臺灣博碩士論文加值系統

維持蛋白質完整功能與結構的恆定，在生物體細胞內扮演著重要角色。這維持恆定的角色大多由同時具備chaperone功能和能量依賴性的寡聚體蛋白酶(oligomeric protease) 來扮演。特別是當生物處在逆境時(如高溫)，這些蛋白酶更凸顯出其重要性，因為在這些逆境下，錯誤摺疊或受損害的蛋白質會大量增加且堆積，造成蛋白質恆定異常。本論文即從台灣烏來溫泉菌－嗜熱短桿菌(Brevibacillus thermoruber WR-249)中選殖出其中一種寡聚體蛋白酶－Lon蛋白酶的基因(Bt-lon)，利用遺傳工程得到大量蛋白質產物後，鑑定其酵素功能及熱穩定性，並進一步探討功能與四級結構之間的關係。
Bt-lon可轉譯出分子量約為88 kDa的Lon蛋白酶(Bt-Lon)。Bt-Lon大致可分成三個功能區(domain)：N端功能區、中間ATP水解酶功能區以及C端蛋白酶功能區，其中ATP水解酶功能區還包含了受質偵測識別功能區(sensor- and substrate- discrimination domain, SSD domain)。首先經實驗證明，Bt-lon是一個受熱誘導的熱休克蛋白基因。接著，實驗證明Bt-Lon 是一個具有多功能的蛋白酶，包括蛋白質的水解、ATP的水解、chaperone-like功能以及與DNA結合之能力。由部分實驗結果顯示，蛋白質的水解與DNA結合能力的最佳溫度均為50℃，此溫度正是這株嗜熱短桿菌生長的最佳溫度。然而，ATP的水解的最佳溫度則為70℃。後續也顯示蛋白質的水解必須在ATP的存在下才能發揮作用，這確認了Bt-Lon的確是一個ATP依賴性(ATP-dependent)的蛋白酶。而且Bt-Lon對受質的專一性也與大腸桿菌Lon不盡相同。值得一提的是，我們首先提出直接證據證明Lon蛋白酶具有chaperone-like功能，它可有效地抑制變性蛋白質的不規則聚集，且是與Bt-Lon本身的濃度有關而與ATP存在與否無關。在熱穩定性的探討方面，利用CD光譜進行熱變性實驗可得到Bt-Lon的Tm值為71.5℃。更進一步將Bt-Lon與枯草桿菌(Bacillus subtilis) Lon (Bs-Lon)作序列分析的比較，結果顯示Bt-Lon的熱穩定性可能來自蛋白質較堅固，不易受熱變動或者來自有較多的離子鍵或氫建，不過尚待實驗證明。另一方面，也開始對Bt-Lon的四級結構形成的特性作初步的探討。由分析型膠體過濾層析法(analytical gel filtration chromatography)實驗結果顯示，Bt-Lon是一個含六個次單元體的複合體結構。同樣地，從化學連結實驗(chemical crosslinking experiments)也可得到相同結果。同時也以此實驗探討Bt-Lon四級結構形成的過程與性質。結果發現Bt-Lon六元體的形成過程傾向二元體 « 四元體 « 六元體的組合模式。而且也發現維持二元體的主要作用力為疏水性作用力；而整個六元體主要是靠離子交互作用力。
為了進一步了解四級結構與功能之間的關係，我們設計了七個Bt-Lon的刪去突變株蛋白質(truncated mutants)，然後利用這些突變株測試蛋白質的水解、ATP的水解、chaperone-like功能以及與DNA結合之能力。同時利用分析型膠體過濾層析法和分析型超高速離心機(analytical ultracentrifugation)來測定這些突變株四級結構的變化。結果顯示Bt-Lon的N端是形成四級結構的必要條件，而且結果也顯示四級結構形成的正確與否也直接影響其功能的運作。因為去掉N端的Bt-Lon突變株不但無法形成四級結構，而且也失去蛋白質的水解、ATP的水解以及chaperone-like活性的功能。從這些結果顯示四級結構的形成對於整個Bt-Lon功能的執行扮演著著決定性的關鍵角色。出乎意料之外，結果發現Bt-Lon的N端並不會與DNA結合，進一步尋找後發現，Bt-Lon的SSD功能區才是與DNA結合有關。最後利用電腦模擬結構對此SSD功能區新發現的功能做完整的討論。

Protein quality control plays key roles in prokaryotic and eukaryotic cells by depicting cellular surveillance systems of structural and functional integrity of proteins inside a cell. Oligomeric ATP-dependent proteases consisting of the chaperone and the protease machinery carry out such quality control. These energy-dependent proteases are especially important under stress conditions because increasing amounts of misfolding and damaged proteins may accumulate during various stresses.
A thermostable Lon protease from Brevibacillus thermoruber WR-249 (Bt-Lon) has been cloned and characterized. The Br. thermoruber Lon gene (Bt-lon) encodes an 88-kDa protein characterized with an N-terminal domain, a central ATPase domain including an SSD (sensor- and substrate- discrimination) domain, and a C-terminal protease domain. The Bt-lon is a heat-inducible gene and may be controlled under a putative Bacillus subtilis sA-dependent promoter but in the absence of CIRCE (controlling inverted repeat of chaperone expression). Bt-lon was expressed in E. coli and its protein product was purified. Bt-Lon is a multi-functional enzyme and its functions include the degradation of proteins, ATPase and chaperone-like activities, and DNA binding. The optimal temperature of ATPase activity for Bt-Lon is at 70oC, while the optimal temperature of peptidase and DNA-binding activities is 50oC. The peptidase activity of Bt-Lon increases substantially in the presence of ATP. Furthermore, the substrate specificity of Bt-Lon was found to be different from that of E. coli Lon by using fluorogenic peptides as substrates. Notably, this Bt-Lon protein shows the chaperone-like activity by preventing aggregation of denatured insulin B chain in a dose-dependent and ATP-independent manner. In the thermal denaturation experiments, Bt-Lon was found to display an indicator of thermostability value, Tm of 71.5 oC. Sequence comparison with a mesophilic Lon protease shows differences in the rigidity, electrostatic interactions or hydrogen bonding of Bt-Lon relevant to thermostability. Additionally, the native Br. thermoruber Lon protease (Bt-Lon) showed a hexameric structure as revealed by analytical gel filtration chromatography, and its nature of oligomerization was investigated. The chemical crosslinking experiments revealed that the oligomerization of Bt-Lon proceeds through a dimer « tetramer « hexamer assembly model. Our results also showed that hydrophobic interactions may play important roles in the dimerization of Bt-Lon and that ionic interactions are mainly responsible for hexamer assembly.
To identify the roles of individual domains in the oligomerization process and the functional activities, seven truncated mutants of Bt-Lon were designed, expressed and purified. We examined the Bt-Lon mutants using assays that reflect five different aspects of Bt-Lon activity: ATP-independent oligomerization, ATP-dependent proteolysis, ATPase activity, chaperone-like activity, and DNA-binding activity. Our results show that the N-terminal domain is essential for the oligomerization. The truncation of N-terminal domain resulted in the failure of oligomerization and led to the inactivation of proteolytic, ATPase and chaperone-like activities, suggesting that oligomerization of Bt-Lon is a prerequisite for its catalytic and chaperone-like activities. However, the N-terminal region of Bt-Lon is not involved in the interaction with DNA. We further found that the SSD domain with a previously uncertain function is involved in DNA-binding based on gel mobility shift assays (GMSA).

TABLE OF CONTENTS
ABSTRACT……………………………………………………………………iv
中文摘要……………………………………………………………………vi
LIST OF ABBREVIATIONS……………………………………………..viii
INTRODUCTION………………………………………………………………..1
MATERIALS AND METHODS
Bacterial identification and culture conditions………………..10
Bacterial strains, enzymes and chemicals…………………...……10
DNA manipulation and sequence analysis…………………………..11
Molecular cloning of Br. thermoruber Lon gene (Bt-lon).……….11
Heat-shock experiments and Northern blotting……………………..12
Plasmid construction of Bt-Lon and its truncated mutants for expression…...........13
Expression and purification of Bt-Lon and its truncated mutants….13
Peptidase and protease assays………………………………...…..14
ATPase assays……………………………………………………...…..16
Assay of chaperone-like activity…………...……………………..16
Antibody preparation and Western blot analysis……………………17
Gel mobility shift assays (GMSA)……………………………….....17
Calculation of DNA binding cooperativity……………………....18
Tryptophan fluorescence……………………………………..……..19
Circular dichroism……………………………………………………..19
Thermal denaturation and unfolding transition…………………..19
Analytical gel filtration chromatography………………………...20
Chemical crosslinking assays………………………………...……..20
Analytical ultracentrifugation……………………..……….……..21
Homology modeling of Bt-Lon-SSD…..…………..……………….....21
RESULTS
PART I
Identification of a gene encoding Lon protease from Brevibacillusthermoruber WR-249…………………………….……..24
Molecular cloning and analysis of Br. thermoruber Lon gene (Bt-lon)….....................................................24
Analysis of promoter and heat-induced transcription of Bt-lon…..………..............................................25
.
PART II
Biochemical characterization and thermal stability of the recombinant Bt-Lon……………………………...............…..26
Characterization of proteolytic and ATPase activities of Bt-Lon……….…...............................................26
Characterization of chaperone-like activity of Bt-Lon………….28
Characterization of DNA-binding activity of Bt-Lon………………29
Thermal stability………………………………………………….…....32
PART III
Structure-Function relationship of Bt-Lon……………………....34
Oligomerization of Bt-Lon under various conditions……………..34
Design of Bt-Lon mutants for structure-functional studies…...35
Oligomerization state of Bt-Lon and its mutants……………....36
Proteolytic and ATPase activities of Bt-Lon mutants……….....38
Chaperone-like activity of Bt-Lon mutants……………………....38
The DNA-binding activity of Bt-Lon and its mutants…………....39
DISCUSSION
Domain features of Bt-Lon…………………….……………………....41
Bt-Lon is a heat shock protein…………………………………....41
The discrepancy between peptidase and ATPase activity in optimum temperature………………………………………………......43
Substrate specificity and catalytic mechanism of Bt-Lon…....43
Characterization of chaperone-like activity of Bt-Lon…………44
Physiological roles of multi-functional Bt-Lon………..…....46
Thermal stability……………….…………………………………....47
Nature of Bt-Lon oligomerization………………………..……....49
The N-terminal domain of Lon proteases is essential for oligomerization….50
Oligomerization of Bt-Lon is a prerequisite for its catalytic and chaperone- like activities…………………………….…......52
The N-terminal domain may possess functions other than oligomerization...53
SSD domain and DNA binding………………………...……....55
FIGURES……………………………………...…………………........57
TABLES………………………………………………………...……....120
REFERENCES…………………………………………………..………..125
APPENDIX………………………….……………………...………..…137

Adachi, T., H. Yamagata, N. Tsukagoshi and S. Udaka (1990). "Use of both translation initiation sites of the middle wall protein gene in Bacillus brevis 47." J. Bacteriol. 172(1): 511-513.
Akiyama, Y., T. Yoshihisa and K. Ito (1995). "FtsH, a membrane-bound ATPase, forms a complex in the cytoplasmic membrane of Escherichia coli." J. Biol. Chem. 270(40): 23485-23490.
Amerik, A., V. K. Antonov, A. E. Gorbalenya, S. A. Kotova, T. V. Rotanova and E. V. Shimbarevich (1991). "Site-directed mutagenesis of La protease. A catalytically active serine residue." FEBS Lett. 287(1-2): 211-214.
Appel, R. D., A. Bairoch and D. F. Hochstrasser (1994). "A new generation of information retrieval tools for biologists: the example of the ExPASy WWW server." Trends Biochem. Sci. 19(6): 258-260.
Barakat, S., D. A. Pearce, F. Sherman and W. D. Rapp (1998). "Maize contains a Lon protease gene that can partially complement a yeast pim1-deletion mutant." Plant Mol. Biol. 37(1): 141-154.
Barnett, M. E., A. Zolkiewska, M. Zolkiewski, M. Kessel, A. Ginsburg and M. R. Maurizi (2000). "Structure and activity of ClpB from Escherichia coli. Role of the amino-and -carboxyl-terminal domains." J. Biol. Chem. 275(48): 37565-37571.
Bergqvist, S., M. A. Williams, R. O''Brien and J. E. Ladbury (2002). "Reversal of halophilicity in a protein-DNA interaction by limited mutation strategy." Structure (Camb) 10(5): 629-637.
Bochtler, M., C. Hartmann, H. K. Song, G. P. Bourenkov, H. D. Bartunik and R. Huber (2000). "The structures of HsIU and the ATP-dependent protease HsIU-HsIV." Nature 403(6771): 800-805.
Botos, I., E. E. Melnikov, S. Cherry, A. G. Khalatova, F. S. Rasulova, J. E. Tropea, M. R. Maurizi, T. V. Rotanova, A. Gustchina and A. Wlodawer (2004a). "Crystal structure of the AAA+ [alpha] domain of E. coli Lon protease at 1.9 A resolution." Journal of Structural Biology 146(1-2): 113-122.
Botos, I., E. E. Melnikov, S. Cherry, J. E. Tropea, A. G. Khalatova, F. Rasulova, Z. Dauter, M. R. Maurizi, T. V. Rotanova, A. Wlodawer and A. Gustchina (2004b). "The catalytic domain of E.coli Lon protease has a unique fold and a Ser-Lys dyad in the active site." J. Biol. Chem. 279: 8140-8148.
Bowie, J. U., R. Luthy and D. Eisenberg (1991). "A method to identify protein sequences that fold into a known three-dimensional structure." Science 253: 164-170.
Chakravarty, S. and R. Varadarajan (2002). "Elucidation of factors responsible for enhanced thermal stability of proteins: a structural genomics based study." Biochemistry 41(25): 8152-8161.
Charette, M. F., G. W. Henderson, L. L. Doane and A. Markovitz (1984). "DNA-stimulated ATPase activity on the lon (CapR) protein." J. Bacteriol. 158(1): 195-201.
Charette, M. F., G. W. Henderson, F. J. Kezdy and A. Markovitz (1982). "Molecular mechanism for dominance of a mutant allele of an ATP-dependent protease." J. Mol. Biol. 162(2): 503-510.
Charette, M. F., G. W. Henderson and A. Markovitz (1981). "ATP hydrolysis-dependent protease activity of the lon (capR) protein of Escherichia coli K-12." Proc. Natl. Acad. Sci. U. S. A. 78(8): 4728-4732.
Chen, M. Y., G. H. Lin, Y. T. Lin and S. S. Tsay (2002). "Meiothermus taiwanensis sp. nov., a novel filamentous, thermophilic species isolated in Taiwan." Int. J. Syst. Evol. Microbiol. 52(5): 1647-1654.
Chin, D. T., S. A. Goff, T. Webster, T. Smith and A. L. Goldberg (1988). "Sequence of the lon gene in Escherichia coli. A heat-shock gene which encodes the ATP-dependent protease La." J. Biol. Chem. 263(24): 11718-11728.
Chung, C. H. and A. L. Goldberg (1981). "The product of the lon (capR) gene in Escherichia coli is the ATP-dependent protease, protease La." Proc. Natl. Acad. Sci. U. S. A. 78(8): 4931-4935.
Chung, C. H. and A. L. Goldberg (1982). "DNA stimulates ATP-dependent proteolysis and protein-dependent ATPase activity of protease La from Escherichia coli." Proc. Natl. Acad. Sci. U. S. A. 79(3): 795-799.
Chung, C. H., L. Waxman and A. L. Goldberg (1983). "Studies of the protein encoded by the lon mutation, capR9, in Escherichia coli. A labile form of the ATP-dependent protease La that inhibits the wild type protease." J. Biol. Chem. 258(1): 215-221.
Deuerling, E., B. Paeslack and W. Schumann (1995). "The ftsH gene of Bacillus subtilis is transiently induced after osmotic and temperature upshift." J. Bacteriol. 177(14): 4105-4112.
Dong, G., C. Vieille and J. G. Zeikus (1997). "Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme." Appl. Environ. Microbiol. 63(9): 3577-3584.
Dougan, D. A., A. Mogk and B. Bukau (2002). "Protein folding and degradation in bacteria: to degrade or not to degrade? That is the question." Cell Mol. Life Sci. 59(10): 1607-1616.
Ebel, W., M. M. Skinner, K. P. Dierksen, J. M. Scott and J. E. Trempy (1999). "A conserved domain in Escherichia coli Lon protease is involved in substrate discriminator activity." J. Bacteriol. 181(7): 2236-2243.
Farahbakhsh, Z. T., Q. L. Huang, L. L. Ding, C. Altenbach, H. J. Steinhoff, J. Horwitz and W. L. Hubbell (1995). "Interaction of alpha-crystallin with spin-labeled peptides." Biochemistry 34(2): 509-516.
Fischer, H. and R. Glockshuber (1993). "ATP hydrolysis is not stoichiometrically linked with proteolysis in the ATP-dependent protease La from Escherichia coli." J. Biol. Chem. 268(30): 22502-22507.
Flanagan, J. M. and M. C. Bewley (2002). Protein quality control in bacterial cells: Integrated networks of chaperones and ATP-dependent proteases. Genetic engineering: principles and methods V.1-. J. K. Setlow and A. Hollaender. New York., Plenum Press: 17-47.
Fu, G. K. and D. M. Markovitz (1998). "The human LON protease binds to mitochondrial promoters in a single-stranded, site-specific, strand-specific manner." Biochemistry 37(7): 1905-1909.
Fu, G. K., M. J. Smith and D. M. Markovitz (1997). "Bacterial protease Lon is a site-specific DNA-binding protein." J. Biol. Chem. 272(1): 534-538.
Fukui, T., T. Eguchi, H. Atomi and T. Imanaka (2002). "A membrane-bound archaeal Lon protease displays ATP-independent proteolytic activity towards unfolded proteins and ATP-dependent activity for folded proteins." J. Bacteriol. 184(13): 3689-3698.
Gajiwala, K. S. and S. K. Burley (2000). "Winged helix proteins." Curr. Opin. Struct. Biol. 10(1): 110-116.
Gerth, U., A. Wipat, C. R. Harwood, N. Carter, P. T. Emmerson and M. Hecker (1996). "Sequence and transcriptional analysis of clpX, a class-III heat-shock gene of Bacillus subtilis." Gene 181(1-2): 77-83.
Gill, R. E., M. Karlok and D. Benton (1993). "Myxococcus xanthus encodes an ATP-dependent protease which is required for developmental gene transcription and intercellular signaling." J. Bacteriol. 175(14): 4538-4544.
Goff, S. A., L. P. Casson and A. L. Goldberg (1984). "Heat shock regulatory gene htpR influences rates of protein degradation and expression of the lon gene in Escherichia coli." Proc. Natl. Acad. Sci. U. S. A. 81(21): 6647-6651.
Goldberg, A. L. (1992). "The mechanism and functions of ATP-dependent proteases in bacterial and animal cells." Eur. J. Biochem. 203(1-2): 9-23.
Goldberg, A. L., R. P. Moerschell, C. H. Chung and M. R. Maurizi (1994). "ATP-dependent protease La (lon) from Escherichia coli." Methods Enzymol. 244: 350-375.
Gottesman, S. (1996). "Proteases and their targets in Escherichia coli." Annu. Rev. Genet. 30: 465-506.
Gottesman, S. and M. R. Maurizi (1992). "Regulation by proteolysis: energy-dependent proteases and their targets." Microbiol. Rev. 56(4): 592-621.
Guo, F., M. R. Maurizi, L. Esser and D. Xia (2002). "Crystal structure of ClpA, an Hsp100 chaperone and regulator of ClpAP protease." J. Biol. Chem. 277(48): 46743-46752.
Ha, J. H., M. W. Capp, M. D. Hohenwalter, M. Baskerville and M. T. Record, Jr. (1992). "Thermodynamic stoichiometries of participation of water, cations and anions in specific and non-specific binding of lac repressor to DNA. Possible thermodynamic origins of the "glutamate effect" on protein-DNA interactions." J. Mol. Biol. 228(1): 252-264.
Hall, T. A. (1999). " BioEdit : a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT." Nucleic Acids Symp. Ser. 41,: 95-98.
Haney, P. J., J. H. Badger, G. L. Buldak, C. I. Reich, C. R. Woese and G. J. Olsen (1999). "Thermal adaptation analyzed by comparison of protein sequences from mesophilic and extremely thermophilic Methanococcus species." Proc. Natl. Acad. Sci. U. S. A. 96(7): 3578-3583.
Hawley, D. and W. McClure (1983). "Compilation and analysis of Escherichia coli promoter DNA sequences." Nucl. Acids. Res. 11(8): 2237-2255.
Hecker, M., W. Schumann and U. Volker (1996). "Heat-shock and general stress response in Bacillus subtilis." Mol. Microbiol. 19(3): 417-428.
Helmann, J. (1995). "Compilation and analysis of Bacillus subtilis sigma A-dependent promoter sequences: evidence for extended contact between RNA polymerase and upstream promoter DNA." Nucl. Acids. Res. 23(13): 2351-2360.
Huang, H. C., M. Y. Sherman, O. Kandror and A. L. Goldberg (2001). "The molecular chaperone DnaJ is required for the degradation of a soluble abnormal protein in Escherichia coli." J. Biol. Chem. 276(6): 3920-3928.
Huffman, J. L. and R. G. Brennan (2002). "Prokaryotic transcription regulators: more than just the helix-turn-helix motif." Curr. Opin. Struct. Biol. 12(1): 98-106.
Ikai, A. (1980). "Thermostability and aliphatic index of globular proteins." J. Biochem. (Tokyo) 88(6): 1895-1898.
Ito, K., S. Udaka and H. Yamagata (1992). "Cloning, characterization, and inactivation of the Bacillus brevis lon gene." J. Bacteriol. 174(7): 2281-2287.
Jaenicke, R. and G. Bohm (1998). "The stability of proteins in extreme environments." Curr. Opin. Struct. Biol. 8(6): 738-748.
Kenney, T. J., P. A. Kirchman and C. P. Moran, Jr. (1988). "Gene encoding sigma E is transcribed from a sigma A-like promoter in Bacillus subtilis." J. Bacteriol. 170(7): 3058-3064.
Kessel, M., M. R. Maurizi, B. Kim, E. Kocsis, B. L. Trus, S. K. Singh and A. C. Steven (1995). "Homology in structural organization between E. coli ClpAP protease and the eukaryotic 26 S proteasome." J. Mol. Biol. 250(5): 587-594.
Kessel, M., W. Wu, S. Gottesman, E. Kocsis, A. C. Steven and M. R. Maurizi (1996). "Six-fold rotational symmetry of ClpQ, the E. coli homolog of the 20S proteasome, and its ATP-dependent activator, ClpY." FEBS Lett. 398(2-3): 274-278.
Krzywda, S., A. M. Brzozowski, C. Verma, K. Karata, T. Ogura and A. J. Wilkinson (2002). "The crystal structure of the AAA domain of the ATP-dependent protease FtsH of Escherichia coli at 1.5 A resolution." Structure (Camb) 10(8): 1073-1083.
Kubo, M. and T. Imanaka (1988). "Cloning and nucleotide sequence of the highly thermostable neutral protease gene from Bacillus stearothermophilus." J. Gen. Microbiol. 134: 1883-1892.
Lanzetta, P. A., L. J. Alvarez, P. S. Reinach and O. A. Candia (1979). "An improved assay for nanomole amounts of inorganic phosphate." Anal. Biochem. 100(1): 95-97.
Laskowski, R. A., M. W. MacArthur, D. S. Moss and J. M. Thornton (1993). "PROCHECK: a program to check the stereochemical quality of protein structures." J. Appl. Cryst. 26: 283-291.
Laue, T. M., Shah, B., Ridgeway, T. M., and Pelletier, S. L. (1992). Analytical Ultracentrifugation in Biochemistry and Polymer Science. S. E. Harding, Rowe, A. J., and Horton, J. C. Cambridge, United Kingkom, The Royal Society of Chemistry: 90-125.
Leonhard, K., A. Stiegler, W. Neupert and T. Langer (1999). "Chaperone-like activity of the AAA domain of the yeast Yme1 AAA protease." Nature 398(6725): 348-351.
Lin, T. H., T. Quinn, M. Walsh, D. Grandgenett and J. C. Lee (1991). "Avian myeloblastosis virus reverse transcriptase. Effect of glycerol on its hydrodynamic properties." J. Biol. Chem. 266(3): 1635-1640.
Liu, T., B. Lu, I. Lee, G. Ondrovicova, E. Kutejova and C. K. Suzuki (2004). "DNA and RNA binding by the mitochondrial Lon protease is regulated by nucleotide and protein substrates." J. Biol. Chem. 279(14): 13902-13910.
Lu, B., T. Liu, J. A. Crosby, J. Thomas-Wohlever, I. Lee and C. K. Suzuki (2003). "The ATP-dependent Lon protease of Mus musculus is a DNA-binding protein that is functionally conserved between yeast and mammals." Gene 306: 45-55.
Lupas, A. (1996). "Coiled coils: new structures and new functions." Trends Biochem. Sci. 21(10): 375-382.
Lupas, A. (1997). "Predicting coiled-coil regions in proteins." Curr. Opin. Struct. Biol. 7(3): 388-393.
Lupas, A. N. and J. Martin (2002). "AAA proteins." Curr. Opin. Struct. Biol. 12(6): 746-753.
Maurizi, M. R. (1992). "Proteases and protein degradation in Escherichia coli." Experientia 48(2): 178-201.
Maurizi, M. R. and C. C. Li (2001). "AAA proteins: in search of a common molecular basis. International Meeting on Cellular Functions of AAA Proteins." EMBO Rep. 2(11): 980-985.
Maurizi, M. R., S. K. Singh, M. W. Thompson, M. Kessel and A. Ginsburg (1998). "Molecular properties of ClpAP protease of Escherichia coli: ATP-dependent association of ClpA and ClpP." Biochemistry 37(21): 7778-7786.
McDonald, J., A. Grasso and L. Rejto (1999). "Patterns of temperature adaptation in proteins from Methanococcus and Bacillus." Mol. Biol. Evol. 16(12): 1785-1790.
Mogk, A., C. Schlieker, C. Strub, W. Rist, J. Weibezahn and B. Bukau (2003). "Roles of individual domains and conserved motifs of the AAA+ chaperone ClpB in oligomerization, ATP hydrolysis, and chaperone activity." J. Biol. Chem. 278(20): 17615-17624.
Nakamura, S., T. Tanaka, R. Yada and S. Nakai (1997). "Improving the thermostability of Bacillus stearothermophilus neutral protease by introducing proline into the active site helix." Protein Eng. 10(11): 1263-1269.
Neuwald, A. F., L. Aravind, J. L. Spouge and E. V. Koonin (1999). "AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes." Genome Res. 9(1): 27-43.
Nicholls, A. (1992). GRASP: Graphical Representation and Analysis of Surface Properties.New York, Columbia University.
Nishio, Y., Y. Nakamura, Y. Kawarabayasi, Y. Usuda, E. Kimura, S. Sugimoto, K. Matsui, A. Yamagishi, H. Kikuchi, K. Ikeo and T. Gojobori (2003). "Comparative complete genome sequence analysis of the amino acid replacements responsible for the thermostability of Corynebacterium efficiens." Genome Res 13(7): 1572-1579.
Niwa, H., D. Tsuchiya, H. Makyio, M. Yoshida, K. Morikawa, S. Krzywda, A. M. Brzozowski, C. Verma, K. Karata, T. Ogura and A. J. Wilkinson (2002). "Hexameric ring structure of the ATPase domain of the membrane-integrated metalloprotease FtsH from Thermus thermophilus HB8." Structure (Camb) 10(10): 1415-1423.
O''Brien, R., B. DeDecker, K. G. Fleming, P. B. Sigler and J. E. Ladbury (1998). "The effects of salt on the TATA binding protein-DNA interaction from a hyperthermophilic archaeon." J. Mol. Biol. 279(1): 117-125.
Ogura, T. and A. J. Wilkinson (2001). "AAA+ superfamily ATPases: common structure--diverse function." Genes Cells 6(7): 575-597.
Oh, J. Y., Y. M. Eun, S. J. Yoo, J. H. Seol, I. S. Seong, C. S. Lee and C. H. Chung (1998). "LonR9 carrying a single Glu614 to Lys mutation inhibits the ATP-dependent protease La (Lon) by forming mixed oligomeric complexes." Biochem. Biophys. Res. Commun. 250(1): 32-35.
Pace, C. N. (1990). "Measuring and increasing protein stability." Trends Biotechnol. 8(4): 93-98.
Parsell, D. A., A. S. Kowal and S. Lindquist (1994). "Saccharomyces cerevisiae Hsp104 protein. Purification and characterization of ATP-induced structural changes." J. Biol. Chem. 269(6): 4480-4487.
Parsell, D. A. and S. Lindquist (1993). "The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins." Annu. Rev. Genet. 27: 437-496.
Petsko, G. A. (2001). "Structural basis of thermostability in hyperthermophilic proteins, or "there''s more than one way to skin a cat"." Methods Enzymol. 334: 469-478.
Phillips, T. A., R. A. VanBogelen and F. C. Neidhardt (1984). "lon gene product of Escherichia coli is a heat-shock protein." J. Bacteriol. 159(1): 283-287.
Rasulova, F. S., N. I. Dergousova, N. N. Starkova, E. E. Melnikov, L. D. Rumsh, L. M. Ginodman and T. V. Rotanova (1998). "The isolated proteolytic domain of Escherichia coli ATP-dependent protease Lon exhibits the peptidase activity." FEBS Lett. 432(3): 179-181.
Rep, M., J. M. van Dijl, K. Suda, G. Schatz, L. A. Grivell and C. K. Suzuki (1996). "Promotion of mitochondrial membrane complex assembly by a proteolytically inactive yeast Lon." Science 274(5284): 103-106.
Riethdorf, S., U. Volker, U. Gerth, A. Winkler, S. Engelmann and M. Hecker (1994). "Cloning, nucleotide sequence, and expression of the Bacillus subtilis lon gene." J. Bacteriol. 176(21): 6518-6527.
Rotanova, T. V., E. E. Mel''nikov and K. B. Tsirul''nikov (2003). "Catalytic dyad Ser-Lys at the active site of Escherichia coli ATP-dependent Lon-proteinase." Bioorg Khim 29(1): 97-99.
Roudiak, S. G., A. Seth, N. Knipfer and T. E. Shrader (1998). "The lon protease from Mycobacterium smegmatis: molecular cloning, sequence analysis, functional expression, and enzymatic characterization." Biochemistry 37(1): 377-386.
Roudiak, S. G. and T. E. Shrader (1998). "Functional role of the N-terminal region of the Lon protease from Mycobacterium smegmatis." Biochemistry 37(32): 11255-11263.
Rudyak, S. G., M. Brenowitz and T. E. Shrader (2001). "Mg2+-linked oligomerization modulates the catalytic activity of the Lon (La) protease from Mycobacterium smegmatis." Biochemistry 40(31): 9317-9323.
Rudyak, S. G. and T. E. Shrader (2000). "Polypeptide stimulators of the Ms-Lon protease." Protein Sci. 9(9): 1810-1817.
Sali, A. and T. L. Blundell (1993). "Comparative protein modelling by satisfaction of spatial restraints." J. Mol. Biol. 234: 779-815.
Sambrook, J. and D. W. Russell (2001). Molecular cloning: a laboratory manual. 3rd edn,Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press.
Schmidt, R., A. L. Decatur, P. N. Rather, C. P. Moran, Jr. and R. Losick (1994). "Bacillus subtilis lon protease prevents inappropriate transcription of genes under the control of the sporulation transcription factor sigma G." J. Bacteriol. 176(21): 6528-6537.
Schuck, P. (2000). "Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling." Biophys. J. 78(3): 1606-1619.
Schwede, T., J. Kopp, N. Guex and M. C. Peitsch (2003). "SWISS-MODEL: an automated protein homology-modeling server." Nucl. Acids. Res. 31(13): 3381-3385.
Senear, D. and M. Brenowitz (1991). "Determination of binding constants for cooperative site-specific protein-DNA interactions using the gel mobility-shift assay." J. Biol. Chem. 266(21): 13661-13671.
Sharp, R. J., P. W. Riley and D.White (1992). Heterotrophic thermophilic Bacilli. Thermophilic Bacteria. J. K. Kristjansson. Boca Raton, Florida, CRC Press: 20-50.
Sherman, M. and A. L. Goldberg (1992). "Involvement of the chaperonin dnaK in the rapid degradation of a mutant protein in Escherichia coli." EMBO J. 11(1): 71-77.
Shotland, Y., S. Koby, D. Teff, N. Mansur, D. A. Oren, K. Tatematsu, T. Tomoyasu, M. Kessel, B. Bukau, T. Ogura and A. B. Oppenheim (1997). "Proteolysis of the phage lambda CII regulatory protein by FtsH (HflB) of Escherichia coli." Mol. Microbiol. 24(6): 1303-1310.
Singh, S. K., R. Grimaud, J. R. Hoskins, S. Wickner and M. R. Maurizi (2000). "Unfolding and internalization of proteins by the ATP-dependent proteases ClpXP and ClpAP." Proc. Natl. Acad. Sci. U. S. A. 97(16): 8898-8903.
Singh, S. K., J. Rozycki, J. Ortega, T. Ishikawa, J. Lo, A. C. Steven and M. R. Maurizi (2001). "Functional domains of the ClpA and ClpX molecular chaperones identified by limited proteolysis and deletion analysis." J. Biol. Chem. 276(31): 29420-29429.
Smith, C. K., T. A. Baker and R. T. Sauer (1999). "Lon and Clp family proteases and chaperones share homologous substrate-recognition domains." Proc. Natl. Acad. Sci. U. S. A. 96(12): 6678-6682.
Sonezaki, S., K. Okita, T. Oba, Y. Ishii, A. Kondo and Y. Kato (1995). "Protein substrates and heat shock reduce the DNA-binding ability of Escherichia coli Lon protease." Appl. Microbiol. Biotechnol. 44(3-4): 484-488.
Stahlberg, H., E. Kutejova, K. Suda, B. Wolpensinger, A. Lustig, G. Schatz, A. Engel and C. K. Suzuki (1999). "Mitochondrial Lon of Saccharomyces cerevisiae is a ring-shaped protease with seven flexible subunits." Proc. Natl. Acad. Sci. U. S. A. 96(12): 6787-6790.
Starkova, N. N., E. P. Koroleva, L. D. Rumsh, L. M. Ginodman and T. V. Rotanova (1998). "Mutations in the proteolytic domain of Escherichia coli protease Lon impair the ATPase activity of the enzyme." FEBS Lett. 422(2): 218-220.
Sterner, R. and W. Liebl (2001). "Thermophilic adaptation of proteins." Crit. Rev. Biochem. Mol. Biol. 36(1): 39-106.
Suzuki, C. K., K. Suda, N. Wang and G. Schatz (1994). "Requirement for the yeast gene LON in intramitochondrial proteolysis and maintenance of respiration." Science 264(5161): 891.
Tojo, N., S. Inouye and T. Komano (1993). "The lonD gene is homologous to the lon gene encoding an ATP-dependent protease and is essential for the development of Myxococcus xanthus." J. Bacteriol. 175(14): 4545-4549.
Tsou, C. L. (1986). "Location of the active sites of some enzymes in limited and flexible molecular regions." Trends Biochem. Sci. 11: 427-429.
Tsou, C. L. (1993). "Conformational flexibility of enzyme active sites." Science 262(5132): 380-381.
Twining, S. S. (1984). "Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes." Anal. Biochem. 143(1): 30-34.
van Dijl, J. M., E. Kutejova, K. Suda, D. Perecko, G. Schatz and C. K. Suzuki (1998). "The ATPase and protease domains of yeast mitochondrial Lon: roles in proteolysis and respiration-dependent growth." Proc. Natl. Acad. Sci. U. S. A. 95(18): 10584-10589.
van Dyck, L., D. A. Pearce and F. Sherman (1994). "PIM1 encodes a mitochondrial ATP-dependent protease that is required for mitochondrial function in the yeast Saccharomyces cerevisiae." J. Biol. Chem. 269(1): 238-242.
Van Melderen, L., M. H. Thi, P. Lecchi, S. Gottesman, M. Couturier and M. R. Maurizi (1996). "ATP-dependent degradation of CcdA by Lon protease. Effects of secondary structure and heterologous subunit interactions." J. Biol. Chem. 271(44): 27730-27738.
Vasilyeva, O. V., K. B. Kolygo, Y. F. Leonova, N. A. Potapenko and T. V. Ovchinnikova (2002). "Domain structure and ATP-induced conformational changes in Escherichia coli protease Lon revealed by limited proteolysis and autolysis." FEBS Lett. 526(1-3): 66-70.
Vieille, C., K. L. Epting, R. M. Kelly and J. G. Zeikus (2001). "Bivalent cations and amino-acid composition contribute to the thermostability of Bacillus licheniformis xylose isomerase." Eur. J. Biochem. 268(23): 6291-6301.
Vieille, C. and G. J. Zeikus (2001). "Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability." Microbiol. Mol. Biol. Rev. 65(1): 1-43.
Voskuil, M. I., K. Voepel and G. H. Chambliss (1995). "The -16 region, a vital sequence for the utilization of a promoter in Bacillus subtilis and Escherichia coli." Mol. Microbiol. 17(2): 271-279.
Walker, J. E., M. Saraste, M. J. Runswick and N. J. Gay (1982). "Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold." EMBO J. 1(8): 945-951.
Wang, N., S. Gottesman, M. C. Willingham, M. M. Gottesman and M. R. Maurizi (1993). "A human mitochondrial ATP-dependent protease that is highly homologous to bacterial Lon protease." Proc. Natl. Acad. Sci. U. S. A. 90(23): 11247-11251.
Watanabe, S., T. Muramatsu, H. Ao, Y. Hirayama, K. Takahashi, M. Tanokura and Y. Kuchino (1999). "Molecular cloning of the Lon protease gene from Thermus thermophilus HB8 and characterization of its gene product." Eur. J. Biochem. 266(3): 811-819.
Waxman, L. and A. L. Goldberg (1985). "Protease La, the lon gene product, cleaves specific fluorogenic peptides in an ATP-dependent reaction." J. Biol. Chem. 260(22): 12022-12028.
Waxman, L. and A. L. Goldberg (1986). "Selectivity of intracellular proteolysis: protein substrates activate the ATP-dependent protease (La)." Science 232(4749): 500-503.
Wickner, S., M. R. Maurizi and S. Gottesman (1999). "Posttranslational quality control: folding, refolding, and degrading proteins." Science 286(5446): 1888-1893.
Yura, T., H. Nagai and H. Mori (1993). "Regulation of the heat-shock response in bacteria." Annu. Rev. Microbiol. 47: 321-350.
Zehnbauer, B. A., E. C. Foley, G. W. Henderson and A. Markovitz (1981). "Identification and purification of the Lon+ (capR+) gene product, a DNA-binding protein." Proc. Natl. Acad. Sci. U. S. A. 78(4): 2043-2047.
Zhu, J. and S. C. Winans (2001). "The quorum-sensing transcriptional regulator TraR requires its cognate signaling ligand for protein folding, protease resistance, and dimerization." Proc. Natl. Acad. Sci. U. S. A. 98(4): 1507-1512.
Zolkiewski, M., M. Kessel, A. Ginsburg and M. R. Maurizi (1999). "Nucleotide-dependent oligomerization of ClpB from Escherichia coli." Protein Sci. 8(9): 1899-1903.
Zuber, U. and W. Schumann (1994). "CIRCE, a novel heat shock element involved in regulation of heat shock operon dnaK of Bacillus subtilis." J. Bacteriol. 176(5): 1359-1363.