Genetic selection for functional designed proteins

Main area:Molecular biology
Target group:Biochemistry, Biology, Molecular Biomedicine
Educational level:Masters, Bachelor
Project description:

In nature, proteins are able to take on a huge range of functions both as structural entities and as enzymes, the latter being the most versatile and specific chemical catalysts known. Since the 1960s it has been understood that the protein structure is solely defined by its amino acid sequence. Following this realization, a huge effort has been made to predict protein structure from sequence, however, only recently are the daunting theoretical and computational challenges relating to this problem beginning to yield.

The emerging ability to predict structure from sequence opens the intriguing possibility of approaching the inverse problem of finding an amino acid sequence compatible with a predefined structure. Considering the diverse functional properties of proteins, the theoretical and practical implications of being able to build protein structure at will are tremendous.

Contemporary tools of chemistry and molecular biology allow protein sequences to be routinely generated from synthetic DNA and recent advances in the computational methods have made it possible in a few cases to design completely new proteins with specific and novel structures.

We are currently examining the virtues and limitations of the RosettaDesign software for design and re-design of protein structure. Re-design aims to follow in the footsteps of nature and design structures, from scratch and without involving prior knowledge of specific sequences to obtain a well folded protein of similar structure to that found in nature. On the other hand a long-term goal will be do design sequence that, at will, take up desired structures, not necessarily resembling any found in nature. For the time being, we are focusing on re-design.

In our current re-design project we are testing a large library of designed sequences to analyze the template-dependence for the design software. Some of these sequences fold into proteins with well-defined tertiary structure and we are currently investigating them using various biochemical and biophysical tools.

To be able to efficiently identify folded sequences we are developing a model system for genetic screening and selection for protein fold independent on enzymatic activity of the design target protein. In this system the folding of the target protein is linked to an enzymatic activity which is genetically selectable.

We hope that such genetic systems will enable us to select for improved designs based on the survival of E.coli that carry more efficiently folding and stable designed proteins. Such systems should also be applicable for the improvement of thermal stability of marginally stable natural proteins.

This work is carried out in close collaboration with Martin Willemoes and Kresten Lindorff-Larsen.
Methods used:Molecular cloning and mutagenesis, western blot, genetic selection
Project home page:
Supervisor(s): Jakob Winther