Amino Acid Selective Isotope Labeling Research Articles

Amino-acid selective isotope labeling enables simultaneous overlapping signal decomposition and information extraction from NMR spectra

Signal overlapping is a major bottleneck for protein NMR analysis. We propose a new method, stable-isotope-assisted parameter extraction (SiPex), to resolve overlapping signals by a combination of amino-acid selective isotope labeling (AASIL) and tensor decomposition. The basic idea of Sipex is that overlapping signals can be decomposed with the help of intensity patterns derived from quantitative fractional AASIL, which also provides amino-acid information. In SiPex, spectra for protein characterization, such as 15N relaxation measurements, are assembled with those for amino-acid information to form a four-order tensor, where the intensity patterns from AASIL contribute to high decomposition performance even if the signals share similar chemical shift values or characterization profiles, such as relaxation curves. The loading vectors of each decomposed component, corresponding to an amide group, represent both the amino-acid and relaxation information. This information link provides an alternative protein analysis method that does not require “assignments” in a general sense; i.e., chemical shift determinations, since the amino-acid information for some of the residues allows unambiguous assignment according to the dual selective labeling. SiPex can also decompose signals in time-domain raw data without Fourier transform, even in non-uniformly sampled data without spectral reconstruction. These features of SiPex should expand biological NMR applications by overcoming their overlapping and assignment problems.

Amino Acid Selective 13C Labeling and 13C Scrambling Profile Analysis of Protein α and Side-Chain Carbons in Escherichia coli Utilized for Protein Nuclear Magnetic Resonance.

Amino acid selective isotope labeling is an important nuclear magnetic resonance technique, especially for larger proteins, providing strong bases for the unambiguous resonance assignments and information concerning the structure, dynamics, and intermolecular interactions. Amino acid selective 15N labeling suffers from isotope dilution caused by metabolic interconversion of the amino acids, resulting in isotope scrambling within the target protein. Carbonyl 13C atoms experience less isotope scrambling than the main-chain 15N atoms do. However, little is known about the side-chain 13C atoms. Here, the 13C scrambling profiles of the Cα and side-chain carbons were investigated for 15N scrambling-prone amino acids, such as Leu, Ile, Tyr, Phe, Thr, Val, and Ala. The level of isotope scrambling was substantially lower in 13Cα and 13C side-chain labeling than in 15N labeling. We utilized this reduced scrambling-prone character of 13C as a simple and efficient method for amino acid selective 13C labeling using an Escherichia coli cold-shock expression system and high-cell density fermentation. Using this method, the 13C labeling efficiency was >80% for Leu and Ile, ∼60% for Tyr and Phe, ∼50% for Thr, ∼40% for Val, and 30-40% for Ala. 1H-15N heteronuclear single-quantum coherence signals of the 15N scrambling-prone amino acid were also easily filtered using 15N-{13Cα} spin-echo difference experiments. Our method could be applied to the assignment of the 55 kDa protein.

A simple guide to the structural study on membrane proteins in detergents using solution NMR

Abstract NMR-based structural studies on membrane proteins are appreciated quite challenging due to various reasons, generally including the narrow dispersion of NMR spectra, the severe peak broadening, and the lack of long range NOEs. In spite of the poor biophysical properties, structural studies on membrane proteins have got to go on ,considering their functional importance in biological systems. In this review, we provide a simple overview of the techniques generally used in structural studies of membrane proteins by solution NMR, with experimental examples of a helical membrane protein, caveolin 3. Detergent screening is usually employed as the first step and the selection of appropriate detergent is the most important for successful approach to membrane proteins. Various tools can then be applied as NMR specializedtechniques in solution that include sample deteuration, amino-acid selective isotope labeling, residual dipolar coupling, and paramagnetic relaxation enhancement.

Escherichia coli auxotroph host strains for amino acid-selective isotope labeling of recombinant proteins.

Enrichment of proteins with isotopes such as (2)H, (15)N, and (13)C is commonly carried out in magnetic resonance and vibrational spectroscopic characterization of protein structures, mechanisms, and dynamics. Although uniform isotopic labeling of proteins is straightforward, efficient labeling of proteins with only a selected set of amino acid types is often challenging. A number of approaches have been described in the literature for amino acid-selective isotope labeling of proteins, each with its own limitations. Since Escherichia coli represents the most cost-effective and widely used host for heterologous production of foreign proteins, an efficient method to express proteins selectively labeled with isotopes would be highly valuable for these studies. However, an obvious drawback is misincorporation and dilution of input isotope labels to unwanted amino acid types due to metabolic scrambling in vivo. To overcome this problem, we have generated E. coli auxotroph strains that are compatible with the widely used T7 RNA polymerase overexpression systems and that minimize metabolic scrambling. We present several examples of selective amino acid isotope labeling of simple and complex proteins with bound cofactors, as an initial guide for practical applications of these E. coli strains.

One-step amino acid selective isotope labeling of proteins in prototrophic Escherichia coli strains

Amino acid selective isotope labeling is a useful approach to simplification of nuclear magnetic resonance (NMR) spectra of large proteins. Cell-free protein synthesis offers essentially unlimited flexibility of labeling patterns but is labor-intensive and expensive. In vivo labeling is simple in principle but generally requires auxotrophic strains, inhibitors of amino acid synthesis, or complex media formulations. We describe a simple procedure for amino acid selective labeling of proteins expressed in prototrophic Escherichia coli strains. Excellent labeling selectivity was achieved for histidine, lysine, methionine, and alanine. Simplicity and robustness of this protocol make it a useful tool for protein NMR.

Cell‐free synthesis of membrane subunits of ATP synthase in phospholipid bicelles: NMR shows subunit a fold similar to the protein in the cell membrane

NMR structure determination of large membrane proteins is hampered by broad spectral lines, overlap, and ambiguity of signal assignment. Chemical shift and NOE assignment can be facilitated by amino acid selective isotope labeling in cell-free protein synthesis system. However, many biological detergents are incompatible with the cell-free synthesis, and membrane proteins often have to be synthesized in an insoluble form. We report cell-free synthesis of subunits a and c of the proton channel of Escherichia coli ATP synthase in a soluble form in a mixture of phosphatidylcholine derivatives. In comparison, subunit a was purified from the cell-free system and from the bacterial cell membranes. NMR spectra of both preparations were similar, indicating that our procedure for cell-free synthesis produces protein structurally similar to that prepared from the cell membranes.

A rapid and robust method for selective isotope labeling of proteins

Amino-acid selective isotope labeling of proteins offers numerous advantages in mechanistic studies by revealing structural and functional information unattainable from a crystallographic approach. However, efficient labeling of proteins with selected amino acids necessitates auxotrophic hosts, which are often not available. We have constructed a set of auxotrophs in a commonly used Escherichia coli expression strain C43(DE3), a derivative of E. coli BL21(DE3), which can be used for isotopic labeling of individual amino acids or sets of amino acids. These strains have general applicability to either soluble or membrane proteins that can be expressed in E. coli. We present examples in which proteins are selectively labeled with 13C- and 15N-amino acids and studied using magic-angle spinning solid-state NMR and pulsed EPR, demonstrating the utility of these strains for biophysical characterization of membrane proteins, radical-generating enzymes and metalloproteins.

Amino acid-selective isotope labeling of proteins for nuclear magnetic resonance study: Proteins secreted by Brevibacillus choshinensis

Here we report the first application of amino acid-type selective (AATS) isotope labeling of a recombinant protein secreted by Brevibacillus choshinensis for a nuclear magnetic resonance (NMR) study. To prepare the 15N-AATS-labeled protein, the transformed B. choshinensis was cultured in 15N-labeled amino acid-containing C.H.L. medium, which is commonly used in the Escherichia coli expression system. The analyses of the 1H- 15N heteronuclear single quantum coherence (HSQC) spectra of the secreted proteins with a 15N-labeled amino acid demonstrated that alanine, arginine, asparagine, cysteine, glutamine, histidine, lysine, methionine, and valine are suitable for selective labeling, although acidic and aromatic amino acids are not suitable. The 15N labeling for glycine, isoleucine, leucine, serine, and threonine resulted in scrambling to specific amino acids. These results indicate that the B. choshinensis expression system is an alternative tool for AATS labeling of recombinant proteins, especially secretory proteins, for NMR analyses.

A simple method for amino acid selective isotope labeling of recombinant proteins in E. coli

A simple and user-friendly method of labeling protein selectively with amino acids in vivo is introduced. This technique does not require the use of transaminase-deficient or auxotrophic strains. By manipulating the product feedback inhibitory loops of the E. coli amino acid metabolic pathways and, if necessary, by using enzyme inhibitors, proteins were labeled efficiently in vivo even with amino acid types that are central to the metabolic pathways, such as glutamine. The sequential backbone resonance assignment of the Neh2 domain of Nrf2 transcriptional factor, an intrinsically disordered protein with high spectral degeneracy, was achieved using this labeling method.

NMR analysis of in vitro-synthesized proteins without purification: a high-throughput approach

A cell-free protein expression system was established that provides protein samples of adequate concentration and purity for direct NMR analysis. The Escherichia coli peptidyl–prolyl cis– trans isomerase PpiB was expressed in this system with dual amino acid-selective isotope labeling to identify the NMR signal from the active site-residue Arg87. Addition of the substrate succinyl-Ala-Ala-Pro-Phe- p-nitroanilide selectively shifted its 15N-HSQC cross peak, confirming binding to the active site. As cell-free protein expression provides high yields of protein per unit mass of labeled amino acid and sample handling is minimal, this strategy presents an exceptionally inexpensive and rapid approach to protein analysis.