Designing and Evolving New Proteins in the Hecht Lab

Basically everything life does — photosynthesis, metabolism, vision, neural impulses, hormonal signaling, DNA replication — is mediated, at the molecular level, by proteins. Billions of years of evolution have led to millions of different proteins, many catalyzing life-sustaining chemical reactions which could not otherwise occur. But the number of functions natural proteins do have is small potatoes compared to the number they could.

But the number of functions natural proteins do have is small potatoes compared to the number they could.

On the third floor of Frick Laboratory, the lab of Professor Michael Hecht (who many know as Master of Forbes College) is trying to expand what proteins can do by making their own. Proteins are chains of amino acids, whose 20 different side-chains in sequence determine the way that the protein folds. The side-chain of a given amino acid can carry a positive or a negative charge (making it a “charged” side-chain), or both charges (“polar”), or neither (“non-polar”). Proteins fold in water because the hydrophilic amino acids — those with charged or polar side-chains –—prefer to point towards water while the hydrophobic amino acids — those with non-polar side-chains — prefer to point towards each other. This is the same principle as the separation of oil and water, but with the “oil” and “water” molecules strung together on one microscopic chain.McIntosh_Hecht_PNG

If the lab chose sequences at random, most would be unordered and wouldn’t fold, so Hecht wanted to limit the sequences to those which were likely to fold. One of the most common motifs in protein structure is the alpha-helix, where proteins make two turns in the space of about seven amino acid links. Hecht decided to make seven-amino-acid patterns of hydrophilic and hydrophobic amino acids, thus collecting all the hydrophobic side-chains on one side of the helix and encouraging them to clump together to form four-helix bundles.

Professor Michael Hecht

Professor Michael Hecht

“It went from a question about molecules to a question about life — is life unique? Are there alternate versions of life?”

Hecht’s students inserted the DNA for more than a million different patterned proteins into auxotrophs — E. coli with single genes deleted in order to prevent them from growing in a nutrient-poor (“minimal media”) environment. Most of these deleted genes code for proteins which help synthesize nutrients that the minimal media lacks. So when cells with the deletions and the code for the patterned protein grew, it meant that the patterned protein not only folded into a stable, soluble protein — a massive feat in itself — but that the new, synthetic protein performed the reaction that the E. coli needed to stay alive! Hecht still considers this his most exciting result, saying “It went from a question about molecules to a question about life — is life unique? Are there alternate versions of life?” But even though they “rescued” the auxotrophs, these proteins didn’t perform as well as their natural partners — could we do better?

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E. coli expressing varying amounts of GFP

Ryan Kitko

In order to further investigate this evolution from “promiscuous” to specialized proteins, the Hecht lab has recently been examining whether the specialized proteins are more rigid due to more specific, ordered interactions among the hydrophobic side-chains in the core. It stands to reason that proteins which are less flexible in terms of function should have less flexible structures as well, in order to be better fits for the molecules they need to bind for their specific function. Answering this question takes a two-pronged approach. A graduate student in the lab is evolving proteins for stability, rather than function, by attaching green fluorescent protein (GFP) to one end — proteins which fold better are less likely to interfere with the folding of the GFP, so these cells glow brighter under blue light. To look for interactions that are nearly impossible to see in real proteins, a recent undergraduate thesis used computer simulations of a few proteins to track which amino acids seem to be interacting closely, and whether less promiscuous proteins had more interactions.

Another direction the lab is taking also uses computers, this time in designing proteins with a completely different type of secondary structure — “beta-sandwiches”, which involve two “sheets” of alternately hydrophobic and hydrophilic amino acids.

So how do you make a protein do what you want? You mimic evolution — and learn a bit about the very beginnings of life in the process.

About The Author

My time in Princeton is split between Frick labs and running around breaking things (and eardrums) with the Band. Outside the bubble, I hike (OA and otherwise) and write the occasional ridiculous poem.