Project 1: Learning Tolerance From Proteins

The sequence of a natural protein is just one of a large subset of allowed sequences that result in a functional molecule. While a sequence has been optimized for function, it also must be an evolvable sequence - one that can easily mutate in response to evolutionary pressures without radical perturbation of structure or function. An optimal protein is one that not only is functional but also is well connected to neighboring sequences, giving mutational pathways for adaptation to traverse. Our lab will investigate the extent of these limitations using novel computational protein design algorithms, bioinformatic tools and protein library screening methods. Large scale mutagenesis studies of proteins have demonstrated a remarkable malleability of a protein sequence to change without disruption of structure. This begs the question - if proteins are so accommodating to mutation, why is the de novo design of proteins challenging?
The goal of our research is to understand the molecular underpinnings of mutational tolerance and apply them to problems in protein and drug design. De novo designed proteins with significant sequence plasticity are optimal starting points for engineering functional active sites. Additionally. understanding how mutations accumulate helps predict how pathogens evolve drug-resistance, giving us the opportunity to anticipate viral evolution. We will study sequence malleability in three ways - (1) develop de novo design methods that computationally search the accessible sequences of a given fold for those that are optimally tolerant to mutations, (2) map the mutational tolerance of the small protein signaling domains through high throughput screening of large libraries of mutations and (3) explore the existing sequence variability of HIV protease for estimating the extent of possible mutations in order to abet drug design efforts.

Project 2: Design of Heterochiral Proteins

Tremendous progress has been made in the rational and computational de novo design of proteins with novel structure and function. Many of the tools used in design are parameterized using the wealth of structural information in the Protein Data Bank (PDB). It would be useful to extend these methods to the design of synthetic folding polymers - foldamers. However, it is important to ask the question: How generally applicable is our accumulated structural and physical understanding of protein design?

Examples of aperiodic heterochiral motifs. L-Ala in green. D-Ala in orange

We address this question by developing protein structure inspired computational methods towards the design of novel protein-like molecules where backbone stereochemistry is variable using a protocol that concurrently optimizes both structure and sequence. Nature occasionally makes use of short heterochiral peptides in the design of antimicrobials with novel secondary structures. Using chemical peptide synthesis, it is possible to build much larger molecules, extending the potential for heterochiral peptides to the design of new tertiary folds unprecedented in nature. We will design and characterize novel heterochiral polypeptide folds for stability and structure. The project consists of three major aims:

One aim is to develop computational methods for designing heterochiral peptides using tools derived from de novo protein design. Goals include the development of backbone and sidechain energy potentials for rapid and accurate scoring of potential structures. These potentials will then be used to build a heterochiral peptide fragment library of secondary structure elements. Elements from the library can then be assembled into larger tertiary folds.

Additionally, we plan to explore the rules of capping interactions in heterochiral peptides. In natural proteins, capping interactions stabilize helices and prevent fraying of the termini. A novel bent-helix structure predicted from simulations will be designed and synthesized. Based on modeling studies, a series of hinge capping residues that bridge the bent helix ends will be engineered and evaluated for stability and structural specificity.

Finally, we will focus on the design of novel tertiary folds using our simulation methods. A heterochiral bundle consisting of alpha left and alpha right-helices will be designed and characterized. The design will be extended to the molecular recognition of the Lac-repressor tetramerization domain, with the intention of disrupting the protein-protein interface by competing heterochiral interactions.