Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - Protein Lego (Protein design from sub-domain sized fragments)

Teaser

Proteins are ubiquitous, very diverse, and participate in virtually every cellular process. Nature has generated this impressive set of proteins using mechanisms such as recombination of smaller, sub-domain sized protein fragments that serve as building blocks in a Lego-like...

Summary

Proteins are ubiquitous, very diverse, and participate in virtually every cellular process. Nature has generated this impressive set of proteins using mechanisms such as recombination of smaller, sub-domain sized protein fragments that serve as building blocks in a Lego-like manner. Based on these observations we propose a rational design strategy in which new functional proteins are build from fragments of existing proteins. With this approach we aim to tackle a long-standing goal in biochemistry, namely the design of complex, custom- made proteins.
Initial experiments that recombine fragments from the same as well as from different folds have been successful in creating new proteins. Moreover, the utilized fragments contribute their unique functional properties to the protein chimeras, which is a tremendous advantage of using existing subunits for the design. Here, we aim to generalize this approach. We will identify common structural fragments and classify them based on their associated functions. We will build stable hybrid proteins from different folds, transfer functional sites associated with particular fragments, and thereby learn about general design rules.
The proposed approach offers a rigorous test for the identification of minimal determinants of protein structure and function. It simultaneously allows us to test our understanding of protein evolution and will have profound implications on the current view of structural classification and interactions. And lastly, the development of a reliable methodology for the design of complex proteins will be very valuable for synthetic biology and bioengineering approaches.

Work performed

Implementation of the proposed actions has progressed as follows in the five project parts:
Project A: Sensitive sequence-based homology searches were performed and a database was set-up of a database with the results. We implemented a web interface and are now running final tests before its release and publication.
Project B: PBP- and flavodoxin-like fold fragments have been combined successfully revealing some needed improvements at the interface. Optimizations in a second-round of designs are currently being tested.
Project C: The evolutionary relationship of HemD and flavodoxin-like proteins has been elucidated and a probably evolutioary path proposed. Hybrid constructs combining both folds have also progressed and function transfer is currently being characterized
Project D: A first round of designs combining two ligand-binding fragments productively are ongoing.
Project E: first routines have been implemented. Scoring is currently being developed.

Final results

We expect to release the Fuzzle database, a global survey of inter-fold similarities in which data can be browsed at different levels of complexity (overview of all folds against all as well as sequence alignments and structural superpositions of individual pairs) and that serves as resource of choosing protein fragments as building blocks for hybrid designs. This will include information on a fragments\' contribution to the function of the natural parent.
We further expect to have shown hybrid designs at different levels of complexity and based on this experience to provide general instructions for hybrid protein design.
In addition computational tools for the selection of building blocks and interface optimization will be provided.