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Project Y

Project Y

 

 

Name:

Prof. Dr. D.J. Manstein

 

Institution:

Department of Biophysical Chemistry

 

Telephone:

0049-511-532-3700

 

Email:

mansteinmpimf-heidelberg.mpg.de

 

Research focus:

The objective of my work is the identification and characterization of molecular motors and proteins that regulate dynamic changes of cytoskeletal and membranous structures. The coordinated generation of movement and force is essential for basic processes such as cell division, phagocytosis, chromosome segregation, muscle contraction, and axonal transport. Elucidation of the molecular mechanisms underlying motile events is of significance with respect to health related issues, such as cell-mediated immune response, wound healing, and the invasion of healthy tissue by malignant tumor cells.

 

Using transient kinetics in combination with cell biological, molecular genetic, and structural approaches, current work has led to the complete structural and functional characterization of several unconventional myosins, dynamin-related proteins, and regulators of small GTPases. Collaborative projects are in progress on Chara corallina myosin XI, the role of PEVK repeats in titin, the characterization of a novel microtubule binding protein, and the development of a high throughput method for the determination of atomic structures by X-ray crystallography.

 

Project:

Work on dynamin will be focused on establishing a direct functional assay for dynamin activity, determination of the X-ray structure of full length dynamin, improvements of the current EM reconstructions of the dynamin ring complex by the use of cryo-methods (collaboration with Dr. Dean Madden, Dartmouth), generation of an atomic model of the dynamin ring complex by combining the high-resolution X-ray crystallography data and the EM maps. The Dictyostelium work is complemented by work on mammalian dynamin, which has led to the crystallization of the full-length protein and a complete native data set has been collected (collaboration with Drs F. Jon Kull, Dartmouth)

 

Work on molecular motors is the extension of a long-standing, highly successful program on characterising the fundamental properties of myosin motors using Dictyostelium myosin II as the archetypical motor. Site-directed mutagenesis and transient kinetics were combined to dissect essential features of the myosin motor like actin binding, nucleotide, binding, and coupling of conformational information between different functional regions of the motor. Engineering principals were applied to the study, design and generation of biological motors. This work has led to the development of tools and reagents that facilitate the expression, purification, and molecular genetic characterisation of complex molecules such as myosin and dynamin family members.

 

Myosins and myosin fragments were specifically designed and optimised for use in kinetic, structural, and cell biological studies. The engineered step size-change of myosins with artificial amplifier domains, consisting of spectrin-like repeats, marked a significant advance in our ability to selectively modify the functional properties of molecular motor. In particular, the artificial amplifier domain work opens the way for the design, production and characterization of mechanically competent constructs of members of the >17 classes of unconventional myosins. The overproduction of unconventional myosins is otherwise difficult and has rarely been achieved. So far, we have produced five members of myosin classes I, VII, and XI in Dictyostelium. Functional constructs for the functional characterization of the myosins in vitro and in vivo were obtained in each case. The concept of producing single peptide motors with artificial amplifier domains was tested with three unconventional myosins. All three were produced in a functional form, moving F-actin with velocities in the range from 0.2 - 2 m/s. The constructs were successfully used to dissect the effects of Ser/Thr phosphorylation in a surface loop in the actin-binding site (TEDS-site) on motor activity.

 

Modified motor proteins are potentially useful for nano- or biotechnological applications. To demonstrate that motor proteins are truly contenders for such applications, key functional properties, such as processivity, direction of movement, and interaction with polymeric tracks are being addressed in my laboratory and many others. Uniquely, the approach I developed, of linking extended, rigid amplifier domains to molecular engines and enzymes in general allows single molecule mechanical measurements of conformational changes associated with transitions between states.

 

As more and more genomes are sequenced, there is a growing backlog of high-resolution crystal structures that need to be determined. The myosin fusion system that was developed in my laboratory is likely to be useful in this endeavour. The 2.8 Å structure of two actinin repeats and the 2.3 Å structure of a dynamin GTPase domain was solved via molecular replacement using only the 761 amino acid myosin catalytic domain. Therefore, the 240 amino acid residue actinin and 315 residue dynamin structures are entirely free of model phase bias normally introduced when using molecular replacement methods. As large quantities of myosin fusions can be expressed and easily purified in Dictyostelium, and as these fusion proteins tend to crystallize readily, we are applying this system to other small or medium sized proteins and protein domains in order to determine their three-dimensional structures.

 

Genetic and biochemical techniques are used to identify new interaction partners for motor proteins, proteins involved in membrane trafficking, and centrosomal proteins. To accelerate the process of identifying new interaction partners for proteins of interest, a myosin-fusion-system for isolating interacting proteins or protein binding partners was developed. A cDNA-library, fused to the 3'-end of DNA encoding a myosin motor domain (MMD), is generated. Expression-vectors were created for the production of the resulting fusion constructs in Dictyostelium and other eukaryotic system. After the transfer of clonal transformants to microtiter-plates, the MMD-fusions are purified to homogeneity by a series of simple pipeting and wash steps. Next, the isolated proteins are probed with the bait-protein of choice fused to a reporter protein like luciferace or -galactosidase. The myosin-fusion-system gives fast and easy access to the purified protein from a small number of cells (typically ~105), all steps can be automatized, and it is thus ideally suited for high-throughput screens. On a smaller scale, the system can be employed to verify interaction partners detected in 2-hybrid screens.

 

Total internal reflection fluorescence microscopy (TIRFM) provides a powerful tool for the identification of interacting proteins and the basis for the development of new in vitro assay systems for cytoskeletal proteins and proteins involved in membrane trafficking. Single Cy3-ATP and GFP molecules can be visualized in our TIRFM set-ups, which enabled us to extend the kinetic characterization of myosins and dynamins to the single molecule level. The single molecule approaches are complemented by transient kinetic methods for the study of protein-protein and protein-ligand interactions on the ms - sec time scale. These transient kinetic methods can be applied to a broad structure function study of the members of the myosin family and a large range of other proteins.

 

Group members:

Prof. Dr. D.J. Manstein, Prof. Dr. J. Alves, Prof. Dr. C. Urbanke, PD Dr. H. Wolfes, Dr. E. Korenbaum, Dr. I. Chizhov, Dr. R. Kownatzki, Dr. U. Curth, Dr. J. Greipel

 

Methods:

  • Steady state and transient kinetics
  • Protein production and purification
  • Protein design and engineering
  • Multiphoton laser scanning microscopy
  • Video-enhanced 5D-microscopy
  • Evanescent field multicolor fluorescence microscopy for single molecule detection.
  • Analytical ultra-centrifugation.
  • Light scattering o Spectroscopic techniques

 

Key References:

  1. Kollmar, M. Dürrwang, U., Kliche, W., Manstein, D.J., & Kull, F.J. (2002) "Crystal structure of the motor domain of a class-I myosin". EMBO J. 21, 2517-25
  2. Klockow, B., Tichelaar, W., Madden, D., Niemann, H.H., Akiba, T., Hirose, K., & Manstein, D.J. (2002) "The dynamin A ring complex: molecular organization and nucleotide-dependent conformational changes". EMBO J. 21, 240-250.
  3. Niemann, H.H., Knetsch, M.L.W. Scherer, A., Manstein, D.J., & Kull, F.J. (2001) "Crystal structure of a dynamin GTPase domain" EMBO J., 21, 5813-5821.
  4. Toomre, D. & Manstein, D.J. (2001) "Lighting up the cell surface with evanescent wave microscopy" Trends Cell Biol., 11, 298-303.
  5. Knetsch, M.L.W., Schäfers, N., Horstmann, H. & Manstein, D. J. (2001) "A Bcr/Abr-related protein controls the organization of a tubolocisternal organelle, F-Actin dynamics, and cytokinesis in Dictyostelium" EMBO J., 20, 1620-1629.
  6. Ruff, C., Furch, M., Brenner, B., Manstein, D.J. & Meyhöfer, E. (2001) "Single-molecule tracking of myosins with genetically engineered amplifier domains" Nature Struct. Biol., 8, 226-229.

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