Research Interests
Synthetic Biology is an actively growing field within the life sciences combining elements from biology and engineering with the aim to design and create life forms with new, unprecedented properties and functions. Synthetic biologists have increased the coding potential of several organisms to allow genetic incorporation of additional “unnatural” amino acids into proteins. These unnatural amino acids have unique chemical or biophysical properties or carry naturally occurring (post-translational) modifications and are therefore fascinating new tools to investigate cellular processes.
Past Projects
Genetically encoding post-translational modifications in E. coli
N(epsilon)-acetylation of lysine is a reversible post-translational modification with a regulatory role that rivals that of phosphorylation in eukaryotes. We have developed a general strategy for genetically encoding the site-specific incorporation of N(epsilon)-acetyllysine in recombinant proteins produced in Escherichia coli via the evolution of an orthogonal N(epsilon)-acetyllysyl-tRNA synthetase/tRNACUApair (1). Using this tool we have developed a general method for the production of homogeneously and site-specifically acetylated recombinant histones (2). From these histones we have reconstituted histone octamers, nucleosomes and nucleosomal arrays bearing defined acetylated lysine residues to investigate the impact of these modifications on chromatin structure and function.
Similarly we have devised a strategy to produce proteins containing 3-nitrotyrosine residues (3). This modification is caused by nitrogen containing radicals in vivo and is a marker for various diseases, such as arteriosclerosis or Parkinson’s disease.
Ribosome Evolution
In vivo incorporation of unnatural amino acids by amber codon suppression is limited by release factor-1–mediated peptide chain termination. Orthogonal ribosome-mRNA pairs function in parallel with, but independent of, natural ribosomes and mRNAs. Hence, they have lost their indispensability and have become evolvable. We have evolved an orthogonal ribosome (Ribo-X) with improved tRNACUA-dependent decoding of amber codons placed in orthogonal mRNAs (4). By combining Ribo-X, orthogonal mRNAs and orthogonal aminoacyl-tRNA synthetase/tRNA pairs in Escherichia coli, we could increase the efficiency of site-specific unnatural amino acid incorporation from ~ 20% to >60% on a single amber codon and from <1% to >20% on two amber codons. This technology should minimize the functional and phenotypic effects of truncated proteins in experiments that use unnatural amino acid incorporation to probe protein function in vivo.
Current and Future Projects
The general aim of the lab is the application of tools from Synthetic Biology to the study of protein function.
1) New protein labeling chemistries
The central property of life is its dynamic behaviour. How can we explore the dynamic nature of cellular processes? The temporal resolution of the technology must be of the same quality as the process under investigation. Various types of spectroscopy could be used, i. e. NMR, IR or fluorescence spectroscopy, but the complexity of the sample demands the introduction of site-specific labels into the molecules of interest. Our aim is to use the incorporation of unnatural amino acids to create methods for the site-specific labelling of proteins, which is presently a limiting factor using standard methods of protein chemistry.
2) Chromatin Dynamics
A single human cell contains 2 meters of DNA confined within a tiny nucleus perhaps 10 µm in diameter. Hence, the cell has to condense its genome 200,000-fold while maintaining its accessibility. Nature has solved this packaging problem by wrapping DNA around nucleosomes and into highly compacted chromatin. This, however, imposes a physical barrier to all DNA transactions and a variety of dynamic processes have evolved to deal with this problem. The main biological focus of the lab is the investigation of these dynamic processes by using tools derived from Synthetic Biology, especially the incorporation of probes and post-translational modifications such as unnatural amino acids.
3) Lysine Acetylation
The important physiological role of this post-translational modification is becoming more and more apparent as proteomic surveys reveal a vast number of acetylated proteins in pro- and eukaryotes. The ability to produce site-specifically acetylated recombinant proteins allows the exploration of the impact of this modification on protein function. We are especially interested in the role of lysine acetylation in the regulation of dynamic cellular processes and its underlying mechanisms.
1. Neumann, H., Peak-Chew, S.Y. & Chin, J.W. Genetically encoding N(epsilon)-acetyllysine in recombinant proteins. Nat Chem Biol 4, 232-4 (2008).
2. Neumann, H. et al. A method for genetically installing site-specific acetylation in recombinant histones defines the effects of H3 K56 acetylation. Mol Cell 36,153-63 (2009).
3. Neumann, H., Hazen, J.L., Weinstein, J., Mehl, R.A. & Chin, J.W. Genetically encoding protein oxidative damage. J Am Chem Soc 130, 4028-33 (2008).
4. Wang, K., Neumann, H., Peak-Chew, S.Y. & Chin, J.W. Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion. Nat Biotechnol 25, 770-7 (2007).