miércoles, 20 de junio de 2018

In-Cell NMR

Download PDF Copy

By P Surat, PhD

The two main techniques to probe the structure of a molecule at an atomic level are X-ray crystallography and nuclear magnetic resonance (NMR). In-cell NMR is used to study the structure of molecules and metabolites inside a cell at an atomic resolution.

Image Credit: nobeastsofierce / Shutterstock

The need for in-cell NMR

X-ray crystallography requires the samples to be a diffracting crystal, while NMR involves investigating the structure of molecules in aqueous solution at room temperature.

Different protein-protein and protein-ligand interactions can be studied using solution NMR. Cryo-EM or electron microscopy is another tool used to study the structures at a great resolution.

However, there is an increasing need to observe and measure properties of molecules in their native state inside the cells, as different procedures of isolation and purification of molecules may alter its state.

Thus, techniques to probe the ‘true cell biological state’ of a molecule inside a cell in its native environment are being studied and employed, and in-cell NMR is just one method which can do this.

In-cell NMR technique

In this technique, NR-active isotopes are expressed or added in the cell. These isotopes include 15N, 13C, and recently 19F is also being used. After expression of these isotopes, the cells are placed in a magnetic field and the resonance frequency, or the frequency at which particular nuclei absorb and re-emit electromagnetic radiation, is measured. This information is used to derive the electronic structure of the molecule.

Expression of isotopes in cells

Prokaryotes

In-cell NMR was started with expressing NMR isotopes inside E.coli cells. The expression of labeled molecules inside cells involves growing them in labeled media, but this generates strong background signals.

Another issue which emerged during early in-cell NMR was that labeled proteins showed a broad resonance, which was difficult to distinguish from one another. One of the reasons for this is that bacteria have a short lifespan after which they burst open. This leads to spilling of the labeled molecules into the media, which then generates broad spectra and huge noise.

Thus, during in-cell NMR the cell has to be intact to prove that the molecues are in their true biological state. In bacteria, isotopic proteins can be expressed by transforming the cells with expression vectors which encode for the protein of interest.

Eukaryotes

As the bacterial systems are not suitable to study eukaryotic proteins, in-cell NMR has been performed in eukaryotic cells has also been developed. However, overexpressing proteins in eukaryotic cells presents several challenges. The main techniques for this purpose are either to overexpress the protein or injection of a protein in to a cell.

Protein injection provides more advantages as the levels of protein can be controlled and thus, background signals generated during overexpression can be reduced. There are two other methods to introduce protein of interest in the eukaryotic cells; one is the use of cell-penetrating peptides, or CPPs, where labeled protein and a peptide which can puncture and enter the cell are conjugated.

Using this method, high quality NMR data was recorded from the cells. Another method is the use of pore forming bacterial toxins which puncture the cell membrane and make it permeable to modified proteins.

Effect of crowding on in-cell NMR

Macromolecular crowding refers to a high concentration of macromolecules in the cytoplasm. This phenomenon leads to changes in the folding properties and weak interactions of proteins, which in turn affect the NMR spectra. It was observed that several proteins which show an intrinsically disordered structure in solution exhibit a partially folded structure inside cells.

Similarly, weak interactive forces between the molecules can also stabilize certain conformations. Thus, crowding can influence the folding landscape and conformation of the protein in in vivo conditions which can lead to changes in in-cell NMR signals.

Studying protein folding and in-cell interactions

In-cell NMR can also be used to study protein-protein interactions and protein maturation. Protein interactions govern the post-translational modifications, translocation todifferent cellular compartments, and degradation of proteins inside a cell. Such protein-protein or protein-drug interactions have also been studied using in-cell NMR.

One research group investigated the various stages of maturation of a protein by measuring the in-cell NMR signatures using the intermediate steps. In-cell NMR has also been used to study protein translocation and the effect of local environmental changes within different sub-cellular compartments on the structure of proteins.

STINT

One of the techniques of in-cell NMR is called STINT, which is used to study the protein-protein interactions. In this technique, the proteins are expressed in a time-dependent manner. . STINT generates a time-lapsed video of in-cell NMR signals to provide information on how the structure of proteins changes in response to protein-protein interactions.

Reviewed by Maho Yokoyama, Ph.D.