Seeing Proteins Inside Cells

IN-CELL nuclear magnetic resonance spectroscopy (NMR) offers great promise for studying proteins in their natural biological environment—the cell. Two research groups in Japan have now boosted the method's prospects by using it to obtain the structure of an in-cell protein and to study proteins in mammalian somatic cells, both for the first time.

The studies could open the door to a broader understanding of how proteins perform their biological functions inside cells, in both health and disease.

Volker Dötsch, now at the University of Frankfurt, and coworkers developed in-cell NMR as a way to study proteins at work (J. Am. Chem. Soc. 2001, 123, 2446). A wide range of studies have since been carried out on the conformations, dynamic motions, and binding interactions of in-cell proteins. But no one has been able to obtain NMR spectra of proteins in mammalian somatic cells or solve the NMR structure of an in-cell protein.

Yutaka Ito of Tokyo Metropolitan University and coworkers have now obtained the first three-dimensional protein structure determined exclusively from NMR data obtained from living cells. They find that the structure of the metal-binding protein TTHA1718 in bacterial cells is slightly different from that in vitro (Nature 2009, 458, 102).

Obtaining in-cell NMR structures "will be especially important to study protein conformations that you cannot isolate in vitro," such as in-cell forms of protein oligomers that may play a role in Alzheimer's disease, Dötsch comments.

A drawback of NMR is that it is a low-sensitivity technique, so structure determination requires days of analysis, whereas NMR-analyzed cells stay alive for only a few hours. To obtain their structure, Ito and coworkers reduced analysis time significantly by using techniques such as nonlinear sampling, methyl group labeling, and automated structure calculation.

In addition, Hidehito Tochio and Masahiro Shirakawa of Kyoto University and coworkers have accomplished the first in-cell NMR study of proteins in mammalian somatic cells (Nature 2009, 458, 106). In-cell NMR has been limited to prokaryotic cells, which can be engineered to express labeled proteins at NMR-analyzable levels, and to frog eggs, which are big enough to be injected with labeled proteins.

Mammalian somatic cells can't express proteins at high levels and are too small for injections. So the researchers labeled the proteins they wanted to observe and linked them to cell-penetrating peptides, which carry them through cell membranes and release them in the cell. Their study shows how FKBP12 protein interacts with immunosuppressants and that ubiquitin exchanges protons more quickly inside cells than outside.

The two papers' advances "were inevitable," says former Dötsch grad student Zach Serber of Amyris Biotechnologies, in Emeryville, Calif., "but someone had to roll up their sleeves and do the hard work to make it possible." And the two Japanese teams have now done that.