ABSTRACT
The recent development of "in-cell NMR" techniques by two independent groups has demonstrated that NMR spectroscopy can be used to characterize the conformation and dynamics of biological macromolecules inside living cells. In this article, we describe different methods and discuss current and future applications as well as critical parameters of this new technique. We show experimental results, compare them with traditional in vitro experiments, and demonstrate that differences between the in vitro and the in vivo state of a macromolecule exist and can be detected and characterized.
Subject(s)
Escherichia coli/metabolism , Nuclear Magnetic Resonance, Biomolecular/methods , Proteins/metabolism , Amino Acids/chemistry , Humans , In Vitro Techniques , Protein ConformationABSTRACT
Our recently developed in-cell NMR procedure now enables one to observe protein conformations inside living cells. Optimization of the technique demonstrates that distinguishing the signals produced by a single protein species depends critically on protein overexpression levels and the correlation time in the cytoplasm. Less relevant is the selective incorporation of (15)N. Poorly expressed proteins, insoluble proteins, and proteins that cannot tumble freely due to associations within the cell cannot yet be observed. We show in-cell NMR spectra of bacterial NmerA and human calmodulin and discuss limitations of the technique as well as prospects for future applications.
Subject(s)
Bacterial Proteins/chemistry , Escherichia coli/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , Amino Acids/chemistry , Bacterial Proteins/metabolism , Calmodulin/chemistry , Calmodulin/metabolism , DNA-Directed RNA Polymerases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Escherichia coli/metabolism , Humans , Nitrogen Isotopes , Protein Conformation , Rifampin/pharmacologyABSTRACT
We have started to develop new NMR pulse sequences that detect carbon magnetization during the acquisition period. These experiments have become possible with the recent introduction of cryogenic probe heads. We show that a careful design of these carbon-detected experiments can at least partially compensate for the inherent lower sensitivity of carbon detection compared to proton detection. We discuss potential applications of carbon detection and demonstrate a deconvolution technique that removes the effects of carbon-carbon couplings from the spectra.
Subject(s)
Biopolymers/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , Carbon Isotopes , Magnetics , Molecular Conformation , Motion , Nucleic Acids/chemistry , Proteins/chemistryABSTRACT
We describe a procedure that allows for very efficient identification of amino acid types in proteins by selective 15N-labeling. The usefulness of selective incorporation of 15N-labeled amino acids into proteins for the backbone assignment has been recognized for several years. However, widespread use of this method has been hindered by the need to purify each selectively labeled sample and by the relatively high cost of labeling with 15N-labeled amino acids. Here we demonstrate that purification of the selectively 15N-labeled samples is not necessary and that background-free HSQC spectra containing only the peaks of the overexpressed heterologous protein can be obtained in crude lysates from as little as 100 ml cultures, thus saving time and money. This method can be used for fast and automated backbone assignment of proteins.
