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The Cordes lab “Physical and Synthetic Biology” specializes in the development and application of novel spectroscopy and imaging techniques that allow to map structure and function of biomolecules and (bio)chemical processes in space and time. For this the group uses a combination of optical techniques (single-molecule fluorescence spectroscopy & super-resolution imaging) with nanoscale sensors, i.e., fluorescent probes. The Cordes group follows a question-driven approach to gain insight into the molecular mechanisms of membrane transport and molecular motors (area 1), as well as chemical reactions and catalysis (area 2). Finally we are active in developing fluorescent probes and biophysical assays to characterize (bio)chemical processes and structures in vitro and in vivo (area 3).

 

Research Area 1: Molecular mechanisms of membrane transport

Membrane transport proteins play crucial roles in numerous cellular processes. Despite their importance, all proposed molecular models for transport are based on indirect evidence due to the inability of classical biophysical and biochemical techniques to directly visualize structural dynamics. This lack of data to validate mechanistic models for transport concerns both primary and secondary active transporters. The Cordes group is using single-molecule tools to decipher the molecular mechanisms of transport of these complex machines directly. This novel biophysical research area will support the development of new strategies against pathogenic bacteria or multi-drug resistant cancer cells.

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1) M. de Boer, et al. „Conformational and dynamical plasticity in substrate-binding proteins underlies selective transport in ABC importers”, eLife online (2019).

2) F. Husada & K. Bountra, et al. „Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET”, The EMBO Journal (2018) e100056.

3) A. Aminian Jazi, et al. „Single-molecule Förster-resonance energy transfer using temporal separation of fluorescent signals by caged fluorophores “, Biochemistry 56 (2017) 2031-2041.

4) G. Gouridis, et al. „Conformational Dynamics in Substrate-Binding Domains Influence Transport in the ABC Importer GlnPQ“, Nature Structural and Molecular Biology 22 (2015) 57-64.

 

Research Area 2: Novel approaches to unravel fundamental principles in chemistry and catalysis

We further use our expertise and techniques to understand mechanisms of “classical” chemical and catalytic problems. Especially single-molecule tools might play an important role in understanding the fundamental principles of catalytic activity. Often less than 1% of the molecules in homogenous catalysis or active sites in heterogenous catalysis fully dominate the outcome of a chemical reaction seen at a macroscopic level. Given the importance of mechanistic insights for the development of chemical reactions, it is surprising that the application of single-molecule and single-particle fluorescence microscopy is not yet common. Our group explores the use of sensitive fluorescence microscopy and time-resolved spectroscopy techniques to chemical and catalytic problems and their reaction mechanisms.

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1) Graupner et al. "Photoisomerization of Hemithioindigo compounds: Combining Solvent-and Substituent Effects into an Advanced Reaction Model", Chemical Physics 515 (2018) 614-621.

2) T. Cordes & S. A. Blum: „Opportunities and Challenges in Single-Molecule and -Particle Fluorescence Microscopy for Mechanistic Studies of Chemical Reactions“, Nature Chemistry 5 (2013) 993-998.

3) T.H. Herzog et al. „The photochemical ring opening reaction of chromene as seen by transient absorption and fluorescence spectroscopy”, Photochemical & Photobiological Sciences 12 (2013) pages 1202-1209.

 

Research Area 3: Development of new spectroscopy and microscopy methods

In relation to the mechanistic (bio)chemical focus, the PSB group develops experimental tools based on time-resolved spectroscopy, photophysics and photochemistry.

Probe development, spectroscopy & biolabelling

Fluorescence emission has evolved to an indispensable tool in the life sciences, e.g., as a general contrast mechanism for imaging, biochemical assays, medical screening, or DNA-sequencing. The merits of these applications and their information content are not limited by physical instruments, but by the performance of the employed fluorescent reporters. These intrinsically suffer from transient excursions to dark states limiting signal height and stability as well as from irreversible photochemical destruction (“photobleaching”) that restricts their observation time. The Cordes lab is developing novel fluorophores with self-healing or other functional properties imparted by the covalent linkage of e.g., photostabilizers to the fluorophore. These vastly improved properties have proven to be crucial for advanced fluorescence applications and super-resolution microscopy (STED & STORM). Current projects focus on optimization of chemical linkage of photostabilizer-dye conjugates and their application in live-cells.

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1) J. H. Smit, et al. „On the impact of competing intra-and intermolecular triplet-state quenching on photobleaching and photoswitching kinetics of organic fluorophores“, PhysChemChemPhys 21 (2019) 3721-3733.

2) J. H. M. van der Velde, et al. „Self-healing dyes for super-resolution fluorescence microscopy”, JPhysD 52 (2018) 034001

3) J. H. M. van der Velde, et al. „A Simple and Versatile Design Concept for Fluorophore Derivatives with Intramolecular Photostabilization“, Nature Communications 7:10444 (2016).

4) N. C. Robb, et al. „Single-molecule FRET reveals the influenza A virus promoter RNA in pre-initiation and initiation conformations“, Nucleic Acids Research 44 (2016) 10304-10315.

5) P. Tinnefeld & T. Cordes: „’Self-Healing Dyes’ – Intramolecular Stabilization of Organic Fluorophores“, Nature Methods 9 (2012) 426-427.

 

Novel biophysical assays
 

The Cordes group is using their photophysical expertise to develop novel biophysical tools to answer mechanistic questions in molecular biology. In typical single-molecule assays used for structural mapping of protein complexes, only a single distance is determined per experiment. One of the recent developments used a combination of photophysical (or photochemical) properties to obtain a multi-dimensional assay with higher information content: While a fluorescence resonance energy transfer process (FRET) reports on the distance (changes) between the two fluorophores the novel assay simultaneously allows to detect the presence of and distance to another yet unlabelled biomolecule via protein-induced fluorescence enhancement. Alternatively, we recently explored the use of caged fluorophores for smFRET experiments.

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1) E. Lerner & T. Cordes et al. „Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer “, Science 359 (2018).

2) A. Aminian Jazi, et al. „ Single-molecule Förster-resonance energy transfer using temporal separation of fluorescent signals by caged fluorophores “, Biochemistry 56 (2017) 2031-2041.

3) E. Ploetz, E. Lerner, et al. „PIFE and FRET as synergetic multi-scale molecular rulers“, Scientific Reports 6:33257 (2016).

4) E. Lerner, E. Ploetz, et al. „A quantitative theoretical framework for PIFE-FRET“, Journal of Physical Chemistry B 120 (2016) 6401-6410.

5) J. Hohlbein, et al. „Alternating Laser Excitation: Single-Molecule FRET and Beyond“, Chemical Society Reviews 43 (2014) 1156-1171.