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Biophysik

In unsere Arbeitsgruppe untersuchen wir biologische Makromoleküle, wie z.B. Proteine oder Proteinkomplexe, hauptsächlich mit spektroskopischen oder mit mikroskopischen Techniken. Dabei ist ein wichtiges Ziel unserer Studien, Strukturänderungen von Proteinen während ihrer biologischen Funktion (z.B. enzymatische Reaktionen, Signalübertragung) oder bei der Proteinfaltung zu verfolgen. Die zeitlich aufgelösten Strukturänderungen helfen uns die molekularen Grundlagen dieser Prozesse genauer zu verstehen. Für unsere Studien spielen Fluoreszenzmethoden eine zentrale Rolle, weil diese Techniken die Beobachtung von Einzelmolekülen ermöglichen. Einzelmolekülmessungen spielen immer dann eine zentrale Rolle wenn sich Prozesse nicht synchronisieren lassen und/oder das Ensemble der biologischen Moleküle in unserer Messprobe eine strukturelle Heterogenität aufweist. Im Folgenden sind einige unserer aktuellen Projekte etwas detaillierter dargestellt.

 

 

Introduction: the research field

One of the major goals in biology is to understand the function of proteins and of macromolecular complexes in their cellular context. In order to reach this goal, different and typically complementary measuring techniques have to be applied. Fluorescence based methods offer the possibility to measure protein properties and interactions with a high sensitivity and selectivity. The advent of bright and more photostable fluorescent dyes and an enormous methodical and technical improvement of high resolution fluorescence spectroscopy and microscopy enabled studies on proteins even at a single molecule level. If one wants to measure sample parameters in ensemble, the investigated processes have to be synchronized which is often difficult or some times even impossible to achieve. By employing single molecule studies asynchronous processes (e.g., protein folding) can be studied in more detail. The advantage of this technique is given by the fact that it provides information on the distribution of parameters characterizing the protein. From bulk measurements only mean values of these parameters can be extracted. The most straightforward approach to study biomolecules in the cellular context is to measure them directly inside (living) cells. This approach is often limited by the assortment of fluorescent dyes, by the accessibility of the target molecule, and by the ability to manipulate the sample. Another promising approach is to study proteins outside the cell in an environment which mimics relevant features of the cell. In the case of single molecule methods these (in vitro) assays typically consist of a low concentration of immobilized proteins or protein-complexes performing their biological activity, for example by interacting with their corresponding binding partners. By choosing a proper labeling configuration, conformational changes of the protein of interest can be monitored in an ongoing biochemical process. An increasing number of interesting and promising results demonstrate that single molecule fluorescence methods in combination with in situ measurements on complex sample arrangements provide a powerful tool to elucidate functional details of proteins and biological nano-machines. Our focus in this respect is outlined in the following, where the major topics currently investigated in my group are described in more detail.

 

 

Topic 1: Tracing folding/unfolding pathways of proteins

A.) Multi-domain protein folding

The unique and well defined native structure of most proteins is a fundamental requirement for proper functioning of these biological macromolecules. The ability to build up and to maintain this native structure is an intrinsic property of the polypeptide chain itself, and is already determined by the amino acid sequence. Since the unfolded state of proteins is rather dynamic and structurally very heterogeneous, the folding process itself exhibits a heterogeneity which is inherently difficult to study with classical ensemble techniques. In this respect single molecule techniques became of increasing importance in recent years because they allow for independent analysis of signals from different subpopulations. In particular single molecule Förster resonance energy transfer (smFRET) is a well suited method to study structural and dynamical properties of individual proteins. Förster resonance energy transfer between two fluorophores bound at different positions at one and the same protein can be used to measure the spatial extension of the protein and related changes thereof upon folding or unfolding. In particular studies on single molecule level offer the advantage to characterize unfolded state populations under conditions where the native state is dominating the ensemble. In numerous studies on structural and dynamical properties, freely diffusing or surface immobilized proteins were analyzed with smFRET at different concentrations of chemical denaturants. Most of these studies have been performed with small and often single domain proteins, while only recently larger multi-domain proteins were analyzed in detail. We extended smFRET studies on larger multi-domain proteins by investigating native and unfolded states of phosphoglycerate kinase (PGK) from yeast (Mw: 44.7 kDa).

Fig. 1: Phosphoglycerate kinase labeled with a fluorescent dye in each domain (left panel) and corresponding FRET efficiency histograms for different states during unfolding (right panel).

Here the effect of domain interactions on folding rates and the succession of specific folding events are of particular interest. In order to elucidate details of the folding or unfolding pathways, typically multiple intra-molecular distances have to be analyzed. For multi-domain proteins label positions can be chosen in a way that either, distances within individual domains are measured or in another way that inter-domain distances are monitored (the latter are shown in Fig. 1). In the end both types of distances have to be measured to obtain a full picture of the complete folding process.

In addition to steady state measurements at equilibrium conditions (with freely diffusing proteins, see Fig. 1) we want to perform time-resolved measurements for extended observation times. In this respect PGK is a promising candidate since the respective transitions are rather slow, which in principle allows to detect intermediate states on the corresponding folding pathway with reasonable counting statistics. A detailed analysis of slower or less frequent processes as part of the folding/unfolding transition will require protein immobilization. For this purpose we established encapsulation techniques by using stable unilamellar vesicles (Polymersomes composed of synthetic triblock copolymers) with a size in the order of 100 – 300 nm. With the encapsulation of single proteins in surface tethered nano-cavities we focus mainly on two aspects:

· Monitoring unfolding/refolding transitions of encapsulated multi-domain proteins in the absence of aggregation which otherwise do not refold (e.g. a-amylases).

· Applying time resolved single molecule fluorescence studies using either photo-induced electron transfer (PET) or Förster resonance energy transfer (FRET) to follow structural changes upon unfolding/refolding transitions. The particular advantage of this approach is to watch individual proteins for extended observation times (> ~ms), which is not possible with freely diffusion proteins in a confocal microscope.

 

Fig. 2: Scheme and properties of the protein encapsulation assay.

Applied techniques: single molecule fluorescence spectroscopy (PET, FRET and anisotropy studies), circular dichroism (CD) spectroscopy, time resolved confocal scanning and wide-field microscopy, Fluorescence correlation spectroscopy (FCS)

Methodical developments: We have demonstrated in an application on protein folding, that surface-tethered polymerosomes are suitable to perform time-resolved single molecules studies with encapsulated proteins.

Selected Publications: Rosenkranz et al. (2010) ChemPhysChem, in press; Rosenkranz et al., (2009), ChemBioChem, 10, 702; Fitter, (2009), Cell. Mol. Life Sci., 66, 1672

Collaborations: W. Meier (Universität Basel, Switzerland)

 


B.) Monitoring protein synthesis and co-translational folding

It is well known that protein folding in the cell is very different from a typical in vitro refolding scenario which is generally used for protein folding studies. The major difference is related to the fact that in the cell the nascent polypeptide chain as synthesized by the ribosome starts already to fold during synthesis and chain elongation. Therefore, we started studies on co-translational protein folding using time resolved single molecule fluorescence microscopy. In a first series of experiments we tethered single ribosomes to a surface and monitored the proper functionality of our in vitro transcription/translation system by following the time-course of single in-situ synthesized green fluorescence proteins (GFP) using a wide field fluorescence microscope. The ambitious objective of this project is to follow folding events of individual polypeptides and proteins while they are associated with ribosomes during translation by employing FRET (co-translational folding).

Fig. 3: (Left panel) Schematic view of a 50S subunit of a ribosome tethered to the surface of a cover slide. (Right panel) In the case of GFP synthesis one can measure the time until GFP starts to fluoresce which includes the time for polypeptide synthesis, for protein folding, and for GFP maturation (i.e . chromophore formation).

Applied techniques: Confocal scanning and wide-field microscopy, FCS, FCCS

Methodical developments: In order to watch surface tethered single molecules while performing their biological functions an imaging chamber was used which enabled us to start the process of biosynthesis by filling in a reaction buffer. Concurrently we measured the fluorescence by using a fast laser-based Z-drift compensator in order to keep the beam focused on the glass surface during longer observation times (> 20 minutes).

Selected Publications: Katranidis et al., (2009), Angew. Chem. Int. Edit., 48, 1758, Collaborations: A. Katranidis and G. Büldt (ICS-5, FZ Jülich), K, M. Gerrits & W. Stiege (RiNA, Berlin)

 

Topic 2: How structural fluctuations control enzymatic activity

Biological macromolecules, such as proteins share a structural complexity which is also reflected in a complex dynamical behavior. In particular fast stochastic fluctuations on a pico- and nanosecond time-scale are relevant to overcome energy barriers which are given by the energy landscape of the biomolecules. These fluctuations can determine the kinetics of transitions between conformational (intermediate) states. In addition, these fluctuations contribute significantly to the conformational entropy of a biopolymer, and are therefore important for protein stability, protein folding and protein-ligand interactions. Beside rather unspecific and fast stochastic local fluctuations on the picosecond timescale, in particular for larger proteins with two or more domains, large-scale rearrangements and movements of domains relative to each other are often directly related to numerous protein functions. One example in this respect is given by phosphoglycerate kinase, where large scale motions facilitate the transfer of atomic groups and increase the efficiency of transfer reactions (Fig. 4). We studied the domain movements in the absence and in the presence of substrate molecules and characterized the amplitudes and the characteristic time scales of the corresponding movements. Besides studies using neutron Spin-Echo spectroscopy (performed by Dr. R. Biehl and Prof. D. Richter, ICS-1) we currently employ complementary to this single molecule FRET.

 

Fig. 4: (Left panel) Model of the PGK structure including both substrates (spheres) bound to the enzyme. A hinge-bending movement facilitates phospho-transfer between both substrate molecules. (Upper right panel) FRET efficiency related parameter obtained from intensity based and life time based calculations are displayed for single molecule events (bursts) in a 2D-plot. The displacement of the native state population from the red static FRET line gives information about the amplitude of fast domain movements. (Lower right panel) A diffusion corrected donor intensity autocorrelation reveals the essential time constants of the corresponding dynamical events.

Applied techniques: Microscale Thermophoresis, Dynamic light scattering (DLS), single molecule FRET, FCS

Selected Publications : Fitter (2003) Biophys. J 84, 3924; R. Inoue et al., (2010) Biophys. J, 99, 2309

Collaborations: R. Biehl & D. Richter (ICS-1, FZ Jülich)

 

 

Topic 3: Membrane protein interactions studied in lipid bilayers

The cell membrane is not only physically separating intracellular organelles and the extracellular environment but also facilitate diverse cellular functions. For example specific proteins embedded in the cell membrane can act as receptors that allow cells to communicate with each other. To fulfill these functions often larger oligomeric protein complexes need to be formed in the cell membrane or cytosolic proteins have to be anchored to the membrane. Instead of studying theses processes directly in the cell, which is often not possible, we perform studies with giant unilamellar lipid vesicles (GUV) which mimic a cell membrane to a certain extent. The most important characteristics of a GUV is given by the large size (~10-40 mm) which allows studies with extreme low protein concentrations (molar protein/lipid ratio of about 1:1,000,000) and special application with confocal microscopes, and the fact that the diffusion of integral membrane proteins as well as protein-protein or protein-lipid interactions is almost unperturbed.

Applied techniques: fluorescence spectroscopy (FRET), time resolved absorption spectroscopy, time resolved confocal fluorescence spectroscopy and wide-field imaging, dual-focus FCS.

Methodical developments: A photoreceptor (NpSRII) and its related transducer (NpHtrII) were incorporated into the lipid membrane of giant unilamellar vesicles (GUV). Intermolecular binding of both membrane proteins was estimated by measuring the lateral diffusion of fluorescently labeled proteins using dual-focus fluorescence correlation spectroscopy (Fig. 5).

Fig. 5: (A) Schematic side view of a surface tethered GUV with a laser beam focus (blue) intersecting the GUV. (B) Obtained two-dimensional diffusion coefficients are given as a function of the cylindrical radius of the diffusing particles (in particular the photoreceptor SRII and transducer HtrII). In blue the diffusion coefficient of the formed complex (HtrII-SRII) is shown.

 

Selected Publications: Enderlein et al. (2004), Current Pharmaceutical Biotechnology 5, 155; Kriegsmann et al. (2009), BBA-Biomembranes, 1788, 522; Kriegsmann et al., (2009), ChemBioChem (2009),10, 1823-1829

Collaborations: M. Engelhard (MPI, Dortmund), J. Enderlein (Universität Göttingen)

 

 


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