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terça-feira, outubro 15, 2013

“Close to a miracle”

Researchers are debating whether function or structure first appeared in primitive peptides

By Rajendrani Mukhopadhyay

Proteins traverse the width and breadth of cells to carry signals and cargo from one end to another, package and replicate DNA, build scaffolds to give cells their shapes, break down and take up nutrients, and so much more. But how often do we stop to ask: How did these diverse and sophisticated molecular machines come to be?

Despite proteins' profound impact on life, their origin is not well understood. What caused a string of amino acids to start doing something? Or are strings of amino acids inherently programmed to do things? These are questions with which researchers in the protein-origin field have been grappling.

Researchers have a better grasp of the processes of selection and evolution once a function appears in a peptide. “Once you have identified an enzyme that has some weak, promiscuous activity for your target reaction, it’s fairly clear that, if you have mutations at random, you can select and improve this activity by several orders of magnitude,” says Dan Tawfik at the Weizmann Institute in Israel. “What we lack is a hypothesis for the earlier stages, where you don’t have this spectrum of enzymatic activities, active sites and folds from which selection can identify starting points. Evolution has this catch-22: Nothing evolves unless it already exists.”

Where’s the starting point?

For more than a decade, researchers have been probing the protein-origin question using molecular biology and computer models. The group led by Michael Hecht of Princeton University has made libraries of proteins that are not derived from existing proteins that have undergone millennia of Darwinian selection. Hecht and colleagues made one particular library that contained more than a million polypeptide chains composed of hydrophobic and hydrophilic residues. They demonstrated that, after being expressed in Escherichia coli, the simple polypeptides were capable of folding.

With these folded sequences, Hecht and colleagues next tested if these entities were capable of performing any biochemical function, such as binding small molecules and cofactors and catalyzing reactions. “They don’t do them well, but they do them well above background noise,” says Hecht.

After that, Hecht’s group turned to E. coli strains deleted for genes that provide essential functions for survival. The investigators transformed these strains with their peptide library and found that a couple of their polypeptides were able to rescue the E. coli and let them grow on minimal medium. “Our proteins — made from scratch and never (having) been through evolution — can provide a life-sustaining function,” Hecht says.

In silico experiments complement data from bench-based experiments. Jeffrey Skolnick and Mu Gao at the Georgia Institute of Technology designed homopolypeptides and collapsed them using a structure prediction algorithm. They then selected sequences at random that were proteinlike when matched to folds found in the Protein Data Bank. They found that each cavity in the artificial structures had a match in real proteins. Plus, there weren’t that many cavities. The cavities had the inherent capacity to bind small molecules and other ligands. “You show in a system, which was simply proteinlike but there is no selection for function, that you got a lot of properties — the binding sites, the geometries, the protein-protein interfaces. This would suggest the system fundamentally has the capacity to engage in function. Maybe it’s crummy function, but it’s still function,” says Skolnick. “This is telling you the systems are primed to do biochemistry.”




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