State of the Art
To understand protein function and the formation of stable and dynamic protein networks in biological systems, a detailed knowledge of the structure and association dynamics of proteins and their complexes is required. Researchers in this research area of CIPSM are world-leading experts in protein structure and networks. Efforts can be expanded into the most promising future directions, including high-end single-particle cryo-electron microscopy, hybrid methods in structural biology, the analysis of transient multicomponent complexes, robotic genetic screens in yeast at a genome scale, novel mass spectrometry and proteomics techniques, and systems biology approaches such as combined experimental and modelling investigations of bacterial models.
At present, groups of CIPSM investigate interactions between multiprotein complexes that govern essential processes associated with eukaryotic genomes. Multiprotein complexes and molecular machines are involved in processes such as gene transcription and RNA maturation, replication of the genome and chromosome segregation, modification of the genome by DNA recombination, and preservation of the genome by DNA repair (Carell, Hopfner). Detailed insights into the function, structure and regulation of the underlying multiprotein complexes is crucial for understanding processes that govern genome expression and maintenance in normal and malignant cells.
In the future, the interest will however shift to an understanding of the multiple functional and structural interactions between these complexes, which coordinate genome-associated processes in space and time. A combined genetic, biochemical, and structural dissection of protein networks will be carried out. To this end, it will be necessary to bring together expertise and state-of-the art technologies in both, structural biology and protein network analysis, in a unique way. This is not an easy task, but is manageable in the Munich area.
Contributions the groups make at present
Outstanding recent scientific achievements include the X-ray structure solution of a 0.6 MegaDalton RNA polymerase enzyme with substrate nucleic acids and a regulatory factor (Cramer), trapping, structural and functional analysis of ATP-dependent motor proteins in functional states (Hopfner), high-resolution imaging of a programmed ribosome-polypeptide chain complex with an associated regulatory particle (Beckmann), rapid methods for the structural characterization of protein-peptide and protein-nucleic acid complexes and their dynamics (Kessler, Sattler), a structural mechanism for RNA degradation by the exosome (Conti), detailed imaging of nuclear pore complexes within their natural environment in the cell by electron tomography (Baumeister), biochemical and genetic dissection of the protein degradation pathway in yeast (Jentsch), a system biology description of signaling networks in halophilic models (Oesterhelt), and the proteomic characterization of the flux into and out of entire organella by mass spectrometry (Mann).
In the following we will describe in some detail the current contributions the groups make. Half of the groups work on the structure of protein complexes, and thus provide the basis for understanding functional protein networks (Cramer, Hopfner, Beckmann, Kessler, Sattler, Conti, Baumeister). The establishment and study of protein networks is the main subject of investigation of the other groups. Protein networks will be analyzed in ideally suited eukaryotic (S. cerevisiae, Jentsch, Mann, Sträßer; Drosophila, Foerstemann) and prokaryotic model organisms (Jung, Oesterhelt).
The Cramer lab recently made seminal contributions to our understanding of the central engines of gene transcription, the RNA polymerases, and their large functional complexes with nucleic acids and additional proteins. To elucidate the mechanisms of transcription and its regulation, the laboratory mainly uses X-ray crystallography to determine three-dimensional structures of multicomponent complexes. Biochemical and genetic methods provide complementary functional insights in vitro and in vivo. A recent success is the completion of the atomic three-dimensional structure of RNA polymerase II, a 0.5 MDa-molecular machine with 12 protein subunits.
The Hopfner lab studies the structural mechanism of DNA repair networks and nucleic acid quality control. They could recently determine the first crystal structure of a SWI2/SNF2 remodelling factor and its complex with DNA. SWI2/SNF2 enzymes remodel nucleosomes or disrupt other DNA-protein complexes in order to create accessible DNA in many cellular processes such as DNA double-strand break repair and gene regulation. Along another line, the Hopfner lab could recently elucidate the structure and function of the exosome, a large molecular assembly that degrades RNA in RNA quality control, maturation and turnover processes.
The Beckman lab focuses on protein targeting and translocation across membranes. Since large and dynamic macromolecular complexes are at the heart of these processes, they employ cryo-electron microscopy (cryo-EM) with single particle analysis to obtain structural information. Highlights include the structure solution of key complexes of protein sorting. The first structures of the inactive and active 80S ribosome in complex with the translocon revealed the spatial arrangement of the components when acting together. They could also solve the structure of a targeting complex consisting of an active eukaryotic ribosome and signal recognition particle (SRP), and now routinely obtain EM reconstructions at a resolution of 10 Å or better.
The Kessler group developed many tools that make NMR a method of choice for systems which are difficult to crystallize, involving transient interactions or dynamic complexes. NMR allows monitoring changes in structure and conformational dynamics, e.g. induced by phosphorylation or small molecules. The Kessler group used NMR to study the cooperative binding of p53, the gate keeper of the genome which is mutated in 50% of all cancers. A library of 400 fluorinated small organic compounds has been established and used for NMR screening to develop potent inhibitors for the riboflavin synthase using the fragment based approach. The Bavarian NMR center at the TUM contains a large number of high-end NMR spectrometers, such as the 900 MHz NMR spectrometer, which was installed as the third instrument of this type worldwide. Many different proteins, peptides and their interactions have been studied to analyse structure, dynamics and conformational changes in proteins in solution (e.g. studying chaperon interactions with the Baumeister and Buchner group). This collaboration with the groups in area B will be continued.
The Sattler group (supposed successor of Prof. Kessler) has provided unique structural insights into key aspects of molecular recognition involved in RNA interference and spliceosome assembly. As the first reported structural biology of RNA interference they discovered that the Argonaute PAZ domain is a novel fold involved in the recognition of the characteristic 3’ ends of small interfering RNAs. They have also elucidated the structural basis for molecular recognition events during the assembly of the spliceosome.
The Conti laboratory is interested in the molecular mechanisms that govern the transport of nuclear proteins and RNAs from their site of synthesis to their site of function. In particular, they want to understand the cross talk of the molecular machineries that coordinate nucleo-cytoplasmic transport with upstream and downstream processes, such as the links between RNA export and its metabolism.
The Baumeister lab has unique expertise in electron tomography (ET). ET is uniquely suited to obtain three-dimensional (3-D) images of large pleomorphic structures, such as supramolecular assemblies, organelles, or even whole cells. Technological advances (namely computer-controlled transmission electron microscopes and large area CCD cameras) have enabled them to develop automated data acquisition procedures.
The Jung lab has a long-standing interest in stimulus perception and signal transduction of E. coli. The work is focused on the molecular mechanisms of different membrane-integrated sensor kinases, which respond to increased osmolarity, low pH, or increased cell density (quorum sensing). They are the leading group in the characterization of these transmembrane sensors in vitro as they are able to purify and reconstitute these proteins in proteoliposomes in a functional active form.
The Jentsch laboratory has a long-standing interest in protein modification by the ubiquitin system and related pathways. Jentsch cloned the genes for the enzymes of the ubiquitin-conjugation pathway, and found that in yeast ubiquitylation mediates numerous functions, including abnormal protein degradation, heavy metal tolerance, ER-associated protein degradation (ERAD), signal transduction, cell cycle progression, and DNA repair. They also showed that proteasomes are responsible for the degradation of ubiquitylated proteins in vivo, and described how proteins are delivered to the proteasome and identified the crucial factors of this proteasomal escort pathway.
The Mann laboratory is a leading group in the field of proteomics and systems biology. Proteomics is the large-scale characterization of the proteins expressed in a cell, tissue or organism. While there are many different approaches to proteomics, one of the most powerful and most rapidly developing ones is mass spectrometry-based proteomics. The department has a very interdisciplinary background ranging from physics, chemistry and computer science to biology and medicine. At the heart of the scientific interest stands the desire to unravel the dynamic changes in cellular and organella protein composition and networks. Our laboratories have developed methods in quantitative proteomics, which can be used in measuring changes in phosphorylation with single amino acid resolution and on a large scale. They have recently quantified close to 4000 phosphorylation sites in vivo as a response to growth factor signaling.
The Oesterhelt department is devoted to a large extent to systems biology of halophilic archaea. The study of protein structure, protein interactions and protein networking that lead into the new discipline of systems biology are key elements in future developments of the biosciences. Beside the use of genome sequencing, DNA array production and a complete set of mass spectroscopic technologies, the department develops general methods for membrane protein expression, purification, crystallization and structural determination.
The Niessing lab studies motor proteins that are essential for the establishment of cell polarity, unequal distribution and differentiation of cells, and for embryonic development. Recently Dierk Niessing showed that the RNA-binding transporter She2 is a novel type of a RNA-binding protein. Based on this, the laboratory aims to gain a mechanistic understanding of myosin function with a multidisciplinary approach, involving X-ray crystallographic, biophysical, in vitro, and in vivo studies to understand mechanisms of specific cargo-selection, complex assembly, and cargo translocation of myosin cargo-transport complexes.
The Sträßer lab studies coupling events in the eukaryote gene expression pathway. In eukaryotes, essential steps of gene expression occurring after the transcription process itself are the processing of the pre-mRNA and the transport of the mature mRNA from the nucleus to the cytoplasm. Katja Sträßer has recently identified a protein complex that couples transcription to mRNA export, TREX, which consists of nine protein subunits.
The Förstemann lab will be established at the Gene Center during 2006 and will work on small RNA-mediated gene control, which has received widespread attention as a technology to transiently ablate the expression of any gene via small interfering RNA (siRNA) transfection into cultured cells. MicroRNAs play important roles in development, memory formation and potentially carcinogenesis. Klaus Foerstemann discovered that in Drosophila, the novel protein Loquatius is required for assembly of miRNAs into functional RNA-protein complexes and for germ-line stem cell maintenance.