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.