Persönlicher Status und Werkzeuge

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Bei Interesse an einer Bachelor-, Master- oder einer Doktorarbeit wenden Sie sich bitte an Prof. Dr. Langosch.

 


Bachelor/Master Thesis Topics

 

We are looking for students with a relevant background (Biology/Biochemistry/Biology/Biotechnology/Bioengineering, etc.) who are interested in learning or improving programming skills.

As a multi-disciplinary lab, we have several innovative computational projects aiming to understand the interaction or dynamics of membrane proteins. These projects often involve collaboration with the TUM bioinformatic or physics departments.

 

For projects involving bioinformatic sequence analysis, please contact Mark Teese.

For projects involving molecular dynamics simulations, please contact Alexander Götz.

Studentische Arbeiten im Bereich Molekulardynamik

 

 

Forschungspraktikum:


Unequivocal identification of the homodimer interfaces of four human transmembrane domains

 

Background: Single-span membrane proteins take part in countless biological processes. In many cases, their function has been demonstrated to require dimerization via their transmembrane domains (TMDs). There are very few crystal structures of single-pass transmembrane proteins, which limit our understanding of dimerization principles, for instance sequence dependence.

Scanning mutagenesis base on ToxR system (Fig.1) is a commonly used assay to determine TMD dimerization interfaces in vivo [1, 2]. It relies upon an intracellular ToxR domain fused to the TMD of interest, which is expressed in Escherichia coli cells. The dimerization of the TMD region brings the ToxR domains together, which then act as a transcription up regulator, increasing the expression of a reporter gene (e.g. GFP, β-galactosidase, or chloramphenicol acetyl transferase). The expression of the reporter gene therefore correlates to TMD affinity.

Figure 1: The ToxR reporter activator system. The dimerization be driven by TMDs results in the transcriptional activation of ctx promoter in E. coli, inducing the expressing of a gene encoding a reporter enzyme. The amount of reporter enzyme activity is therefore a measure of the strength of the dimerization [3].

 

To find interface residues of known homodimers, each residue is mutated individually, and the ToxR assay used to find “mutation sensitive” positions. This scanning mutagenesis approach identifies key interface residues, but also residues important in secondary structure and membrane insertion. Once the key interfacial residues are identified, however, the exact 3D structure of the dimer can be postulated. The polyansky lab has created an automated program, PREDDIMER [4], that gives as output a number of possible dimer structures. Based on scanning mutagenesis data, the best fitting dimer structure can be chosen.

 

Aim:

Your aim is to determine whether mutation sensitive residues are part of an interface, or instead, are important for secondary structure or membrane insertion.

 

Tasks:

1.    Examine the TMDs and available scanning mutagenesis data

2.    Create further mutations at selected positions

3.    Do ToxR assay to record the effect of these mutants on dimerization.

4.    Check effect of selected mutants on membrane insertion with a “PD28 assay”

5.    Doing the two types of control experiments, western blot and PD 28 assay, to make sure the observed disruption was due to the effect on dimerization.

6.    Analyze data, identify true interface residues.

7.    Choose the most likely dimer structure from the PREDDIMER output.

 

Methods:

-       molecular biology

-       optimized Q5 scanning mutagenesis method

-       primer design and PCR

-       degenerate codon design

-       protein biochemistry

-       amino acid functions, properties and interaction mechanisms

-       protein 2° structure concepts and analysis

-       protein 4° structure concepts and analysis

-       microbiology

-       E. coli -based ToxR assay

-       data analysis

-       python data processing and figure creation

 

Method overview:

Q5 mutagenesis to replace residues at specific positions, measuring strength of dimerization with ToxR assay, confirm positive mutant by DNA sequencing, prove the correct insertion of ToxR proteins with PD 28 assay, check ToxR protein expression level with western blot, analyze data by exist python script.

 

Expected results:

1.    Identify mutation sensitive residues that are not at the interface

2.    Identify new mutation sensitive residues

 

Expected impact:

By distinguishing between different types of mutation sensitive residues, the true interface can be found. This allows us to predict the role of natural human variants in disease, predict the dimer structure of the TMD, and to comment on the general properties of TM interface residues.

 

What we offer:

1.    Excellent training opportunities in a highly collaborative research environment.

2.    We provide a broad range of experimental methods and techniques.

 

Requirements:

1.    TUM student in a related discipline e.g. Molecular biotechnology or biology

2.    Designed for Forschungspraktikum, bachelor student or master student

3.    A good academic background in biochemistry and molecular biology.

4.    Essential lab skills

5.    Good communication and writing skills in English.

6.    Motivation to solve complex biological problems

 

Working Language

English and German

 

Language of Dissertation

English

 

 

References:

1.    Langosch, D., Brosig, B., Kolmar, H. & Fritz, H.J. Dimerisation of the glycophorin A transmembrane segment in membranes probed with the ToxR transcription activator. J Mol Biol 263, 525-30 (1996).

2.    Lawrie, C.M., Sulistijo, E.S. & MacKenzie, K.R. Intermonomer hydrogen bonds enhance GxxxG-driven dimerization of the BNIP3 transmembrane domain: roles for sequence context in helix-helix association in membranes. J Mol Biol 396, 924-36 (2010).

3.    Jan Kirrbach(2012).Mapping the Human Bitopic Membrane Proteome for Self-Interacting Transmembrane Helices.(Doctoral thesis,Technische Universität München , Freising,Germany).

4.    Polyansky, A.A., Volynsky, P.E. & Efremov, R.G. Multistate organization of transmembrane helical protein dimers governed by the host membrane. J Am Chem Soc 134, 14390-400 (2012).

5.    Elazar, A. et al. Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane. Elife 5(2016).