Research
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The approaches that the group uses to address the complex problems presented by Nature are necessarily interdisciplinary.
Some of our thinking remains chemical, but we use techniques taken from molecular biology and protein chemistry to biophysics as well as mechanistic organic chemistry or microengineering.
Enzymes are a prime example where a better understanding is necessary to pass the most severe test: that of making catalysts rivalling the efficiency of natural systems. Enzymes define the chemistry of life and have evolved to perform biological tasks with exquisite specificity and are amazingly efficient. Biomolecules are processed in seconds by enzymes, although their reactivity may be a million years. This tremendous power of enzymes corresponds to huge rate accelerations of over 1021. However, despite much progress we still have no comprehensive understanding of enzyme action – and can rarely reproduce their power.
To this end we study natural enzymes to delineate their mechanisms in detail.
We also make and analyse synthetic enzyme models, e.g. derivatised polymers that tell us how primordial enzymes may have been assembled.
In addition to mechanistic studies in the conventional way, where we look in great detail at one system, we are also getting accustomed to the synthesis, assay and analysis of large molecular ensembles. We try to design such combinatorial experiments so that mechanistic insight can be gained by systematic analysis. Especially in the biomedical sciences, where an enormous diversity of genes or proteins is involved such an approach can be the only way to find out the rules in complex descriptor spaces.
For example, in directed evolution libraries of in proteins or polymeric enzyme models are employed to give mechanistic insight into how enzyme catalysis is brought about. Similarly problems like the molecular recognition features of drug delivery reagents for DNA transfection lead to insight into the cell biology of such processes. In all these projects it will be the systematic analysis of the enormous amounts of data generated that will ultimately lead to an understanding of fundamental chemical and biological problems.
Many projects have a strong technical component, because considerable technical challenges are involved in the way diversity is brought about and probed. One example is a new high-throughput screening system in which emulsion microdroplet reactors with picolitre volumes are handled in microfluidic devices.
We hope that this platform technology will enable us to characterise libraries with similar precision to the macroscopic scale.
A number of projects are undertaken in collaborations with partners in Cambridge and elsewhere. For example, we study methods to use microfluidic droplets as picolitre reactors for chemical and biological experimentation (http://www-microdroplets.ch.cam.ac.uk) with collaborators at the Chemistry Department and at Imperial College. European collaborations exist in the context of Marie-Curie networks, e.g. ENEFP (on directed evolution), ProSA (on protein-protein interactions) or PhosChemRec (Recognition and Cleavage of Biological Phosphates).
Selected literature:
Catalytic promiscuity
• Olguin et al.; Efficient Catalytic Promiscuity in an Enzyme Superfamily: An Arylsulfatase Shows a Rate Acceleration of 1013 for Phosphate Monoester Hydrolysis, J. Am. Chem. Soc., 2008, 130, 16547–16555.
• Babtie et al. F. Hollfelder; Efficient Catalytic Promiscuity for Chemically Distinct Reactions, Angew. Chem., 2009, 81 (1), 302-306.
• B. Villiers and F. Hollfelder; Mapping the limits of substrate specificity of the adenylation domain of TycA, ChemBioChem, 2009, 10 (4), 671-682.
Enzyme Mechanism
• Baxter et al. MgF3--Formation in an Enzyme Active Site: A Trojan Horse Transition State Analogue, 2006, Proc. Nat. Acad. Sci., 103, 14732-14737.
• S. Jonas et al A New Member of the Alkaline Phosphatase Superfamily With a Formylglycine Nucleophile: Structural and Kinetic Characterization of Phosphonate Monoester Hydrolase/Phosphodiesterase from Rhizobium leguminosarum. J. Mol. Bio., 2008, 384,120-36.
Gene transfection
• Hufnagel et al. Fluid Phase Endocytosis Contributes to Transfection of DNA by PEI-25. Mol. Therapy 2009, 17(8):1411-7.
Enzyme Models
• Avenier, F.; Hollfelder, F. Combining Medium Effects and Cofactor Catalysis: Metal-coordinated Synzymes Accelerate Phosphate Transfer by 108. Chem. Eur. J. 2009, available online
Directed Evolution
• V. Stein, I. Sielaff, K. Johnsson and F. Hollfelder A Covalent Chemical Genotype-Phenotype Linkage for in vitro Protein Evolution, 2007, ChemBioChem., 2191-4.
• Villiers, B. R. M.; Stein, V.; Hollfelder, F., USER friendly DNA recombination (USERec): a simple and flexible near homology-independent method for gene library construction Protein Eng Des Sel 2009
• Stein, V.; Hollfelder, F. An efficient method to assemble linear DNA templates for in vitro screening and selection systems. Nucleic Acids Res 2009 available online
• H. Leemhuis, K. P. Nightingale and F. Hollfelder Directed Evolution of a Histone Acetyltransferase: Enhancing Thermostability, whilst Maintaining Catalytic Activity and Substrate Specificity, 2008, FEBS Journal, 275, 5635-47.
Microfluidic Droplets
• Schaerli et al. Continuous-flow Polymerase Chain Reaction in Microfluidic Microdroplets: Amplification of Single DNA Copies, Anal. Chem. 2009, 81, 302-6.
• Huebner et al. Quantitative detection of protein expression in single cells using droplet microfluidics. Chem Commun 2007, 28(12):1218-20.
• Shim et al Simultaneous determination of gene expression and enzymatic activity in individual bacterial cells in microdroplet compartments. J. Am. Chem. Soc. 2009, available online
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