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«Max-Planck-Institut für Biochemie Abteilung Strukturforschung Biologische NMR-Arbeitsgruppe STRUCTURAL AND FUNCTIONAL STUDIES ON PHOTOACTIVE ...»

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Technische Universität München

Fakultät für Organishe Chemie und Biochemie

Max-Planck-Institut für Biochemie

Abteilung Strukturforschung

Biologische NMR-Arbeitsgruppe





Pawel Smialowski Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation.

Vorsitzender: Univ.-Prof. Dr. Dr. A. Bacher

Prüfer der Dissertation:

1. apl. Prof. Dr. Dr. h.c. R. Huber

2. Univ.-Prof. Dr. W. Hiller Die Dissertation wurde am 10.02.2004 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 17.03.2004 angenommen.


Parts of this thesis have been or will be published in due course:

Markus H. J. Seifert, Dorota Ksiazek, M. Kamran Azim, Pawel Smialowski, Nedilijko Budisa and Tad A. Holak Slow Exchange in the Chromophore of a Green Fluorescent Protein Variant J. Am. Chem. Soc. 2002, 124, 7932-7942.

Markus H. J. Seifert, Julia Georgescu, Dorota Ksiazek, Pawel Smialowski, Till Rehm, Boris Steipe and Tad A. Holak Backbone Dynamics of Green Fluorescent Protein and the effect of Histidine 148 Substitution Biochemistry. 2003 Mar 11; 42(9): 2500-12.

Pawel Smialowski, Mahavir Singh, Aleksandra Mikolajka, Narashimsha Nalabothula, Sudipta Majumdar, Tad A. Holak The human HLH proteins MyoD and Id–2 do not interact directly with either pRb or CDK6.

FEBS Letters (submitted) 2004.



{I}-S NOE, nuclear Overhauser effect on nucleus S by saturating nucleus I 1D, one dimensional 2D, two dimensional 3D, three dimensional 4FW, 4-fluoro-tryptophan 5FW, 5-fluoro-tryptophan 6FW, 6-fluoro-tryptophan CBCA(CO)NH, 13Cβ-13Cα-(13C')-15N-1HN correlation CSA, chemical shift anisotropy DD, dipole-dipole f, NOE, enhancement factor FID, free induction decay GFP, green fluorescent protein H-D, exchange, hydrogen-deuterium exchange hetNOE, heteronuclear Overhauser effect HNCA, 1HN-15N-13Cα correlation HNCO, 1HN-15N-13C' correlation HSQC, heteronuclear single-quantum coherence kDa, Kilodalton n, number of β-sheets in a β-barrel NMR, nuclear magnetic resonance NOE, nuclear Overhauser effect NOESY, nuclear Overhauser effect spectroscopy ppm, parts per million R1, longitudinal relaxation rate R2, transversal relaxation rate S, shear number in β-barrels T1, longitudinal relaxation time T2, transversal relaxation time

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TROSY, transverse relaxation-optimized spectroscopy ∆σ, anisotropy of the chemical shift tensor ∆υ NMR, line width η, cross-correlation rate of 15N-labeled CSA and 1H-15N dipolar relaxation τC, overall rotational correlation time τm, mixing time ω, Larmor frequency

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1.1 Cell differentiation and cell cycle

1.1.1 Cell cycle

1.1.2 Interplay between cell differentiation and cell cycle

1.1.3 Helix-loop-helix (HLH) protein family

1.1.4 Id – 2

1.1.5 MyoD

1.1.6 CDK4/6 CDK4 CDK6

1.1.7 pRb

1.1.8 HPV16 virus E7 protein

1.2 Photoactive proteins

1.2.1 Function of GFP

1.2.2 α – PEC and photodynamic light sensing in plants

1.3 NMR (Nuclear Magnetic Resonance)

1.3.1 Protein NMR Principles of nuclear magnetic resonance Larmor precession Making Larmor precession of a nuclear spin observable........17 Energy levels, populations & signal-to-noise Nuclear magnetic resonance Relaxation and NMR linewidths Chemical Shifts J-coupling Nuclear Overhauser effect (NOE) Exchange Larger molecules, relaxation and TROSY The NMR spectrum of a folded protein


2.1 Materials

2.1.1 Chemicals

2.1.2 Enzymes and buffers

2.1.3 Kits and reagents

2.1.4 Oligonucleotides Primers for MyoD mutagenesis Primers for MyoD cloning to different vectors Primers for MyoD gen synthesis Primers for Id – 2 cloning Primers for Id – 2 mutagenesis Primers for E7 gene synthesis Primers for E7 mutagenesis Primers for GFP mutagenesis

2.1.5 Plasmids and constructs Plasmids supplied by the companies Plasmids constructed during the work

2.1.6 Bacterial strains

2.1.7 Peptides

2.1.8 Buffers and media LB medium: Minimal medium (MM) for uniform enrichment with 15N.....39 IPTG stock solution: Antibiotics

2.1.9 Antibodies and other proteins Antibodies Molecular weight marker for SDS-PAGE electrophoresis......41 2.1.10 Other chemicals Protease inhibitors: Isotopically enriched chemicals: Miscellaneous :

2.2 Apparatus


1 ÄKTA explorer 10 purification system Chromatography equipment, columns and media:..................43 2.2.2 NMR spectrometers

2.2.3 Other apparatus

2.3 Consumables

2.4 Methods

2.4.1 DNA techniques Basic cloning techniques Isolation of the plasmids Screening of positive colonies Restriction assays DNA sequencing Cloning to LIC vectors Gen synthesis Site directed mutagenesis In vitro protein synthesis Escherichia Coli transformation

2.4.2 Bacterial culturing Bacterial culture in LB medium: Bacterial culture in MM: Medium for selectively enrichment amino acids

2.4.3 Protein production techniques Protein expression in Escherichia Coli strains Expression in Spodoptera frugiperda strain SF9

2.4.4 Protein purification techniques Cell differentiation and cell cycle proteins GFPuv and mutant proteins

2.4.5 Handling and storing of the proteins

2.4.6 Analytical methods Protein detection Pull down assays Analytical gel filtration

V INDEX 2.4.

6.4 Mass spectroscopy Functional assays of cell cycle and differentiation proteins....61 Optical Spectroscopy NMR NMR diffusion measurements NMR relaxation measurements Other methods

2.4.7 Bioinformatics und calculations


3.1 Cell differentiation and cell cycle proteins

3.1.1 The HLH domain of MyoD or Id–2 does not interact with the pocket domain of pRb Results Discussion

3.1.2 There is no interaction between human or chicken MyoD and CDK6 Results Discussion

3.2 Photodynamic proteins

3.2.1 GFPuv Results Discussion

3.2.2 F fluoro tryptophane GFP Results Discussion

3.2.3 α-PEC (α - subunit of phycoerythrocyanin) Results Discussion





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7.1 CDK4/6 structurally guided alignment

7.2 Bioinformatics und calculations

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1.1 Cell differentiation and cell cycle 1.1.1 Cell cycle The cell cycling process is carefully regulated and responds to the specific needs of a certain tissue or cell type. Normally, in adult tissue, there is a delicate balance between cell death (programmed cell death or apoptosis) and proliferation (cell division) producing a steady state. Disruption of this equilibrium by loss of cell cycle control may eventually lead to tumor development (Sherr 1996).

Figure 1.1.

During cell cycle precisely regulated expression of CDK’s (cyclin dependent kinases), cyclins, and CDK’s inhibitors take place. The lines thickness corresponds to the level of activity of given kinases.

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The cell cycle is, therefore, an alteration of two main processes: A) the "doubling" process (S = synthesis phase) where DNA is synthesized, and B) the "halving" process (M = mitosis phase) where the cell and its contents are divided equally into two daughter cells. The periods between these processes are called gap periods (G phase). Taken together, the cell cycle consists of the different phases listed in table 1.1 (Sandal et al. 2002).

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G0* Temporary or permanent state of cell cycle exit. Postmitotic/terminally differentiated *Exit from the cell cycle at G1, not occurring in every cell cycle.

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The switch, or transition, between phases is a hallmark of the cell cycle, with an extremely accurate timing and order of molecular events. However, if something goes wrong, the cell has several systems for interrupting the cell cycle. These are the quality control points of the cell cycle and are often referred to as checkpoints (Elledge 1996). At checkpoints, there are important mechanisms sensing damaged DNA before the cell enters the S phase (G1 checkpoint) or the M phase (G2 checkpoint) (Figure 1.1). One major molecular hallmark of checkpoint control is where transitions turn off the previous state and promote the future state of the cell cycle (irreversible progression). Loss of checkpoint control results in genomic instability, accumulation of DNA damage, uncontrolled cell proliferation, and, eventually, tumorigenesis. Indeed, this has been implicated in the progression of many human cancers (Elledge 1996; Sherr 2000).


Figure 1.2. Network of protein – protein interactions on the onset of G1 phase of cell cycle serve as a precise switch. If cell is stimulated by myogenic signals from environment it allows passing through G1 restriction point. It accumulates outside signals and cause expression of proteins indispensable for DNA synthesis thought S phase. Function and mode of action of selected proteins involved in the cell cycle regulation are described in text.

The G1-S transition is a highly regulated and important transition in the cell cycle. At this stage, the cell cycle passes a point between the G1 and S phase (restriction point) with an irreversible commitment to a new cycle. Stimulation of cells with mitogens results in cell cycle progression. G1 progression requires sustained cyclin D expression, persisting as long as there are ongoing mitogenic signals. Mitogenic transcription activation of D-type cyclins has been shown to be induced, for instance, by c-Myc, AP-1, and NF-κB transcription factors (Ekholm et al. 2000). However, when mitogens are removed, the level of cyclin D rapidly decreases and the cell is arrested in G1. A similar block in cyclin D-kinase activity and subsequent arrest in G1 phase is achieved by the INK4a family of CKIs (Figure 1.2) (Sherr 1996). Four different members of this family (p16INK4a, p15INK4a, p18INK4a, and


p19INK4a) are known to bind and inhibit CDK4 and CDK6, without affecting other CDKs (Sherr 1996). The INK4a-bound CDKs are not able to complex with cyclin, and stay intact as an INK-CDK heterodimer. In contrast, the Cip/Kip family of CKIs (p27Cip/Kip, p21Cip/Kip, and p57Cip/Kip), which do not inhibit the G1 phase, play a positive role by stabilizing the CDK/cyclin complex (Cavenee et al. 1995; Sherr et al. 1999). Cyclins are unstable proteins and their levels vary throughout the cell cycle. This is due to degradation by the ubiquitin/proteasome pathway when they are not required. CDK activity is inhibited by phosphorylation on specific tyrosine residues, and phosphatase treatment leads to a hyperactive kinase (Heichman et al. 1994). Three different mammalian phosphatases are known, Cdc25 A, B, and C. The regulation of Cdc25A is critical for G1 response to DNA damage. The main role of the CDK4/6 / cyclin D complex in the early progression of G1 is to phosphorylate pRb and thereby promote cell cycle progression. The pRb phosphorylation releases E2F and allows the expression of regulators required for DNA synthesis and S phase progression. E2F triggers expression of proteins like dihydrofolate reductase, thymidine kinase, different DNA polymerases and the late-G1 cyclin E (Renan 1993).

Expression of cyclin E establishes a positive feedback loop of pRb phosphorylation, since cyclin E in complex with CDK2 will continue to phosphorylate pRb (Bishop 1996), contributing to an irreversible transition into the S phase and cell cycle progression, even in the absence of growth factors (Harbour et al. 2000). The biochemical events are primarily phosphorylation, dephosphorylation, and ubiquitination, with the overall mission to either prevent or induce a new cell cycle via the pRb pathway.

1.1.2 Interplay between cell differentiation and cell cycle Cell differentiation is crucial for growth and function of any multicellular organism.

In order to execute cell differentiation on the cellular level there has to be interplay between differentiation signals and the cell cycle. The proteins from the HLH family are crucial for coordination of the differentiation processes with the cell cycle. Large body of evidence indicates that protein – protein interactions are important in pathways leading to and from cell cycle.

1.1.3 Helix-loop-helix (HLH) protein family There are two main categories of the basic helix-loop-helix (bHLH) proteins. The class A bHLH, also known as the E proteins, such as those encoded by differentially spliced

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