In-cell NMR spectroscopy
Introduction
Interactions between biological macromolecules give rise to and regulate biological activity. This activity is manifest through structural dynamics and changes in the macromolecular structures that comprise these interactions [1], [2], [3]. Until recently, mostly in vitro techniques have been used to study macromolecular interactions that govern biological processes under conditions remote from those existing in the cell [4]. With the advent of in-cell Nuclear Magnetic Resonance (NMR) spectroscopy [4], these processes can now be studied within a cellular environment.
In-cell NMR spectroscopy provides atomic level resolution of molecular structures under physiological conditions. NMR-active nuclei in biological macromolecules are extremely sensitive to changes in the chemical environment resulting from specific and non-specific binding interactions with ions, small effector ligands and macromolecules as well as from changes due to biochemical modifications. These interactions alter molecular surfaces and may result in tertiary and quaternary conformational changes, all of which are reflected by changes in the chemical shifts of these nuclei. Thus, by performing NMR spectroscopy on living cells, we can begin to understand the structural underpinning of biological activity.
As the field of in-cell NMR spectroscopy has progressed, the severity of early concerns regarding the validity of in-cell NMR for studying biological macromolecules has abated. Chemical shift differences between the resonance peaks of proteins measured in-cell versus those measured in vitro are small, reflecting the effect of the intracellular environment on the protein structure. Potential pitfalls in the technique are the need for abnormally high, non-physiological concentrations of the labeled target because of low signal intensity, the effects of molecular crowding inherent to the cytosol, the relevance of studying prokaryotic proteins in a eukaryotic intracellular milieux, the viability of cells during data acquisition and the ability to expand in-cell methodology to eukaryotic cells. These potential problems have proven to be more tractable than expected [15], [48]. The results have reaffirmed the power of in-cell NMR spectroscopy to measure changes in structure, resulting from post-translational biochemical modification, interactions with other biological molecules and/or allosteric changes resulting from binding interactions under physiological or near physiological conditions and in determining three-dimensional (3D) structures de novo.
Section snippets
Target labeling
To use NMR spectroscopy to study biological macromolecules in living cells the labeled targets must be easily distinguished from all other species present. Specific isotopic labeling schemes are employed to detect and resolve in-cell NMR protein resonance peaks and to yield the lowest background signals.
Applications
Much of the early work in the field of in-cell NMR utilized E. coli as the host cell. E. coli are easy to handle and grow very rapidly. Proteins are uniformly labeled with NMR-active isotopes, primarily 13C and 15N, and over-expressed to high enough intracellular levels to yield high quality HSQC spectra with little or no interfering background. Furthermore, the ability to selectively label proteins and grow cells in D2O-based media provides the capability to study high molecular weight
Conclusions and future directions
The application of in-cell NMR spectroscopy to cellular structural biology is still in its infancy. The first steps have been taken to demonstrate the feasibility of using in-cell NMR experiments to study proteins and nucleic acids, and their interactions in prokaryotic and eukaryotic cells. In-cell NMR spectroscopy continues to expand thanks to new procedures for introducing labeled proteins into living cells and advancements in instrumentation. Studies utilizing prokaryotic cells have become
Acknowledgments
This work was supported by NIH R01 GM085006-01A2 Grant and American Diabetes Association Career Development Award 1-06-CD-23 to A.S.
Glossary
- 3FY
- 3-fluoro-tyrosine
- α-SYN
- α-synuclein
- A–E
- di-peptide alanine-glutamic acid
- AUIM
- 28 amino acid peptide from ataxin 3
- BSA
- bovine serum albumin
- CI2
- chymotrypsin inhibitor 2
- CK2
- casein kinase 2
- CPP
- cell penetrating peptide
- CPPTat
- cell penetrating peptide from the tat protein of HIV-1
- CYANA
- software package for structure calculation using torsion angle dynamics
- DUB
- deubiquitinating enzymes
- ERK
- extracellular signal-regulated kinase
- FK506
- tacrolimus, an immunosuppressive drug
- FKBP
- FK506 binding protein
- FRAP
- fluorescence
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All authors contributed equally to this work.