Watching protein fate inside cells
July 16, 2012 § Leave a comment
Biochemists are always seeking new tools that will help them peer inside living cells and see how molecules work their magic in a natural environment. As a step toward this goal, Alexander Shekhtman and colleagues at the State University of New York at Albany recently described how to use nuclear magnetic resonance to analyze a protein’s structure in yeast cells in a paper in the Journal of the American Chemical Society.
Cells are like a bustling metropolis with lots of activities going on inside them all at once. It’s hard to re-create all their activities in a test tube or a Petri dish.
Eukaryotic cells have compartments, much like buildings in a city. These compartments have different environments that can cause proteins to change their structure and perhaps even their biological activity. “The mechanisms that regulate protein transport to and storage in these compartments can provide insight into therapeutic treatments,” notes Shekhtman.
To understand how a protein may change its shape and activity as it passed through different compartments, Shekhtman and colleagues studied ubiquitin, a small protein that targets other proteins for recycling and degradation. The investigators used a technique called in-cell NMR in a yeast strain called Pichia pastoris.
In-cell NMR is NMR spectroscopy done on a particular molecule inside a living cell. The molecule has a special tag, the nuclear spin of a non-radioactive isotope of nitrogen, that can be picked up by magnetic resonance. The tag is on the molecule’s backbone so that researchers can see how the molecule’s structure shifts with changes in environment.
To carry out in-cell NMR in yeast, the investigators overexpressed ubiquitin under different conditions. They discovered that ubiquitin is compartmentalized differently based on the metabolic state of the yeast.
When P. pastoris was grown in medium containing methanol as the sole carbon source, ubiquitin was distributed throughout the cytoplasm and in storage bodies. When P. pastoris was grown in medium containing both dextrose and methanol as carbon sources, the protein was largely restricted to intracellular protein storage bodies.
Shekhtman and colleagues repeated their experiments with another protein, beta-galactosidase. “In this case, by isolating the protein storage bodies and lysing them, we recovered fully functional protein, which implied that the structure of the stored protein was not compromised by compartmentalization,” he says. “Previously, the process of compartmentalization into the proten storage bodies was thought to denature or alter the native structure.” He adds that their data indicate that storage of fully functional protein may be a general phenomenon.
The investigators next will do experiments to study protein interactions in yeast. Shekhtman explains that, by carrying out the experiments with eukaryotic proteins in eukaryotic cells, the investigators can observe natural transcriptional, translational and post-translational processes under normal physiological conditions.