Method courses

Analysis of protein transport across mitochondrial membranes

We will generate radiolabelled precursor proteins by in vitro translation and import them into isolated mitochondria. Various analytical tools will then allow us to follow their import and intraorganellar sorting to the respective subcompartiment.

Biophysical characterization of protein stability and molecular interactions by Differential Scanning Fluorimetry (nanoDSF)

NanoDSF is a rapid and precise tool to analyze protein stability and molecular interactions. It requires little amount of protein and can be used for soluble and membrane proteins, for instance for optimization of protein purification protocols, detergent screening or ligand binding analysis. Protein samples can be brought for analysis.

Biophysical characterization of molecular interactions by Microscale Thermophoresis (MST)

MST is a biophysical analysis tool which can be used to characterize biomolecular interactions of proteins, especially protein-protein interactions, in solution. Benefits are the usability for a broad range of binding affinities, little protein consumption, good applicability for membrane proteins, and typically rapid assay development time. Protein samples can be brought for analysis.

Blue native gel electrophoresis

Blue native gel electrophoresis is a powerful tool to separate and characterize protein complexes of biological samples like mitochondria. This technique allows the analysis of the stability, activity and assembly of protein complexes.

Cell-free production of membrane proteins

In contrast to soluble proteins membrane proteins tend to aggregate upon their translation in typical conventional expression systems. We will learn in this course how to overcome this problem using various cell-free translation systems deriving from rabbit reticulocyte lysates or wheat germ lysates. The membrane proteins can be kept in theses systems in competent forms e.g. for insertion into biological membranes or artifical liposomes.

CRISPR-/CAS techniques

Gene knockout using the CRSIPR/CAS system is a powerful technique to silence genes in a quick, reliable and straight-forward fashion. This technique is applicable to various organisms ranging from mammalian cells to bacteria.

Electrophysiology of ion channels and transporters

This theory & hands-on course will focus on protein-mediated ion transport across biological membranes. Particular attention will be given to protein reconstitution strategies and the use of Planar Lipid Bilayer versus Solid-supported membrane electrophysiology techniques. Ion transport will be monitored and selectivity, affinity and pKa profiles will be estimated. Students can bring their own samples whenever appropriate.

The course runs once during the winter semester, at a time to be agreed by the parts involved and once a minimum number of students raise interest in participating.

EPR-spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy is a most useful and important method to study molecular structures and dynamics. Application to biological molecules provides information on chemical and structural changes in course of a reaction.

The course includes: Seminar proving the theoretical background Simulation of spectra to understand their shape Simulation of experimentally measured spectra Introduction to the machine, recording of spectra

The course is carried out upon request, contact your coordinator.

HPLC/MS and GC/MS methods

HPLC is a powerful method to analyze natural compounds. Modern mashines are often connected to UV spectrometer and mass spectrometer. 

In vivo and in vitro localisation of transport proteins

Transport proteins will be stained by fluorescent antibodies. Uptake kinetics and intracellular trafficking will be investigated by confocal microscopy.

In vivo site directed cross-linking methods

Protein-protein interactions drive many cellular processes, but are often only very transient and difficult to identify in vivo. In vivo cross-linking is a powerful method for stabilizing transient and weak interactions in the context of living cells and is also suitable for down-stream processing of samples by mass spectrometry.

Isothermal titration calorimetry to quantify membrane binding of small amphiphilic molecules

ITC has become the gold standard for label-free binding analyses of ligands to receptors. It can also be used for measuring the partitioning of amphiphilic molecules into lipid bilayers, but this requires a special setup and fit model.

Mass spectrometry on transport proteins

Mass spectrometry (MS) is a key technology in proteomic research which enables the systematic identification and quantification of proteins from cells and tissues. Furthermore, advanced MS-based approaches allow for the characterization of post-translational modifications, biomolecular interactions, structures and functions of proteins. In this course, we will introduce modern proteomic workflows as well as explain the operation principles of different MS instrument types and how to apply them for specific research questions. Hands-on exercises on MS data analysis, visualization and interpretation will help to understand the applications, with a focus on membrane proteins and multi-protein complexes.

MD-based thermodynamic calculations

The course introduces classical approaches to biomolecules, such as Monte Carlo and molecular dynamics simulations. We discuss force fields and simulation packages, and methods to compute changes in free energies, such as umbrella sampling and thermodynamic integration.

Membrane dynamics on Giant Liposomes visualized by Confocal microscopy

Synthetic membrane systems, like Giant Liposomes are powerful tools to rebuild cellular processes. Confocal microscopy allows the visualization of receptor protein clustering and membrane invaginations.

Membrane dynamics on supported lipid bilayers visualized by TIRF microscopy

Synthetic membrane systems, like supported lipid bilayers, are powerful tools to rebuild cellular processes. Total internal reflection fluorescence (TIRF) microscopy allows the visualization of protein and lipid clustering, and membrane reorganization in high resolution

Microcalorimetry

Microcalorimetric techniques monitor very small heat responses of a sample after injection of a second component (ITC), during a temperature scan (DSC), pressure change (PPC), or due to intrinsic (e.g., degradation) processes (isothermal calorimetry). The course gives an overview of principles, instruments and applications followed by a short practical training in the lab.

Molecular modeling

The molecular modeling course introduces into computational methods for modeling biomolecules and their interactions. This includes the modeling of protein structures (homology modeling), the modeling of direct interactions (protein and ligand docking) as well as the simulation of the molecular dynamics of proteins, e.g. for modeling of conformational changes of proteins.

Peripheral Peptides and Proteins at Lipid Monolayers

Lipid monolayers are models for membrane surfaces, where the lateral pressure can be controlled. Adsorption of proteins can expand or compress the monolayer depending on the local structure and interaction and on the pre-set lateral pressure.

Protein Structure Determination by X-ray Crystallography

Crystal structures remain the predominant way of investigating and visualizing molecular features in atomic detail. In this method course we have a guided walk-through of a complete structure solution process from a diffraction image to a refined structural model.

Synthetic Biology of Membrane Proteins

Synthetic biology describes the design and engineering of proteins/enzymes with novel properties that are usually not found in nature. It is a very powerful approach to generate novel proteins, delineate evolutionary principles and for understanding the role of protein-protein interactions in biological systems.

The course includes theoretical and experimental parts:

Theoretical Part:

• Background on synthetic biology with special emphasis on membrane proteins
• Recent advances and applications of synthetic membrane proteins.

Experimental Part:

• Experimental design and cloning of genetically fused and truncated version of membrane proteins. As examples a fusion between the P_1B-ATPase type copper transporter CcoI and the periplasmic copper chaperon SenC will be generated. In addition, we will use a reductive approach of synthetic biology by constructing a truncated version of the recently identified cupric reductase CcoG, lacking the periplasmic immunoglobulin domain.
• The functionality of these constructs will be phenotypically analyzed in vivo and in vitro.

Teaching goals: Understanding basic principles for designing synthetic membrane proteins.

Instructor: Dr. Yavuz Öztürk

Duration: 3 days

Participants: 3-4 students

Location: Institute of Biochemistry and Molecular Biology

Time resolved fluorescence anisotropy of membrane probes to monitor membrane order and dynamics

The course gives a short introduction to the principles of fluorescence spectroscopy and its applications in membrane biophysics, covering e.g. steady-state fluorescence spectroscopy, time-resolved fluorescence spectroscopy, anisotropy, quenching, and Förster resonance energy transfer (FRET). Special focus is on time-correlated single photon counting and its advantages over steady-state measurements.