 5.4 (the storage

buffer) before loading. The sample was filtered with 0.2jLim filter and loaded

to the column. TRP would be eluted approximately around 220 ml. The

purity of fractions collected with TRP was analyzed by SDS-PAGE gel.

Fractions with high purity was pooled together and concentrated

30 Materials and Methods

2.15.3 Concentrating TRP as protein stock

The protein fraction were concentrated with Centriprep (Millipore) at

3500rpm at 4°C until OD260 >2.0. The protein stock was stored at -20�C

and storage buffer for maintaining stability.

31 Materials and Methods

2.16 Protein stability

The protein's structural stability was measured by means of circular

dichroism (CD), which is a property of optically active compounds. CD was

measured by Jasco® J810 Spectropolarimeter. CD signal at 222nm was

measured as the difference in absorbance or as ellipticity, expressed in

millidegrees (mdeg) and then the residual molar ellipticity (RME) of the

proteins was calculated.

The CD signal at 222mn was recorded and studied because the signal

changed significantly from folded state to unfolded state, because it reflects

the conformation of the a-helix in the protein (figure 2.2).

The conformational stability requires determining the equilibrium constant

(Ku) and the free energy (AGu(h20)). Therefore it reflects the folding state of

the protein. The conformational stability requires determining the

equilibrium constant and the free energy change of protein unfolding, AG for

the reversible reaction.

A (Hellma® PRECISION made of Quartz SUPRASIL) 0.1cm quartz cuvette

held the sample for CD spectrometric measurement. Before loading samples,

the cuvette was washed with 8M GdnHCl, pH 7.4 to remove protein debris.

32 广-,•.�...‘••.....,.•,.....‘.�.,.�.....W.V.--�.‘���..�.,.™��:‘.,...��..�...... ".,...... ,,,...� �...... ,.,.. ,,...... V. ,.•‘-,.,....,--,,,,..,, ‘v..,,,,,,,,,.v,"-.,, ... .,,,,,,..,,•-,,,,,,..,�.,,,.…………".‘-",?,,.,.� rv 丨 ‘，\ i ： i�”; �\\ I ！ \ I �� \ I P I ...:�� | I \| I \ .ot \\ i 1： ^ I \ i -；叫 \ \ I r I I 劣 \ ~ \ \ ,—��.� i • .、 卜••、•….-.•".•.•‘• ••-•- ••••• 、.‘•.--.••••• • •；一 ""^aflWii：^*^ “…� i \I A\ � X zZ广...... •....•二二—一 I.?： “1 I / \ .. / I i A"" / i “ i \ / I K / 乂 I f 夕 > \� .J‘ X-� / f I .i I { I ^ i i; ^ I ‘ ” .…7

The cuvette was rinsed with lOmM PB, pH 7.4 to remove remaining

GdnHCl.

2.16.1 Chemical Stability

Mutant TRP was diluted to 250|iM by gel filtration buffer. of TRP at

this concentration was added to 450)il of 0.0 to 7.2M of guanidine

hydrochloride (GdnHCl) ladder and mixed well.

Four hundred microlitre buffers with different concentrations of GdnHCl

(0-7.2M) were added to the 0.1cm cuvette for CD spectroscopy measurement

from 260 to 190nm at 25°Cto obtain buffer blank. The TRP mutant proteins

were added to different concentrations of GdnHCl and then CD

measurements of TRP mutant in different concentrations of GdnHCl (0-7.2M)

were obtained, where the signal given by the buffer blank was eliminated.

Full spectra from 260mn to 190nm were only obtained from OM GdnHCl

and 7.2M GdnHCl. Spectra obtained from other GdnHCl concentrations

were from 225 to 215nm.

Chemical Reversibility

This was to ensure the reversible unfolding of mutant TRP in GdnHCl

solution. One hundred microlitre of TRP stock was added to 400jul of 8M

GdnHCl, pH 7.4 solutions. TRP in 6.4M GdnHCl solution was incubated at

33 Materials and Methods

room temperature for 30 minutes for unfolding. Then the lOmM PB, pH 7.4

was added as a 10-fold dilution. Again 30 minutes of incubation were needed.

The final solution contains about 10|liM of TRP in 0.64M GdnHCl, pH 7.4. A

spectrum from 260 to 190nm at 25� waC s obtained. A control was established

by mixing 40|li1 of 8M GdnHCl in lOmM PB with 450^1 lOmM PB and then

10|Lil TRP stock. A spectrum was obtained from scanning the solution

containing lOiuM TRP and 0.64M GdnHCl from 260 to 190mn at 25^C. The

two spectra should coincide to ensure the TRP's chemical reversibility in

GdnHCl.

2.16.2 Thermal Stability

Twenty-Five \aM O f mutant TRPprotein was prepared From the Stock by

diluting the stock protein with the dialysing buffer lOmM PB, lOOmM NaCl,

pH 7.4. The mixture was dialyzed with the dialyzing buffer for 3 hours, 4�C

to replace the storage buffer with the dialyzing buffer. Four hundred

micro litre of TRP in dialyzing buffer was loaded to the 0.1cm cuvette. A full

spectrum of TRP before heating from 260 to 190nm at 25 °C was obtained.

A variable temperature scan was carried out from 25 to 110°C at wavelength

222nm, with temperature change rate l°C/min; CD signal was recorded at

each 0.2°C.

34 Materials and Methods

At 110�, Ca full spectrum (from 260 to 190nm) was obtained to check for the complete unfolding. To prevent prolonged heating, the temperature was immediately lowered to 25°C after full spectrum measurement.

Thermal reversibility

When the sample steadily reached 25�C ,a 5-minute incubation period ensured the protein to reach a steady temperature and complete any possible folding. A full spectrum at 25°C was obtained and compared to the spectrum obtained at the very beginning of the experiment. They should coincide to show their reversibility.

35 Results

Chapter 3 Results

3.1 Construction of mutant TRP genes

In order to study the contribution of charge-charge interaction in TRP, mutant

TRP genes were constructed by site-directed mutagenesis. A total number of 12

mutants were constructed: R39A, R39M, K46A, K46M, E47A，E50A, R54A,

R54M, R39A/E62A, R39M/E62A. Overlapping PCR using mutagenic primer

was applied to carry out the mutagenesis. The amplified mutant TPR genes

fragments were purified and sub-cloned into expression vector pET8c. The

plasmids carrying the mutated gene were screened by their size, restriction

digestion and DNA sequencing.

3.1.1 PCR Mutagenesis

Following the PCR mutagenesis strategy described in Section 2.12.1, twelve

mutants TRP mutant genes were constructed. Typical results of PCR mutagenesis

are shown in Figure 3.1. After PCR mutagenesis, the gene products were loaded

into 2% agarose gel for electrophoresis. Bands with expected size of 0.3 kb were

observed and purified by gene-clean.

36 m . 300-：.

fo®� . . - £ . J ^ -. . �

Figure 3.1 ： PCR mutagenesis of R39A, R39M, E62A, E64A, E47A, E50A, R54A, R54M Overlapping PCR were performed for constructing TRP mutant gene. In the first round PCR reaction, two gene fragments (forward and reverse) per each mutant were amplified. (A) The amplified gene fragments were purified and as the template in second round PCR. 300 bp DNA fragments amplified were the full length mutant TRP gene. Purified from the agarose gel, the mutant gene could then be sub-cloned to an expression vector. Results

3.1.2 Sub-cloning of mutant TRP gene to express vector pET8

Both the mutant TRP gene and the vector pET8c were digested by restriction

enzyme BamHI and Ncol. The digested mutant TRP gene fragments were ligated

with the vector. The ligation product was transformed into E. coli DH5a

competent cells and single colonies were cultured for plasmid amplification by

miniprep. After PCR screening, the plasmid that showed positive result was then

digested by restriction enzymes BamHI and Ncol. The mutant TRP insert (0.3 kb)

and vector were separated. Bands with molecular size around 0.3 kb (mutant TRP

gene insert) and a band with a very large size (vector; >3kbs) were found. The

restriction digestion screening results are shown in F igure 3 .2. The mutations

were further confirmed by DNA sequencing (by Service Team of Biochemistry

Department, CUHK or Tech Dragon Ltd.)

Summary

TRP mutants R39A, R39M, K46A, K46M, E47A, E50A, R54A, R54M,

R39A/E62A, R39M/E62A were successfully constructed. All clones were with

correct mutation.

37 300-

- » ^ - • " I

、浓.11 Iimwii . 二 ^v：-^- V必- � •'•m^mm '' J • �i - • M�

� > --!

300- u^jjjjjmi^^^jjjji^jjjjm^jjjjjjjjjjjij^^^

Figure 3.2 Restriction Digestion Screening of R39A，R39M, E62A, E64A, E47A, E50A, R54A, R54M After PCR mutagenesis, the mutant gene fragment were loaded to 2% gel for electrophoresis and purified out. By digesting the mutant gene and vector, pET8c，they were ligated with the vector and transformed to bacterial host E. coli DH5 a . In order to check if the colonies of the transformation carrying the mutant gene insert, restriction digestion screening was performed. Colonies from the transformation was selected out and the plasmids were purified. The plasmids were then digested by restriction enzyme Ncol and BamHl. A 300 bp band could be seen after loading the digestion product to a 2% gel for electrophoresis if the plasmid contained the mutant gene insert. Results

3.2 Expression and purification of mutant TRP

Twelve mutant proteins were successfully over-expressed in bacterial host E. coli

BL21 (DE3) pLysS. Protein expression was checked by SDS-PAGE (Figure 3.3).

All mutant proteins were purified by ion exchange chromatography (Fig. 3.4a),

heparin affinity chromatography (Fig. 3.4b) and gel filtration (Fig. 3.4c). Results

of R39A were shown as an example for the expression and purification of TRP

mutant proteins (Figure 3.5). After purification, the mutant proteins obtained

were concentrated until OD 280 greater than 2.0 and stored in 20 mM NaOAc,

0.2M Na2S04, pH 5.4 at -20°C.

38 •？w- >:

� / / I.* t^ Before After Pure induction induction TRP marker

Figure 3.3 Expression of mutant TRP R39A TRP mutant R39A cloned in the expression vector pET8c was transformed to E.coli BL21 (DE3) pLysS strain. Colonies of the transformed competent cells were inoculated into 15 ml of M9ZB medium. The culture was grown at 37��with shaking at 280 rpm for 5 hours. Then 10 ml starter culture was transferred to 1 L of M9ZB. The culture was grown until OD 600 reached 0.6 — 1.0. The expression was then induced by adding isopropylthio- /5 -galactoside (IPTG) and the cells were allowed to grow overnight. ’ (.�� � • �,_e��<">;_ ����i “ L � -�‘ “ �) f-^lj“^^RYRCSR^ "-f —,»、： 、二 i-V" Tr.-^ gv-x \ f �-� - - �--—- , .. �

‘ I - 1 、“。々 ：-- I - ；

‘ .1 ... T- 1 _ i -r -1 n v/^ .�V- vl^' i 1 — • r .-".. ； _ L二 .--. T —_ 1 --二二二—:-..--.’"""碑^^•^•^^^•^�^•^•j尸•tfj--....�’,-.«�.� .-.,;;� I ...... 、-一 ....!. . . . i . . i i I I ？ f - __ _ _ - I ！ ..(一，-•。.！ . i } I ！ i 4 .; • •‘ 1 .z ； I i I i - -••.-..^ --A------一3 I… --”——--厂-_ r-_ -广—-— • i 1 ！ ！ 「-- i •• ； • - I ！ • • i ！ 1 ----�--- _- _ -� ------'I - - 1 _

, ； i ； i ‘ ： {...... • '.；.："；.；.：：|：：：：；•-：-：.：：;：;：'；;：|；：：：；"：".：：".：-：：： … ；• 口.1:^—•-....i—•..-一-....…^--•-....“i—..—•^「：……一一-：—••：―….==:::=.:..:::=::|::二二：二 二工 „:„ -L；-—_—i — ； I 4 1 -""'r I ； 1 I …i i 、」:..:：:：二…:.：：-::—::-:二 ^^^^^^^ ：.：!, ^ ； ； ； f j...-。魂-..….-:.:::J：：：：：： :=;：•;- == 二：中E —-1 L—！ } i i f j 1 —{ ‘,‘ 岁卜9 I , ！ .,、•科〕1 1 _ ‘ 1 .丨 .1 .. V. i S 卜• . I j ••• -f--- •• \ •- • 厂：、\/\e 0.4M Nab i ： �

,；i\\ :；‘ ———— .1 . :V�\� : J 丨 / I ^

I ! / w sJ^-f ••：.•.• ：I '.： ；；：••：••：: —:::、.：] ... ： {...... 、一 ..- ；：：：.：.：：••—：.....= /i -•" /-—.广：十——-——.-―-t i—es^-i —厂―：^^^———：―广-二二 ^ ~

/.丨 J I - -—..:::’..-...... -;1 >— -丄一：-\= 十...... ——L—:=::..: 二二二:: j:二二:=|:==:: 二二：：：二 ::=: •:.:：…_ _ ： • _ —t :I3 士： [ 」— i — 1 1 二=::::.1:： - • “ Figure 3.4a. Elution Profile of R39A in HiTrap SP column The red line showed the conductivity of the eluting solution. With a higher concentration of salt, the conductivity increased. The blue line showed the OD280 of the eluting solution, with a higher concentration of protein, the OD280 reading increased. The peak area showed that protein was eluted into those fractions. TRP was eluted at around 0.4M NaCl. \ 一 \

R39A peak

f I ——“ ！ ； • . 1 > • ！; ‘ ^ i ； i • ： / j； I i I f i ‘一 i ； • 1 ； •] • ！ ！ { I ！ _ ‘ ；• M i ！ ； M 1 ！ ！ [ M ！ r ！‘ 丨：：丨丨丨m, ‘丨丨：丨！n|

• ： ^ • ； --�-i--“丄‘^ i J__；__L i. J ！_！ ！

； o>.,； ； I 入 ‘- �. Figure 3.4b. Elution Profile of R39A in HiTrap Heparin column TRP fractions purified from HiTrap sp solumn was pooled together and as the input of the Heparin column. The red line showed the conductivity of the eluting solution. With a higher concentration of salt, the conductivity increased. The blue line showed the OD280 of the eluting solution, with a higher concentration of protein, the OD280 reading increased. The peak area showed that protein was eluted into those fractions. TRP was eluted at around 0.45M NaCl. \ \ \ R39A peak

i A I n

.. ..Hj. ’ i�“; it ��n i i I �! t ! 1 �M ^ 1 1 w �h !

—:丨 I丨；‘： U i - ...... 、...、：<、....— • • ... - t-^—“ . .-:、：-.•""-. •• ‘ ~ -、 i i I I

Figure 3.4c. Elution Profile of R39A in Gel Filtration Superdex 75 column The TRP fraction purified from HiTrap Heparin column was pooled together and concentrated to 5ml. The concentrated protein was used as the input of the Gel Filtration Superdex 75 column. The peak area showed that protein was eluted into those fractions. TRP was eluted at around 200ml ml of running buffer. 丨•n =,

^^^^ �.

% � ^ �M” -

Figure 3.5 Expression and Purification Profile of TRP R39A Summary of the expression and purification of TRP mutant R39A. The protein was expressed in the bacterial host E. coli BL21(DE3) pLysS. After sonication, the mutant protein was found soluble in the supernatant. By centrifugation, the cell pellet was discarded and the supernatant was loaded into HiTrap SP ion exchange column for purification. The mutant protein fractions were pooled and further purified by HiTrap Heparin column. And finally the protein fractions from Heparin column were pooled and concentrated for the purification by gel filtration Superdex-75 column. Results

3.3 Protein stability

The stability of the wild type and mutant TRP was determined by circular

dichroism (CD). The stability of TRP mutants was measured by two

thermodynamic parameters — 1. free energy of unfolding, AGU(h20), and 2.

melting temperature, Tm. Free energy of unfolding was determined by chemical

unfolding experiments at different concentration of denaturant while melting

temperature was determined by thermal unfolding experiment. The unfolding was

followed by CD at 222 nm. Both chemical and thermal unfolding of the TRP

mutants must be reversible in order to calculate the transition between two states,

3.3.1 Free energy of unfolding

AGu(H20) and D1/2 were determined in the chemical unfolding experiments. TRP

mutants were mixed with denaturant guanidine hydrochloride (GdmHCl) at 25�C.

The concentration of GdmHCl was ranging from 0.0 M— 7.2 M with 0.2 M

interval. Four hundred microlitre protein sample in GdmHCl was loaded in the 1

mm cuvette and incubated at 25� Cfor 1 minute. The unfolding of TRP was

monitored by CD signals at 222 nm.

39 Results

During unfolding, some portions of proteins were changed from folded state to unfolded state by heat or by the denaturing chemical.

i.e. Folded state (F)� �Unfolded state (U)

The fraction unfolded of mutant TRP was obtained at various concentration of denaturant. A chemical denaturation graph (Figure 3.6) was obtained by plotting fraction unfolded against the concentration of GdmHCl. The [D]i/2 was obtained from the graph by finding the concentration of GdmHCl where half of the protein population was unfolded.

If the Fraction folded is FF, the Fraction unfolded is Fu,

For FF + Fu = 1 , and the equilibrium constant Ku = Fu / FF ,

Ku 二 Fu/(1-Fu)

Then, AGu was calculated according to:

AGu = -RT In Ku

where R is the gas constant (8.314 J mol"' K'') and T is the absolute temperature.

The AG in the transition state was calculated and plotted against denaturant

concentration [D], in this case [GdmHCl]. To obtain free energy of unfolding in

the absence of denaturant, AG(H20), a linear extrapolation model is assumed:

AG = AG(H20) - m [D]

where m is a measure of the dependence of AG on denaturant concentration, [D].

40 Fraction Unfolded of Chemical Denaturation of TRP R39A

1,2 I

1 — �Z • • -•-��• /, • / / // 0.8 - I

m I TD I ① • 0.6 - I t 0-5 1 S 0.4 -

0.2 - / / 0 — �••• ••• •誦 ^ Figure 3.6 Fraction of TRP R39A unfolded as a function of GdmHCl concentration [D]i/2 was obtained from the graph by finding the concentration _0 2 丨 I�I I I I I I 1 I I 1 I I I�I I I I I I I I I I I I I I I I I I I I I I I I 丨 of GdmHCl where half of the protein population was 0 1 2 3 4rni5 6 7 8 unfolded. L Jl/2 [GdmHCl] Results

In practice, the above process can be simplified by fitting all experimental parameters using the following equations:

y 二 {(yF + mp[D]) + (yu + mu[D]) x exp[m x ([D] 一 [D],/2)/i^7]}/

(l+exp[mx([D]-[D],/2)/i?7]). where 少f and yu are the intercepts of the pre- and post-transition regions, and mp and mu are the slopes of the pre- and post-transition regions.

From the calculations above, AGU(H20) and [D]I/2 were obtained and summarized

in Table 3.1

41 Results

Table3.1 Table showing the comparison of ~Gu(H20) and [DJ 1/2 of the mutants

and the wild type

TRP [DJ1/2 (M) AGu(H20) (kJ/mol) AG(mutant) - AG(WT)

Wild Type 4.45 ± 0.02 48.9 ± 3.0 ------

R39A 4.60 ± 0.01 50.5 ± 3.1 l.6

R39M 4.34 ± 0.003 47.3 ± 2.9 -l.6

E62A 3.98 ± 0.01 43.7 ± 2.7 -5.2

E64A 4.39 ± 0.02 48.2 ± 3.0 -0.7

E47A 4.48 ± 0.02 49.2 ± 3.0 0.3

E50A 4.29 ± 0.01 47.1±2.9 -l.8

R54A 4.39 ± 0.01 48.2 ± 3.0 -0.7

R54M 4.36 ± 0.01 47.9 ± 2.9 -l.0

K46A 4.31±0.01 47.3 ± 2.9 -l.6

K46M 4.28 ± 0.02 47.0 ± 2.9 -l.9

R39A/E62A 4.06 ± 0.02 44.6 ± 2.7 -4.3

R39M/E62A 4.03 ± 0.01 44.3 ± 2.7 -4.6

42 Results

3.3.2 Thermal stability

In thermal unfolding experiments, the melting temperature (Tm) of the mutant

protein was determined. Mutant protein stock was first diluted to 20-25 juM and

dialyzed against 10 mM PB, pH 7.4 for three hours. Measurement of Tm value

requires reversible thermal unfolding of the protein samples. The CD signals at

222 nm were recorded at temperatures ranges from 25� Cto 110� Cat the scan

rate of l°C/minute. Fraction unfolded (Fy) of the protein was obtained and

thermal unfolding graphs were plotted. (Figure 3.7). The Tm results of the

mutant proteins are summarized in table 3.2.

43 Fraction Unfolded of Thermal Unfolding of TRP R39A

‘ > ！ ！

4一 i …i .… …'……�―- i

^ 1丨..…丨.；-jf\--

I 0.6 ： ： ： I ：…………- ！ 0.5 ：^： ^I ： 0 I 1 -……_.……….….…-…… ：………j…1…r…….…-

： 丨 ：I ： 丨！； I 0.2 —— ：i \ — j Figure 3.7 Fraction of TRP R39A unfolded as a ：J function of temperature T^ was obtained from .遂 the graph by finding the temperature where half of

0 - — the protein population was unfolded.

I i i I 20 40 60 80 Tm 100 120 Temperature Results

Table 3.2 - Table showing the comparison of Tm of the mutants and the wild type

TRP Tm rC Tm (mutant) - Tm (wt) TC

Wide Type 94.6 ±0.1 ——

R39A 91.7 ±0.3 -2.9

R39M Precipitated at 88.0 ----

E62A Precipitated at 89.0 ----

E64A 94.3 士 0.3 -0.3

E47A 93.8 ±0.4 -0.9

E50A 94.4 ±0.3 -0.2

R54A 89.6 ±0.4 -5.0

R54M Precipitated in dialysis ----

K46A Precipitated at 85.0 ----

K46M Precipitated at 85.0 ----

R39A/E62A Precipitated at 80.0 ----

R39M/E62A Precipitated at 80.0 —-

44 Results

3.3.3 Summary

After studying the mutant proteins by chemical unfolding experiment and thermal

unfolding experiment, some mutants had lower thermostability when compared

with TRP wild type while some mutants had only little change. The results are

summarized below:

(I) Chemical Unfolding: (The following trend in AGu(H20))

WT > E62A, R39A/E62A，R39M/E62A

In fact, the results could be simplified into two groups:

I R39A, E47A, R54A, R54M, R39M, K46A, E50A and K46M

II E62A, R39A/E62A, and R39M/E62A

In group I, the AGu(H20) of the mutant proteins have similar values when

comparing with that of TRP-WT, while in group II, the AGu(H20)value of E62A,

R39A/E62A AND R39M/E62A has decreased significantly (from 4.2 — 5.1

kJ/mol) (Figure 3.8).

(II) Thermal Unfolding: (The following trend in descending Tm)

WT > R39A, R54A

Only five mutants c ould f inish the thermal unfolding experiment. The r esults

could be simplified into three groups:

I E64A, E47A, E50A

45 ^ Fraction Unfolded (TRP WT) Fraction Unfolded (E62A) -o — Fraction Unfolded (R39A/E62A) -->�-Fraction Unfolded (R39M/E62A)

Fraction Unfolded of Chemical Denaturation of TRP WT, E62A，R39A/E62A，R39M/E62A

1 -

:•广/ fj

�.8- 於 f -

I 0.6 - (i [ c 丨： � i； I Figure 3.8 Comparison of fraction unfolded curve in 0 K J chemical unfolding experiment The unfolding curve of 1 0.4 — I�/ single mutant E62A and double mutant R39A/E62A ^ /f / AND R39M/E62A were left shift when comparing with I' / TRP wild type. The result indicates that these three j ‘� mutants have a lower [D] 1/2 than wild type, and thus they �2 - / are less stable than wild type \ � iJ 0n - ^ 2 f y A X玄 0 各 V . 0 t 资 X • * X -I~I_IIIIII_I__I_II_I_I_I_I_I__I_I__I_I_I_I__I_I__I_I_I__I I I I I I I I I I I I I I 012345678 [GdmHCI] (M) Results

II R39A, R54A

III R39M, E62A, R54M, K46A, K46M, R39A/E62A, R39M/E62A

In group I, mutant proteins have similar results when comparing with TRP wild

type. The T^ of Group II mutants have decreased significantly. (From 2.9°C to

5.0°C) (Figure 3.9).Group III mutants were precipitated and failed to give result

in thermal refolding.

46 «^ Fraction Unfolded (TRP WT) 一Fraction Unfolded (TRP R54A) •Fraction Unfolded (TRP R39A)

Thermal Unfolding of TRP WT, R39A and R54A rj f il I J 、？ ‘4 j » Figure 3.9 Thermal Unfolding Curve The Tm of R39A, R54A have decreased when compared with TRP wild type.

_Q 2 ~I 1 1 1 I I I I I I I I I I I 1 1 1 40 60 80 100 Temperature Discussion

Chapter 6 Discussion

In present study, a ribosomal protein L30e from T. celer (TRP) was used as a model to understand the thermostability of protein. By comparing TRP with its mesophilic counterpart Y RP, w e f ound t hat t hey dif fer g reatly in c onformational s tability. T. celer L30e has a higher melting temperature and a higher AGu than YRP. Structural analyses show that T. celer L30e has more ion pairs than YRP. However, the role of electrostatic interaction has been controversial since it was first proposed by Perutz

& Raidt (1975). It has been argued that solvent exposed salt-bridges do not stabilize protein because the energy that is gained by the electrostatic interactions is offset by the desolvation energy and the entropic cost of fixing two charged side-chains.

However, ion-pair network may be stabilizing due to the synergetic effects among the multiple ion-pair interactions. In addition, more and more findings suggested that salt-bridges increased in the majority of the thermophilic proteins and had significance in thermostability. (Kumar et al. 2000; Lim et al 2001; Karshikoff and

Ladenstein 2001 ； Kumar and Nussinov 2001.)

In order to study the charge-charge interaction and thermostability, mutants of TRP

(R39A, R39M, K46A, K46M, E47A, E50A, R54A, R54M, E62A, E64A,

R39A/E62A, R39M/E62A) were constructed and purified by chromatographic methods for the study of the effect of charge-charge interaction in TRP. By mutating

47 Discussion the charged residue to uncharged residues, alanine and methionine, the charge-charge interaction would be eliminated. The thermostability parameters, Tm and AGu(H20), of the mutants w ere d etermined by thermal and chemical unfolding using guanidine hydrochloride (GdmHCl) as denaturant respectively. The results of AGU(h20) and Tm

were summarized in Table 3.1 and 3.2.

4.1 Effect of R39, K46, E62, E64

In chemical unfolding and thermal unfolding experiments, the mutants of R39 and

E62 have significant changes in stability when compared with TRP wild type. R39A

has decreased in T^ (91.7�C an) d E62A has lowered in free energy of unfolding (43.7

kJ/mol). R39M, E62A, K46A and K46M have precipitated at around 89°C and 85°C

during the thermal unfolding experiments. Therefore, we cannot obtain the Tm from

these mutants. On top of that, the experiment results of E64A were similar to the TRP

wild type.

R39M, E62A, K46A and K46M were precipitated in thermal unfolding experiment.

It may be due to two possibilities. First, the mutation may cause a lowering in

thermostability and have irreversible thermal unfolding. Second, the mutation may

cause a lowering in solubility as a surface charged amino acid residue is mutated to

an uncharged residue. In this case, the mutation might lower the conformational and

48 Discussion chemical stability of the protein and thus the mutants did not give a reversible thermal unfolding result. In addition, the solubility of R39M and K46M have been lowered due to the methionine mutation. Although we could not get quantitative data for these mutants, their precipitation at around 85� Cindicating that R39, K46 and

E62 are critical in stabilizing TRP.

In order to analyze these results, the 3D structure of TRP could be used for assistance

(Figure 4.1). From the 3D structure, we can find that R39, K46, E62 and E64 are

located in the same cluster and are very close to each other. The distance between

R39 and E62 is 4.39人.Since single mutant E64A did not show any significant

change either in thermal or chemical unfolding experiment, we predict that E64 does

not play an important role in stabilizing the TRP (Figure 4.2). However, we believe

that R39, E62 and K46 are critical in stabilizing TRP as their mutants did show some

differences in the chemical unfolding or thermal unfolding. Charged residues R39,

E62 and K46 locate closely to each other in 3D structure, the distance between E62

and R39 is 6.2人，charge-charge interactions are found and thus ion-pair can be

formed between these residues. Ion pairs between these residues have synergic effect

in increasing the stabilizing. The synergetic effects among these multiple lon-pair

interactions are strong enough to stabilize the protein. Therefore, when one of these

charged residues were mutated to alanine or methionine, the charge-charge

49 ‘ 、：.•，.

^ A ”�^^ "广 - hk'-^^^^lb. Figure 4.1 Ribbon diagram of TRP I 戮 • n ^^-^BWr yArg-b4 showing the charged residues under 赢 Jm • study. Mutation at R39 and R54 caused �.導 ^m H >4 ��lower of the Tm of the protein while — ^jf mutation at E62 caused lower the [D]y�.

Arg-39 f

(Lvs-46 \ . 4

i VjL . W—^Jk Figure 4.2 Mutation at Glu-64 did not affect the • Glft-62 m 夕) conformational stability of TRP The experimental result • (^^ip JP^ A j J in both chemical and thermal denaturation of E64A were Glu SO / % �^similar to TRP wild type. According to the diagram, Glu-64 ‘ 7 ' d m m ：參、 詹、-�\ may not involve in forming ion pair network with other z M I charged residues such as R39 and E62. 少肩：臺 Discussion interaction was then interrupted.

50 Discussion

4.2 Double Mutation at R39 and E62

In order to further study the contribution of the charge-charge interactions in thermostability and the interaction between R39 and E62, double mutation experiments were carried out. Two double mutants were constructed. (R39A/E62A,

R39M/E62A) (Figure 4.3)

In thermal unfolding experiment, both double mutants were irreversibly precipitated at around 80�C 5,� Cearlier than the single mutants. Thus, we believe that the double mutations affect the conformational stability as well as the chemical stability to the protein. Although we could not get the T^ of the double mutants, the early precipitation (80�C o)f the double mutants in thermal unfolding may still give some hints that the charge-charge interactions inside the cluster play a very important role in keeping the high thermostability of TRP. Double mutations disrupt the charge-charge interactions and thus the mutant proteins have a lower thermostabiltiy.

In performing the chemical unfolding experiment of the double m utants, we c an

determine the interaction between R39 and E62. The interaction energy, AAGint,

between two residues can be measured. After we measured the AGu of the double

mutant, combining the AGu of the single mutants, the interaction energy between

R39 and E62 can be calculated by:

AAG,nt = AGu(R39A) - AGu(WT) - AG,(R39A/E62A) + AGu(E62A)

51 R39

E64

, / \ ； J ^ Figure 4.3 Double Mutation at R39 and E62 to � - '善\ \ JT"^ R39A/E62A and R39M/E62A Chemical unfolding ^^^ experiment were performed and the interaction / ^ ' i ^ \ energy, AAGint, between R39 and E62 was calculated / \ � { ^^^ The interction energy between these two residues is I C^ X 1、： 、： 0.7kJ/mol, meaning that there are nearly no interaction between these two residues. Discussion

The AAGint between these two residues are 0.7 kJ/mol, indicating that the interaction between R39 and E62 is very weak. In addition, the decrease of the free energy of unfolding of the double mutants was due to E62A only. It means that E62 may have interactions between other charged residues and more double mutants should be constructed for studying the interaction in E62.

4.3 Effect ofR54

For R54, two mutations (R54A and R54M) have been constructed. The thermostability of these two mutants, however, did have differences — R54A

decreased in Tm to 89.6� Cwhile R54M started to precipitate during dialysis, i.e. salt

concentration decreasing.

The difference in thermal unfolding experiment between R54A and R54M may be

due to the different chemical property between alanine and methonine. Arginine has

a long and positively charged hydrophobic side chain. Methonine has uncharged

hydrophobic side chain while alanine is uncharged and does not contain a long side

chain. Mutation to methionine mimics the effect of hydrophobic side chain without

any charge, while mutation to alanine eliminated both effects of positive charge and

hydrophobic side chain. Hydrophobic side-chain on protein surface destabilizes the

protein structure and decrease the protein solubility. Thus the decrease of the

solubility of R54M could account for the fact that it started to precipitate in dialysis

52 Discussion against 10 mM PB, pH 7.4.

To explain the decrease in stability of R54A, the 3D structural of TRP is employed.

R54 is located in helix 3 of TRP, however, there are no nearby charged residues that can form ion-pairs to R54 for stabilizing the thermostability of TRP. The distance between R54 and E47 is 12.07A. Therefore we believed that R54 may play a role for long-range electrostatic interactions and stabilizing TRP. In fact, recent theoretical and experimental studies have highlighted the importance of long-range electrostatic interactions among charged residues on protein thermostability. For example, substitutions of two surface residues (R3E/L66E) are responsible for the differences in stability of a thermophilic cold shock protein and its mesophilic homologues.

Their results suggest that the stabilizing effect is due to the optimization of surface electrostatic interactions (for example, avoiding repulsive contacts between same charges) but not the formation of specific salt-bridges. Comparing TRP and YRP,

TRP clearly has more favorable longer-range electrostatics interactions. Therefore,

R54 may play a role in stabilizing the thermostability of TRP by a long-ranged

interaction.

53 Discussion

4.4 Effect of E47 and E50

The value of the free energy of unfolding and Tm of E47A and E50A were very similar to that of TRP Wild Type. T hus, itis b elieved that E 47 and E50 do no t involve in stabilizing TRP structure. (Fig4.4)

54 厂\

^^ E50 Figure 4.4 Ribbon diagram of TRP helix 3 Mutation were done at \ 7 权7, E50 and R54. The chemical and thermal unfolding experiment M results of E47A and E50A were similar to TRP wild type. R54A has 1 decrease in in thermal unfolding experiment. Discussion

Conclusions and future works

In sum ma 17, we have constructed twelve mutants and measured their conformational stability by two parameters, AGu(H20) and T,”. Thermodynamics measurements show that R39, K46, R54 and E62 play an important role in stabilizing TRP. R39,

E62, K 46 a re m the s ame cluster and are critical for the s tabilization of TRP b y forming lon-pairs network. Ion pairs between these residues have synergistic effect on increasing the stabilizing. The mutation of the above residues can lower the conformational stability of proteins. Double mutation between R39A and E62A was also constructed. The chemical unfolding experiment showed that R32 does not interact to E62. On top of that, we believe that R54 may play a long-range electrostatic interaction among other charged residues for stabilizing the T. celer

L30e. E47, E50 and E64 do not involve in TRP stabilization. Mutants constructed in these sites having similar experimental results with the TRP wild type.

Salt dependency

Salt dependency experiment can be done in the future in order to further confirm the

contribution of charged residues. If electrostatic interactions contribute to the

thermostability of T. celer L30e, a higher salt concentration should destabilize the

protein b y screening t he favorable electrostatic interactions. O n the other hand, a

higher salt concentration, in the case of NaCl, will stabilize the protein by the

55 Discussion

Hofmeister effect (Record et al. 1998).

After finishing the first round single mutation of TRP, more double mutation can be constructed. The interaction energy between two residues will be measured.

At last but not least, the rules of thermostability consist of many factors and the contribution of charge-charge interaction on thermostability is still unresolved. Other charged residues of TRP should be studied to figure out the effect of charge-charge interaction using TRP as model.

56 Reference

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63 Appendix:

Fraction Unfolded of Chemical Denaturation of Fraction Unfolded of Chemical Denaturation of TRP-R39M TRP-R39A r 0.8 / 08 广/

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64 Fraction Unfolded of Chemical Denaturation of Fraction Unfolded of Chemical Denaturation of TRP-E62A — TRP-E64A ‘ 厂’… 厂 / ： /

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65 ！

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66 Fraction Unfolded of Chemical Denaturation of Fraction Unfolded of Chemical Denaturation of TRP-R54A TRP-R54M

1 产••_‘_? 1 •••_•‘’

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67 Fraction Unfolded of Chemical Denaturation of . _ ^ TDD WARA Fraction Unfolded of Chemical Denaturation of 丁 RP-K46A TRP-K46M , 厂。，厂 P 0.6 / ^ /

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68 Fraction Unfolded of Chemical Denaturation of Fraction Unfolded of Chemical Denaturation of TRP-R39A/E62A TRP-R39M/E62A

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70 Fraction Unfolded of Thermal Denaturation of Fraction Unfolded of Thermal Denaturation of TRP-E64A TRP-E47A

•

1 f 1 产

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0.2 I 0.2 J

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Temperature Temperature

71 Fraction Unfolded of Thermal Denaturation of Fraction Unfolded of Thermal Denaturation of TRP-E50A TRP-R54A

1 y 1 - ^^

0.3 / 0.B- f J 9 J ！ I 0-6 / I 0.6 — f ° / c f - / ？ . I § I o i •I I 04 - ， ”.4 I 2 I I I I f % 0.2 / 0.2 i rJ �f

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72

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