The use of 2H, 13C, 15N multidimensional NMR to study the structure and dynamics of proteins
01 Jan 1998-Annual Review of Biophysics and Biomolecular Structure (Annu Rev Biophys Biomol Struct)-Vol. 27, Iss: 1, pp 357-406
TL;DR: Developments in isotopic labeling strategies, NMR pulse sequences, and structure-determination protocols that have facilitated this advance and hold promise for future NMR-based structural studies of even larger systems are reviewed.
Abstract: During the past thirty years, deuterium labeling has been used to improve the resolution and sensitivity of protein NMR spectra used in a wide variety of applications. Most recently, the combination of triple resonance experiments and 2H, 13C, 15N labeled samples has been critical to the solution structure determination of several proteins with molecular weights on the order of 30 kDa. Here we review the developments in isotopic labeling strategies, NMR pulse sequences, and structure-determination protocols that have facilitated this advance and hold promise for future NMR-based structural studies of even larger systems. As well, we detail recent progress in the use of solution 2H NMR methods to probe the dynamics of protein sidechains.
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TL;DR: A rapid and efficient approach for preparing isotopically labeled recombinant proteins is presented and expression yields obtained provide a fourfold to eightfold reduction in isotope costs using simple shake flask growths.
Abstract: A rapid and efficient approach for preparing isotopically labeled recombinant proteins is presented. The method is demonstrated for 13C labeling of the C-terminal domain of angiopoietin-2, 15N labeling of ubiquitin and for 2H/13C/15N labeling of the Escherichia coli outer-membrane lipoprotein Lpp-56. The production method generates cell mass using unlabeled rich media followed by exchange into a small volume of labeled media at high cell density. Following a short period for growth recovery and unlabeled metabolite clearance, the cells are induced. The expression yields obtained provide a fourfold to eightfold reduction in isotope costs using simple shake flask growths.
742 citations
Cites methods from "The use of 2H, 13C, 15N multidimens..."
...Prior attempts at heteronuclear 2H/13C/15N labeling of the Lpp-56 protein resulted in complete loss of protein expression following the prolonged growth in deuterated media using established 2H/13C/15N labeling protocols (Gardner and Kay, 1998)....
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TL;DR: A selective protonation strategy is described that uses [3-2H] 13C α-ketoisovalerate to introduce ( 1H-δ methyl)-leucine and (1H-γ methyl)-valine into 15N, 13C, 2H-labeled proteins, offering enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.
Abstract: A selective protonation strategy is described that uses [3-2H] 13C α-ketoisovalerate to introduce (1H-δ methyl)-leucine and (1H-γ methyl)-valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of ≈100 mg/L α-ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] α-ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-δ1 methyl)-isoleucine (>90% incorporation). 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.
487 citations
TL;DR: Binding of substrate can lead to the repositioning of catalytic groups, effectively bridging the dynamic processes of substrate binding and catalysis.
Abstract: Enzyme catalysis is an inherently dynamic process. Binding and release of ligands is often accompanied by conformational changes, both subtle and dramatic (reviewed more extensively in refs 1 -4), and these conformational changes may be rate-limiting in the overall reaction scheme, for example in dihydrofolate reductase (DHFR), 5 triosephosphate isomerase (TIM), 6,7 and protein kinase A. 8 The catalytic step itself, by its very nature, is dynamic. Atomic fluctuations along the reaction coordinate lead to bonds being broken and new ones being formed. Binding of substrate can lead to the repositioning of catalytic groups, effectively bridging the dynamic processes of substrate binding and catalysis. 2,9
428 citations
TL;DR: Stereo-array isotope labelling (SAIL) is presented, a technique that can overcome many of these problems by applying a complete stereospecific and regiospecific pattern of stable isotopes that is optimal with regard to the quality and information content of the resulting NMR spectra.
Abstract: Nuclear-magnetic-resonance spectroscopy can determine the three-dimensional structure of proteins in solution. However, its potential has been limited by the difficulty of interpreting NMR spectra in the presence of broadened and overlapping resonance lines and low signal-to-noise ratios. Here we present stereo-array isotope labelling (SAIL), a technique that can overcome many of these problems by applying a complete stereospecific and regiospecific pattern of stable isotopes that is optimal with regard to the quality and information content of the resulting NMR spectra. SAIL uses exclusively chemically and enzymatically synthesized amino acids for cell-free protein expression. We demonstrate for the 17-kDa protein calmodulin and the 41-kDa maltodextrin-binding protein that SAIL offers sharpened lines, spectral simplification without loss of information, and the ability to rapidly collect the structural restraints required to solve a high-quality solution structure for proteins twice as large as commonly solved by NMR. It thus makes a large class of proteins newly accessible to detailed solution structure determination.
408 citations
TL;DR: The three-dimensional fold of the 19 kDa (177 residues) transmembrane domain of the outer membrane protein A of Escherichia coli in dodecylphosphocholine (DPC) micelles in solution is determined using heteronuclear NMR.
Abstract: We have determined the three-dimensional fold of the 19 kDa (177 residues) transmembrane domain of the outer membrane protein A of Escherichia coli in dodecylphosphocholine (DPC) micelles in solution using heteronuclear NMR. The structure consists of an eight-stranded beta-barrel connected by tight turns on the periplasmic side and larger mobile loops on the extracellular side. The solution structure of the barrel in DPC micelles is similar to that in n-octyltetraoxyethylene (C(8)E(4)) micelles determined by X-ray diffraction. Moreover, data from NMR dynamic experiments reveal a gradient of conformational flexibility in the structure that may contribute to the membrane channel function of this protein.
392 citations
References
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TL;DR: The MOLSCRIPT program as discussed by the authors produces plots of protein structures using several different kinds of representations, including simple wire models, ball-and-stick models, CPK models and text labels.
Abstract: The MOLSCRIPT program produces plots of protein structures using several different kinds of representations. Schematic drawings, simple wire models, ball-and-stick models, CPK models and text labels can be mixed freely. The schematic drawings are shaded to improve the illusion of three dimensionality. A number of parameters affecting various aspects of the objects drawn can be changed by the user. The output from the program is in PostScript format.
13,971 citations
Additional excerpts
...a) drawn using MOLSCRIPT (79); all other...
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TL;DR: Special efforts were made to allow for appropriate display and analysis of the sets of typically 20-40 conformers that are conventionally used to represent the result of an NMR structure determination, using functions for superimposing sets of conformers, calculation of root mean square distance (RMSD) values, identification of hydrogen bonds, and identification and listing of short distances between pairs of hydrogen atoms.
7,111 citations
2,189 citations
"The use of 2H, 13C, 15N multidimens..." refers background in this paper
...More recently, building on the enhanced sensitivity nongradient methods of Rance and coworkers (14, 126), gradient-based pulse schemes with pathway selection that do not suffer from the above mentioned sensitivity losses have been developed (67, 112, 137, 138)....
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TL;DR: It is shown that by combining both 1H and 13C chemical-shift indices to produce a ‘consensus’ estimate of secondary structure, it is possible to achieve a predictive accuracy in excess of 92%.
Abstract: A simple technique for identifying protein secondary structures through the analysis of backbone 13C chemical shifts is described. It is based on the Chemical-Shift Index [Wishart et al. (1992) Biochemistry, 31, 1647–1651] which was originally developed for the analysis of 1Hα chemical shifts. By extending the Chemical-Shift Index to include 13Cα, 13Cβ and carbonyl 13C chemical shifts, it is now possible to use four independent chemical-shift measurements to identify and locate protein secondary structures. It is shown that by combining both 1H and 13C chemical-shift indices to produce a ‘consensus’ estimate of secondary structure, it is possible to achieve a predictive accuracy in excess of 92%. This suggests that the secondary structure of peptides and proteins can be accurately obtained from 1H and 13C chemical shifts, without recourse to NOE measurements.
2,017 citations
"The use of 2H, 13C, 15N multidimens..." refers background or methods in this paper
...In a highly deuterated, 13C labeled protein the 1δ values calculated from13Cα, 13Cβ and13CO chemical shifts can be used to quickly and qualitatively identify whether a residue is in an α-helix orβ-strand conformation using the Chemical Shift Index (CSI) method (167) after suitable correction for deuterium isotope effects on 13C chemical shifts (see above: “Deuterium Isotope Effects on13C and15N Chemical Shifts”)....
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...of “secondary shift” based identification of secondary structure elements (167)...
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TL;DR: An analysis of the 1H nuclear magnetic resonance chemical shift assignments and secondary structure designations for over 70 proteins has revealed some very strong and unexpected relationships.
1,862 citations