Protein & Enzyme Engineering

Since about 2000, a major focus of our research has been the selection and engineering of a variety of proteins and enzyme catalysts for new, improved or altered biophysical properties such as their folded structure, their thermostability, their binding specificity and affinity, and (in the case of enzymes) their catalytic abilities and substrate specificity. 

In 2002, we carried out what was (for us) a seminal experiment in which we created a fully active, chimeric homing endonuclease capable of recognizing and cleaving a complementary chimeric target site.  As part of that project, we discovered that computational algorithms being developed David Baker’s laboratory at the UW could be successfully applied to such problems, by repacking a protein interface and creating a novel domain packing architecture.  Subsequently, we (the Baker lab and ourselves in various collaborative mixtures) have used similar algorithms with great success to design a novel protein fold, novel protein-protein heterodimer association specificity, and enzyme catalysts with new activities, specificities, and thermostabilities.

Protein and enzyme structure images
  • 2002: Creation of a fully active, chimeric homing endonuclease (H-DreI), using computational design methods to enable protein interface redesign.  Project carried out by Brett Chevalier (Stoddard Lab) in collaboration with Tanja Kortemme (UW; now at UC-San Francisco) and the Monnat Lab (UW).
  • 2003: Creation of a fully artificial protein fold and sequence without the use of information derived from naturally existing protein homologoues.  Structure solved by Greg Ireton (Stoddard Lab) in collaboration with Brian Kuhlman (now at University of North Carolin) and Gautam Dantas (now at Washington University) 
  • 2004: Redesign of protein-protein binding specificity, using the colicin-immunity protein system (an HNH endonuclease related to phage homing endonucleases, also studied in the Stoddard lab). Structures solved by Lukasz Joachimiak, in collaboration with Tanja Kortemme.
  • 2005: Computational thermostabilization of an enzyme catalyst. Project carried out by Aaron Korkegian in the Stoddard Lab.
  • 2006: Structure-based retargeting of endonuclease DNA cleavage specificity.  Structures solved by Django Sussman, in collaboration with Justin Ashworth (Baker Lab, UW).
  • 2008: de novo catalyst design Structures solved by Lindsey Doyle and Jill Bolduc, in collaboration with Lin Jiang (UW).
  • 2009: Enzyme substrate specificity redesign Structures solved by Jill Bolduc, in collaboration with Paul Murphy (UW)
  • 2010: de novo bimolecular catalyst design.  Structures solved by Abigail Lambert, in collaboration with Justin Siegel (UW; now at UC-Davis)
  • 2012: Metalloprotein design.  Structures solved by Ryo Takeuchi, in collaboration with Sagar Khare (UW).
  • 2013: Design with unnatural amino acids.  Structures solved by Jill Bolduc, in collaboration with Jeremy Mills (UW).  Design of novel ligand binding proteins. Structures solved by Lindsey Doyle, in collaboration with Christine Tinberg (UW).
  • 2014: Design of protein-protein interfaces and protein-based antagonists in cell signalling.  Structures solved by Betty Shen, in collaboration with Erik Procko (UW). Engineering of homing endonucleases for targeted X-chromosome disruption and sex ratio bias in mosquitos, conducted by Lindsey Doyle.
  • 2015: Design of Tyr-tRNA synthetase to require an unnatural amino acid (UAA) for functiuon as part of a biosynthetic containment strategy (structures solved by Ryo Takeuchi in collaboration wth Dan Mandell; Harvard University) and Design of alpha-helical tandem repeat proteins with closed architectures (structures solved by Lindsey Doyle and Jazmine Hallinan in collaboration with Phil Bradley at FHCRC).
  • 2016: Computationally designed high specificity inhibitors of Bcl2 family proteins; in collaboration with Stephanie Berger and Eric Procko in the Baker lab at UW).
  • 2017: Characterization of factors that dictate accuracy in the design of small molecular ligand-binding proteins (structures solved by Lindsey Doyle; in collaboration with Jiayi Dou (Baker lab; UW).
  • 2018: Creation and functionalization of a novel protein fold and development of a new fluorescent protein (with Jiayi Dou and Baker lab; UW);  Creation and functionalization of a large engineered cTRP protein with 24 repeats (with Phil Bradley, FHCRC).

Current and Long-Term Efforts to Continue Protein and Enzyme Engineering Work:

We are currently involved in an ongoing set of collaborations with:

  • David Baker's laboratory at the UW, where we are working on the structural and physical basis of ligand binding affinity and specificity, spanning both small molecular targets and larger protein-protein interactions.
  • Phil Bradley's laboratory at the Hutchinson Center, where we are developing a technology and corresponding set of protein scaffolds termed 'cTRPs' ('circular Tandem Repeat Proteins') which can be used as structural scaffolds for the organization and delivery of bioactive protein subunits spanning a wide variety of symmetries, distances and copy number.