Protein Folding in vivo

The vast majority of proteins cannot reversibly refold, meaning that thermodynamics is insufficient to explain how proteins assemble complex molecular architectures from primary amino acid sequences. Instead, cells combine the intrinsic physical properties of nascent protein chains to self-assemble with many levels of biological regulation such as codon usage, translational timing, and RNA/protein features that cue chaperones at specific times. We want to watch proteins fold and observe their intermediates in vivo in order to understand how physics and biology work together to build the world’s most proficient catalysts and molecular machines.

This figure illustrates a number of different ways in which additional 'instructions' in a biological system could help a protein find its native state. mRNA can introduce local traffic controls using fast codons or secondary structures, whilst higher order structures created by operons or the polysome could generate further instructions to individual ribosomes constructing a nascent chain. We are curious to characterize these features and potentially discover unknown mechanisms directing protein folding.

These studies are additionally motivated by a desire to understand the many genetic diseases which are caused by synonymous mutations: mutations which do not change the primary sequence of a protein, but which tamper with instructions to the protein synthesis machinery and can result in a misfolded products. This category includes cystic fibrosis, haemophilia, Celiac’s disease, among others. It is further motivated by the need to understand how cells keep proteomes properly folded (i.e., maintain proteostasis), whose disregulation lead to cancer and ageing.

Cross-Linking Mass Spectrometry

One technique that is able to provide structural information of biomolecules in living cells is cross-linking mass spectrometry (XL-MS). We apply XL-MS to problems in protein folding, but we are also interested in developing new methods and technologies in this growing field of analytical biochemistry which can serve our own research interests as well as those of the broader biochemical community. Cross-linkers enable structural information to get “frozen” into protein molecules through covalent bonds; then next-generation mass-spectrometry using high accuracy mass-analyzers and automated spectral data analysis enables the identification and quantification of cross-linked fragments, pinpointing which amino acids have become cross-linked together. These data provide a contact map, which can act as a blue-print for developing structural models.

We are especially interested in photo-activatable cross-linkers and cross-linking amino acids that can be incorporated into nascent proteins co-translationally.

By inserting cross-linkers in specific locations, initiating cross-linking at specific times, and synchronizing cross-linking to other processes in vivo we can obtain dynamic and functional information about protein structures in their native contexts, in a way that no other structural technique can provide.

A Loopable Translator

Biological translation occurs in a vectorial fashion, from a start codon until a stop codon, which fundamentally limits the potential chain length a protein can reach. In order to design programmable protein materials consisting of long chains with hierarchical structures, we are endeavouring to create a new modality of protein translation that would resemble a computer program. The key element of a computer program is the loop: the ability to perform a set of operations a specific number of times, or until a control event occurs. Analogously, a ribosome that can translate a specific mRNA region a specific number of times before moving on to a new stretch of mRNA would enable the synthesis of large proteins with complex, hierarchical sequences, and would remove the size and repetitiveness constraints placed on proteins by the natural modality of translation.

Novel Fibrous Proteins

By developing a loopable translator that operates in vivo, we are opening the doors to evolve and create new materials in a manner that is currently only facile for small globular proteins.

In particular, we envision the possibility of designing new classes of materials based off "protein meshes." Such materials comprise of micron-length fibrous domains interwoven with control elements (globular domains) that facilitate long-range intra-chain contacts to create a specific three-dimensional object. Because proteins can be secreted co-translationally, we further envision designing microbial communities secreting such proteins as they are being synthesized, which in turn, interact and assemble with one another outside the cells synthesizing them. In this way, bacteria could collaborate to "nanolathe" a macroscopically-sized gene product.

Protein meshes are biological counterparts to MOFs, but have the additional advantages of genetic encodability (so they can be evolved), living assembly (so they can be autonomously controlled by gene circuits), and environmentally sustainability (because they can be readily disassembled/recycled).

Our long-term goal is to be able to eventually replace non-sustainable consumer materials derived from petrochemicals (an increasingly worrying burden on the environment) with protein-derived materials. Protein-based materials would enable society's material culture to be in greater alignment with natural biological cycles, allowing "refuse to become resource."