Proteins can be read with a molecular motor

Individual proteins can be read down to the smallest building block by pulling them like a wire through a nano-opening. Proteins consist of a series of amino acids strung together. The sequence of amino acids determines the function of a protein. Researchers at TU Delft have developed a method to determine this sequence. In doing so, they reused a method that had previously been developed to analyze DNA molecules. The research popped up Thursday in Science.

Proteins are among the most researched biological molecules. However, figuring out the basic structure is still difficult. This is mainly due to the great mutual variety and complexity of proteins and amino acids. There are twenty different amino acids and an amino acid can be positively, negatively or neutrally charged. And proteins are always folded and rolled into complex structures, which you first have to untie completely. Moreover, long after proteins are made, they can get unexpected molecular groups stuck to them. Because of these ‘post-translational’ modifications, biochemists have even less insight into what proteins look like in the cell.

Typically, researchers use a mass spectrometer to determine the amino acid sequence of protein fragments. However, the device costs tons, and it requires expertise to find the amino acid sequence of a complete protein. In addition, large amounts of a single protein are needed. As a result, the device lacks all kinds of rare proteins, which occur in smaller quantities.

Hole in a thin membrane

The Delft researchers made a ‘nanopore’ – a hole of a nanometer in diameter in a thin membrane – through which they pull an unrolled piece of protein thread of 25 amino acids. The researchers measure the extent to which each amino acid blocks the hole – that affects how many other particles can still flow through. The researchers want to map out which degree of blockage is appropriate for which amino acid.

A comparable nanopore has already been used to analyze a DNA molecule, but with proteins it was difficult to pull a strand through the pore slowly enough. The Delft researchers solved this problem by sticking a piece of DNA to the end of the protein. A molecular protein motor uses that DNA as a handle to pull the piece of protein through the hole. This is done step by step, amino acid by amino acid, at the level of a single molecule.

“A major breakthrough,” Giovanni Maglia calls the study. He is professor of chemical biology in Groningen. “What may be the crucial problem in the field has now been solved: the precision with which a protein moves through the nanopore. With this method, I expect that whole proteins can be scanned in the near future. All the puzzle pieces are there.”

A limitation of the new method is that it can only work with proteins up to 25 amino acids in size – only a fraction of the size of most proteins. Still, this is an improvement over the mass spectrometer, which works with even smaller fragments. Moreover, 25 amino acids is already enough to distinguish all human proteins from each other. “I really think this is a breakthrough technology,” says Cees Dekker. He is a university professor at TU Delft and supervisor of the research. “I am also very optimistic that we will be able to distinguish post-translational modifications. We are working on that in follow-up research.” TU Delft has applied for a patent for the invention.