Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR (original) (raw)
Related papers
2001
Nuclear magnetic resonance is an important tool for high-resolution structural studies of proteins. It demands high protein concentration and high purity; however, the expression of proteins at high levels often leads to protein aggregation and the protein purification step can correspond to a high percentage of the overall time in the structural determination process. In the present article we show that the step of sample optimization can be simplified by selective labeling the heterologous protein expressed in Escherichia coli by the use of rifampicin. Yeast thioredoxin and a coix transcription factor Opaque 2 leucine zipper (LZ) were used to show the effectiveness of the protocol. The 1 H/ 15 N heteronuclear correlation two-dimensional NMR spectrum (HMQC) of the selective 15 N-labeled thioredoxin without any purification is remarkably similar to the spectrum of the purified protein. The method has high yields and a good 1 H/ 15 N HMQC spectrum can be obtained with 50 ml of M9 growth medium. Opaque 2 LZ, a difficult protein due to the lower expression level and high hydrophobicity, was also probed. The 15 N-edited spectrum of Opaque 2 LZ showed only the resonances of the protein of heterologous expression (Opaque 2 LZ) while the 1 H spectrum shows several other resonances from other proteins of the cell lysate. The demand for a fast methodology for structural determination is increasing with the advent of genome/proteome projects. Selective labeling the heterologous protein can speed up NMR structural studies as well as NMR-based drug screening. This methodology is especially effective for difficult proteins such as hydrophobic transcription factors, membrane proteins, and others.
2001
Nuclear magnetic resonance is an important tool for high-resolution structural studies of proteins. It demands high protein concentration and high purity; however, the expression of proteins at high levels often leads to protein aggregation and the protein purification step can correspond to a high percentage of the overall time in the structural determination process. In the present article we show that the step of sample optimization can be simplified by selective labeling the heterologous protein expressed in Escherichia coli by the use of rifampicin. Yeast thioredoxin and a coix transcription factor Opaque 2 leucine zipper (LZ) were used to show the effectiveness of the protocol. The 1 H/ 15 N heteronuclear correlation two-dimensional NMR spectrum (HMQC) of the selective 15 N-labeled thioredoxin without any purification is remarkably similar to the spectrum of the purified protein. The method has high yields and a good 1 H/ 15 N HMQC spectrum can be obtained with 50 ml of M9 growth medium. Opaque 2 LZ, a difficult protein due to the lower expression level and high hydrophobicity, was also probed. The 15 N-edited spectrum of Opaque 2 LZ showed only the resonances of the protein of heterologous expression (Opaque 2 LZ) while the 1 H spectrum shows several other resonances from other proteins of the cell lysate. The demand for a fast methodology for structural determination is increasing with the advent of genome/proteome projects. Selective labeling the heterologous protein can speed up NMR structural studies as well as NMR-based drug screening. This methodology is especially effective for difficult proteins such as hydrophobic transcription factors, membrane proteins, and others.
An approach for high-throughput structure determination of proteins by NMR spectroscopy
Journal of biomolecular NMR, 2000
An approach is described for rapidly determining protein structures by NMR that utilizes proteins containing 13C-methyl labeled Val, Leu, and Ile (delta1) and protonated Phe and Tyr in a deuterated background. Using this strategy, the key NOEs that define the hydrophobic core and overall fold of the protein are easily obtained. NMR data are acquired using cryogenic probe technology which markedly reduces the spectrometer time needed for data acquisition. The approach is demonstrated by determining the overall fold of the antiapoptotic protein, Bcl-xL, from data collected in only 4 days. Refinement of the Bcl-xL structure to a backbone rmsd of 0.95 A was accomplished with data collected in an additional 3 days. A distance analysis of 180 different proteins and structure calculations using simulated data suggests that our method will allow the global folds of a wide variety of proteins to be determined.
Current approaches for the study of large proteins by NMR
Journal of Molecular Structure, 2002
An overview of current methods employed for characterizing larger (.25 kDa) proteins by NMR is presented. These techniques include: the attenuation of T 2 relaxation effects by offsetting dipole±dipole and chemical shift anisotropy relaxation mechanisms (TROSY); the extraction of residual dipolar couplings from partially oriented molecules; the elimination of relaxation pathways by incorporating deuterium nuclei into protein samples; the easing of resonance overlap by isotopically labeling only speci®c protein segments; and the decrease of rotational correlation times by dissolving proteins in low viscosity solvents.
High yield expression of proteins in <i>E. coli</i> for NMR studies
Advances in Bioscience and Biotechnology, 2013
In recent years, high yield expression of proteins in E. coli has witnessed rapid progress with developments of new methodologies and technologies. An important advancement has been the development of novel recombinant cloning approaches and protocols to express heterologous proteins for Nuclear Magnetic Resonance (NMR) studies and for isotopic enrichment. Isotope labeling in NMR is necessary for rapid acquisition of high dimensional spectra for structural studies. In addition, higher yield of proteins using various solubility and affinity tags has made protein overexpression cost-effective. Taken together, these methods have opened new avenues for structural studies of proteins and their interactions. This article deals with the different techniques that are employed for over-expression of proteins in E. coli and different methods used for isotope labeling of proteins vis-à-vis NMR spectroscopy.
Annual Review of Biophysics and Biophysical Chemistry, 1991
Over the past 5 to 10 years, NMR (nuclear magnetic resonance) has developed into a powerful method for determining three-dimensional structures of small proteins of about 100 residues or less. The principal geometric information used in these structure determinations resides in short (< 5 ~) approximate interproton distance restraints derived from the observation of nuclear Overhauser effects (NOE) that are proportional to r-6 (97). The stimulus for this rapid progress has stemmed from one technological and two methodological advances, in particular, the advent of high-field NMR spectrometers (500 600 MHz) and the development a whole array of two-dimensional (2D) NMR experiments and of algorithms for converting NMR-derived interproton distances into cartesian coordinates. The basic strategy for NMR structure determination is relatively straightforward and can be divided into three stages: (a) sequential assignment of backbone and side-chain ~H resonances using experiments that demonstrate through-bond (scalar) and through-space (< 5 ~) nectivities; (b) identification of as many through-space nuclear Overhauser (NOE) connectivities as possible that yield a large set of approximate interproton-distance restraints; and (c) calculation of three-dimensional structures on the basis of these distance restraints, supplemented if possible by some torsion-angle restraints derived from coupling constants. The theoretical basis of two-dimensional NMR has been discussed in detail in a book by Ernst et al (47), and the application of these methods to protein structure determination has been comprehensively reviewed (5, 27, 28, 59, 115, 116). However, several exciting developments from over the past year promise to extend the NMR methodology both with respect to the size of proteins that can be studied and to the accuracy and precision of the structure determinations. These include the extension of 2D NMR into 3D and 4D NMR and the use of systematic database searches to obtain numerous torsion-angle restraints on the basis of the experimental NMR data. In
Multidimensional NMR Methods for Protein Structure Determination
IUBMB Life (International Union of Biochemistry and Molecular Biology: Life), 2001
Structural studies of proteins are critical for understanding biological processes at the molecular level. Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for obtaining structural and dynamic information on proteins and protein-ligand complexes. In the present review, methodologies for NMR structure determination of proteins and macromolecular complexes are described. In addition, a number of recent advances that reduce the molecular weight limitations previously imposed on NMR studies of biomolecules are discussed, highlighting applications of these technologies to protein systems studied in our laboratories. IUBMB Life, 52: 291 -302, 2001
Microgram-scale protein structure determination by NMR
Nature Methods, 2007
Using conventional triple-resonance nuclear magnetic resonance (NMR) experiments with a 1 mm triple-resonance microcoil NMR probe, we determined near complete resonance assignments and three-dimensional (3D) structure of the 68-residue Methanosarcina mazei TRAM protein using only 72 lg (6 ll, 1.4 mM) of protein. This first example of a complete solution NMR structure determined using microgram quantities of protein demonstrates the utility of microcoil-probe NMR technologies for protein samples that can be produced in only limited quantities.
Angewandte Chemie International Edition, 2010
Eukaryotic proteins typically have a modular architecture, characterized by multiple structural domains that are connected by flexible linkers. Regulation of cellular processes depends on an interaction network between these individual modules and the formation of a quaternary structure. Dynamic rearrangement, often coupled to ligand binding, is a common feature of these multicomponent systems. While compact and rigid complexes can be efficiently studied using X-ray crystallography, protein complexes or multidomain proteins that involve weak and transient domain interactions should be preferably investigated using solution techniques. A number of NMR spectroscopy studies of protein complexes have been reported in recent years. However, a general protocol is not available, and many applications still rely on NOE-based interdomain distance restraints, which are difficult to obtain and to assign in high-molecular-weight systems.