The latest news from academia, regulators
research labs and other things of interest
Posted: Sep 10, 2014
Protein synthesis and folding analysis on a chip
(Nanowerk News) Misfolded proteins can lead to a variety of diseased states, including Alzheimer’s and Parkinson’s. NIM researchers have now developed a method with which one can synthesize hundreds of proteins and analyze their folding properties at once at the single-molecule level ("From Genes to Protein Mechanics on a Chip").
In their natural state, individual proteins are folded up, like crumpled ribbons or tangled spaghetti. Incorrect protein folding can have deleterious effects on cell functioning, which is why protein folding is an active area of biological research.
Together with their team, biophysicists Prof. Hermann Gaub and Dr. Michael Nash (LMU Munich) have developed what amounts to a miniature protein factory. With this platform, they can synthesize proteins and analyze their folding states directly with the help of an atomic force microscope. The whole system fits onto a microchip that measures 1.5 cm x 4 cm.
This new method allows much higher sample throughput than previous methods, and a one-to-one comparison of protein unfolding forces that may only differ by a small amount. Importantly, the system components they used on the cantilever remain stable over thousands of measurement cycles allowing measurement of many different protein types in a single experiment.
The tension rises
Typical folded proteins are only a few nanometers long, but their folding states can be directly measured using an atomic force microscope. To achieve this, the proteins are designed such that one end binds to a glass slide and the other end specifically docks with a protein attached to a nanometer scale cantilever tip. The cantilever is used to apply miniscule forces that pulls the protein apart. When a specific portion of the protein (i.e., a domain) gives way and unfolds, the detector registers a small jolt in the cantilever position. The data obtained in this way are analyzed to determine specific unfolding patterns of individual protein domains.
Until now, this type of measurement was performed in several steps. Proteins must first be produced and purified, and finally fixed onto a glass slide prior to the measurement. For each protein sample, a different cantilever had to be prepared and calibrated. “Using the standard protocols, we are unable to achieve the required measurement precision to, for example, compare protein mutants with only small differences in unfolding forces,” explains Markus Jobst, on of the authors on the paper. “Many measurements had to be repeated in order to obtain sufficient statistical power.”
The Munich biophysicists found a 2-in-1 solution where each step can be performed on a single chip. Each protein is synthesized in one of over 600 dumbbell-shaped reaction chambers on the chip. Each side of the reaction chamber is separated by a micromechanical valve. On one side of the chamber, the proteins are synthesized from coding DNA. The valve is then opened, and the proteins diffuse to the lower chamber where they are bound to the glass surface. After that, the chip is peeled off and a cantilever is used to measure each protein in the array.
The biophysicists could therefore produce many different proteins and measure them with the same cantilever. That means calibration of the cantilever must only be performed once, and small relative differences between proteins can be reliably detected. Currently they have demonstrated this method with 4 different protein types in parallel, however, given that the chip contains over 600 reaction chambers, in the future many more protein types could be measured.
During development of this system, the biophysicists needed to find a solution to the problem of binding each protein of interest to the cantilever. They extended each protein with a short amino acid sequence called a “Dockerin” and attached its binding partner, a “Cohesin”, to the cantilever. Like LEGO bricks, the Dockerin and Cohesin click together, attaching the protein of interest to the cantilever tip. After unfolding the protein domains, the Dockerin and Cohesin break contact and the cantilever is free to move to the next protein spot. The Cohesin remains attached to the cantilever and correctly folded throughout thousands of measurement cycles, enabling repeated measurement of many protein in an array.
“Our next goal is to use this method to measure larger gene libraries,” said Dr. Michael Nash, looking forward to the future. “We are especially excited to learn in how specific mutations affect the mechanical stability of protein domains.”