7.1.1 CxUb (x = 11; 48; 6,11; 11,48; 11,63; 6,11,48) DNA sequences
C11Ub ATGCAGATTTTTGTTAAAACCCTGACCGGTTGCACCATTACCCTGGAAGTTGAACCGAGC C11C63Ub ATGCAGATTTTTGTTAAAACCCTGACCGGTTGCACCATTACCCTGGAAGTTGAACCGAGC C48Ub ATGCAGATTTTTGTTAAAACCCTGACCGGTAAAACCATTACCCTGGAAGTTGAACCGAGC C11C48Ub ATGCAGATTTTTGTTAAAACCCTGACCGGTTGCACCATTACCCTGGAAGTTGAACCGAGC C6C11Ub ATGCAGATTTTTGTTTGCACCCTGACCGGTTGCACCATTACCCTGGAAGTTGAACCGAGC C6C11C48Ub ATGCAGATTTTTGTTTGCACCCTGACCGGTTGCACCATTACCCTGGAAGTTGAACCGAGC *************** ************ ***************************
C11Ub GATACCATTGAAAATGTGAAAGCCAAAATTCAGGATAAAGAAGGTATTCCGCCGGATCAG C11C63Ub GATACCATTGAAAATGTGAAAGCCAAAATTCAGGATAAAGAAGGTATTCCTCCGGATCAG C48Ub GATACCATTGAAAATGTGAAAGCCAAAATTCAGGATAAAGAAGGTATTCCTCCGGATCAG C11C48Ub GATACCATTGAAAATGTGAAAGCCAAAATTCAGGATAAAGAAGGTATTCCTCCGGATCAG C6C11Ub GATACCATTGAAAATGTGAAAGCCAAAATTCAGGATAAAGAAGGTATTCCTCCGGATCAG C6C11C48Ub GATACCATTGAAAATGTGAAAGCCAAAATTCAGGATAAAGAAGGTATTCCTCCGGATCAG ************************************************** *********
C11Ub CAGCGTCTGATTTTTGCAGGTAAACAGCTGGAAGATGGTCGTACCCTGAGCGATTATAAT C11C63Ub CAGCGTCTGATTTTTGCAGGTAAACAGCTGGAAGATGGTCGTACCCTGAGCGATTATAAT C48Ub CAGCGTCTGATTTTTGCAGGTTGTCAGCTGGAAGATGGTCGTACCCTGAGCGATTATAAT C11C48Ub CAGCGTCTGATTTTTGCAGGTTGCCAGCTGGAAGATGGTCGTACCCTGAGCGATTATAAT C6C11Ub CAGCGTCTGATTTTTGCAGGTAAACAGCTGGAAGATGGTCGTACCCTGAGCGATTATAAT C6C11C48Ub CAGCGTCTGATTTTTGCAGGTTGCCAGCTGGAAGATGGTCGTACCCTGAGCGATTATAAT ********************* ************************************
C11Ub ATTCAGAAGGAAAGCACCCTGCATCTGGTTCTGCGTCTGCGTGGTGGT C11C63Ub ATTCAGTGCGAAAGCACCCTGCATCTGGTTCTGCGTCTGCGTGGTGGT C48Ub ATTCAGAAAGAAAGCACCCTGCATCTGGTTCTGCGTCTGCGTGGTGGT C11C48Ub ATTCAGAAAGAAAGCACCCTGCATCTGGTTCTGCGTCTGCGTGGTGGT C6C11Ub ATTCAGAAAGAAAGCACCCTGCATCTGGTTCTGCGTCTGCGTGGTGGT C6C11C48Ub ATTCAGAAAGAAAGCACCCTGCATCTGGTTCTGCGTCTGCGTGGTGGT ****** ***************************************
Protein sequences
C48Ub MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGCQLEDGRTLSDYN C11C48Ub MQIFVKTLTGCTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGCQLEDGRTLSDYN C6C11C48Ub MQIFVCTLTGCTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGCQLEDGRTLSDYN C11C63Ub MQIFVKTLTGCTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN C11Ub MQIFVKTLTGCTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN C6C11Ub MQIFVCTLTGCTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN ***** **** ************************************ ************
C48Ub IQKESTLHLVLRLRGG C11C48Ub IQKESTLHLVLRLRGG C6C11C48Ub IQKESTLHLVLRLRGG C11C63Ub IQCESTLHLVLRLRGG C11Ub IQKESTLHLVLRLRGG C6C11Ub IQKESTLHLVLRLRGG ** *************
92 7.1.2 Aha75CxUb (x = 6; 11; 27; 29; 33; 48; 63; 11,48) DNA sequences
Aha75C48Ub GGATCTCGTCGTGCATCTGTTGGATCCCAGATTTTTGTTAAAACCCTGACCGGTAAAACC Aha75C11C48Ub GGATCTCGTCGTGCATCTGTTGGATCCCAGATTTTTGTTAAAACCCTGACCGGTTGCACC Aha75C33Ub GGATCTCGTCGTGCATCTGTTGGATCCCAGATTTTTGTTAAAACCCTGACCGGTAAAACC Aha75C29Ub GGATCTCGTCGTGCATCTGTTGGATCCCAGATTTTTGTTAAAACCCTGACCGGTAAAACC Aha75C27Ub GGATCTCGTCGTGCATCTGTTGGATCCCAGATTTTTGTTAAAACCCTGACCGGTAAAACC Aha75C6Ub GGATCTCGTCGTGCATCTGTTGGATCCCAGATTTTTGTTTGTACCCTGACCGGTAAAACC Aha75C63Ub GGATCTCGTCGTGCATCTGTTGGATCCCAGATTTTTGTTAAAACCCTGACCGGTAAAACC Aha75Ub GGATCTCGTCGTGCATCTGTTGGATCCCAGATTTTTGTTAAAACCCTGACCGGTAAAACC Aha75C11Ub GGATCTCGTCGTGCATCTGTTGGATCCCAGATTTTTGTTAAAACCCTGACCGGTTGTACC *************************************** ************ ***
Aha75C48Ub ATTACCCTGGAAGTTGAACCGAGCGATACCATTGAAAATGTGAAAGCCAAAATTCAGGAT Aha75C11C48Ub ATTACCCTGGAAGTTGAACCGAGCGATACCATTGAAAATGTGAAAGCCAAAATTCAGGAT Aha75C33Ub ATTACCCTGGAAGTTGAACCGAGCGATACCATTGAAAATGTGAAAGCCAAAATTCAGGAT Aha75C29Ub ATTACCCTGGAAGTTGAACCGAGCGATACCATTGAAAATGTGAAAGCCTGTATTCAGGAT Aha75C27Ub ATTACCCTGGAAGTTGAACCGAGCGATACCATTGAAAATGTGTGTGCCAAAATTCAGGAT Aha75C6Ub ATTACCCTGGAAGTTGAACCGAGCGATACCATTGAAAATGTGAAAGCCAAAATTCAGGAT Aha75C63Ub ATTACCCTGGAAGTTGAACCGAGCGATACCATTGAAAATGTGAAAGCCAAAATTCAGGAT Aha75Ub ATTACCCTGGAAGTTGAACCGAGCGATACCATTGAAAATGTGAAAGCCAAAATTCAGGAT Aha75C11Ub ATTACCCTGGAAGTTGAACCGAGCGATACCATTGAAAATGTGAAAGCCAAAATTCAGGAT ****************************************** *** *********
Aha75C48Ub AAAGAAGGTATTCCTCCGGATCAGCAGCGTCTGATTTTTGCAGGTTGTCAGCTGGAAGAT Aha75C11C48Ub AAAGAAGGTATTCCTCCGGATCAGCAGCGTCTGATTTTTGCAGGTTGCCAGCTGGAAGAT Aha75C33Ub TGTGAAGGTATTCCTCCGGATCAGCAGCGTCTGATTTTTGCAGGTAAACAGCTGGAAGAT Aha75C29Ub AAAGAAGGTATTCCTCCGGATCAGCAGCGTCTGATTTTTGCAGGTAAACAGCTGGAAGAT Aha75C27Ub AAAGAAGGTATTCCTCCGGATCAGCAGCGTCTGATTTTTGCAGGTAAACAGCTGGAAGAT Aha75C6Ub AAAGAAGGTATTCCTCCGGATCAGCAGCGTCTGATTTTTGCAGGTAAACAGCTGGAAGAT Aha75C63Ub AAAGAAGGTATTCCTCCGGATCAGCAGCGTCTGATTTTTGCAGGTAAACAGCTGGAAGAT Aha75Ub AAAGAAGGTATTCCGCCGGATCAGCAGCGTCTGATTTTTGCAGGTAAACAGCTGGAAGAT Aha75C11Ub AAAGAAGGTATTCCGCCGGATCAGCAGCGTCTGATTTTTGCAGGTAAACAGCTGGAAGAT *********** ****************************** ************
Aha75C48Ub GGTCGTACCCTGAGCGATTATAATATTCAGAAAGAAAGCACCCTGCATCTGGTTCTGCGT Aha75C11C48Ub GGTCGTACCCTGAGCGATTATAATATTCAGAAAGAAAGCACCCTGCATCTGGTTCTGCGT Aha75C33Ub GGTCGTACCCTGAGCGATTATAATATTCAGAAAGAAAGCACCCTGCATCTGGTTCTGCGT Aha75C29Ub GGTCGTACCCTGAGCGATTATAATATTCAGAAAGAAAGCACCCTGCATCTGGTTCTGCGT Aha75C27Ub GGTCGTACCCTGAGCGATTATAATATTCAGAAAGAAAGCACCCTGCATCTGGTTCTGCGT Aha75C6Ub GGTCGTACCCTGAGCGATTATAATATTCAGAAAGAAAGCACCCTGCATCTGGTTCTGCGT Aha75C63Ub GGTCGTACCCTGAGCGATTATAATATTCAGTGTGAAAGCACCCTGCATCTGGTTCTGCGT Aha75Ub GGTCGTACCCTGAGCGATTATAATATTCAGAAGGAAAGCACCCTGCATCTGGTTCTGCGT Aha75C11Ub GGTCGTACCCTGAGCGATTATAATATTCAGAAGGAAAGCACCCTGCATCTGGTTCTGCGT ****************************** ***************************
93 Protein sequences
Aha75C11C48Ub GSRRASVGSQIFVKTLTGCTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGCQLED Aha75C63Ub GSRRASVGSQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLED Aha75C48Ub GSRRASVGSQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGCQLED Aha75C33Ub GSRRASVGSQIFVKTLTGKTITLEVEPSDTIENVKAKIQDCEGIPPDQQRLIFAGKQLED Aha75C29Ub GSRRASVGSQIFVKTLTGKTITLEVEPSDTIENVKACIQDKEGIPPDQQRLIFAGKQLED Aha75C27Ub GSRRASVGSQIFVKTLTGKTITLEVEPSDTIENVCAKIQDKEGIPPDQQRLIFAGKQLED Aha75C11Ub GSRRASVGSQIFVKTLTGCTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLED Aha75Ub GSRRASVGSQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLED Aha75C6Ub GSRRASVGSQIFVCTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLED ************* **** *************** * *** ************** ****
Aha75C11C48Ub GRTLSDYNIQKESTLHLVLRLRM Aha75C63Ub GRTLSDYNIQCESTLHLVLRLRM Aha75C48Ub GRTLSDYNIQKESTLHLVLRLRM Aha75C33Ub GRTLSDYNIQKESTLHLVLRLRM Aha75C29Ub GRTLSDYNIQKESTLHLVLRLRM Aha75C27Ub GRTLSDYNIQKESTLHLVLRLRM Aha75C11Ub GRTLSDYNIQKESTLHLVLRLRM Aha75Ub GRTLSDYNIQKESTLHLVLRLRM Aha75C6Ub GRTLSDYNIQKESTLHLVLRLRM ********** ************
94
7.2 NMR
N-(Propargyl)-4-aminobenzophenone (2)
1H-NMR ((CD3)2CO, 400 MHz)
13C-NMR ((CD3)2CO, 100 MHz)
95
N-(4-Benzoylphenyl)-2-bromo-N-(prop-2-yn-1-yl)acetamide (3, BPBPAA)
1H-NMR (CD3CN, 400 MHz)
13C-NMR ((CD3)2CO, 100 MHz)
96
7.3 Mass spectrometry
C11Ub-PA
C48Ub-PA
97 Aha75Ub
Aha75C48Ub
98 Ub248-PA
C11C48Ub-PA
99 C11C63Ub-PA
Ub2-11,63
Ub
100 Aha75C11Ub-PA
Aha75C48Ub-PA
101 Aha75C11C48Ub-PA
BPBPAA
102 C48Ub-BPBPAA
Ub2-BP48SH
103 Ub2-BP48Bio
104 HRMS High resolution-mass spectrometry Iso T Isopeptidase T
ITPG Isopropyl-β-D-thiogalactopyranosid
LC-MS Liquid chromatography-mass spectrometry MeCN Acetonitrile
NBT Nitro-blue tetrazolium chloride NCL Native chemical ligation
NEt3 Triethylamine
NMM New minimal medium NMR Nuclear magnetic resonance
OD Optical density
OTUB1 Otubain-1
PA Propargyl acrylate
PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline
PTM Post-translational modification
Rf Retention factor
SDS Sodium dodecylsulfate
SPI Selective pressure incorporation
105 SPPS Solid-phase peptide synthesis TCEP Tris-(2-carboxyethyl)phosphine TCS Thrombin cleavage site
THPTA Tris-(3-hydroxypropyltriazolylmethyl)amine TLC Thin layer chromatography
Ub Ubiquitin
UV Ultraviolet
WB Western blot
106
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10 Acknowledgements
I wish to express sincere appreciation to my supervisor, Prof. Dr. Andreas Marx. First of all, I am extremely grateful that he led me to the research field of ubiquitin, which I never imagined I would work with. In this interesting project, I grasped rich experimental skills in molecular biology and biochemistry, which will definitely benefit my future career greatly. I want to thank him for the kind support in financing my research, including the application and extension for DAAD fellowship, the cooperation with other research groups, and the participation in the international conferences.
Moreover, I wish to offer my gratitude to Mr. Marx for his encouragement throughout the development of this project and the great patience in the correction of this thesis.
I would like to give many thanks to Prof. Dr. Martin Scheffner for his co-supervision, special gratitude for the inspirational discussion and suggestion in ubiquitin biology, the reagents and equipment from his lab. I would also like to thank Dr. Michael Kovermann for his chairing in my examination committee, and the cooperation in the NMR studies of ubiquitin. Dr. Florian Stengel and Prof. Dr.
Daniel Summerer are thanked for the suggestions in my research.
I wish to thank all my cooperators in the research, including Joachim Lutz, Matthias Frese, Maike Lehner, Dr. Anna Sladewska-Marquardt, Dr. Andreas Marquardt, Dr. Daniel Schneider, Dr. Tatjana Schneider, Dr. Konrad Bergen, Dr. Daniel Rösner, Andreas Heim, Dr. Yan Wang, Dr. Franziska Mortensen, Fabian Offensperger, Felix Ebner, Luis Valencia, Tobias Longer, and Oliver Hartmann. I do appreciate their support in the experiments and all the discussion.
I would also like to thank my dear colleagues Meng Zheng, Yizhi Yuan, Dr. Audrey Hottin, Sarah Wallrodt, Dr. Jana Balintová, Vanessa Radtke, Dr. Govindan Raghunathan, Heike Kropp, Vlasta Fleischhauer, and many others in Marx group. I really cherish the lovely time in and out of the lab.
I also want to thank my dear friends Yiqun Geng, Qijun Liang, Yuyi Feng, Zhu Li, Weiye Pu, and many more. Thanks for the nice memories together in Konstanz.
Moreover, DAAD is sincerely acknowledged of the PhD fellowship, and Konstanz Research School Chemical Biology of the training courses and the wonderful research atmosphere.
At last, my deepest thanks to my family, especially to my dear parents.