Empirical force-field calculations on biological molecules represent an effective method to obtain atomic detail information on the relationship of their structure to their function. Results from those calculations depend on the quality of the force field. In this manuscript, optimization of the CHARMM27 all-atom empirical force field for nucleic acids is presented together with the resulting parameters. The optimization procedure is based on the reproduction of small molecule target data from both experimental and quantum mechanical studies and condensed phase structural properties of DNA and RNA. Via an iterative approach, the parameters were primarily optimized to reproduce macromolecular target data while maximizing agreement with small molecule target data. This approach is expected to ensure that the different contributions from the individual moieties in the nucleic acids are properly balanced to yield condensed phase properties of DNA and RNA, which are consistent with experiment. The quality of the presented force field in reproducing both crystal and solution properties are detailed in the present and an accompanying manuscript (MacKerell and Banavali, J Comput Chem, this issue). The resultant parameters represent the latest step in the continued development of the CHARMM all-atom biomolecular force field for proteins, lipids, and nucleic acids. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 86–104, 2000

All-atom empirical force field for nucleic acids: I. Parameter optimization based on small molecule and condensed phase macromolecular target data

The expression of the phase 2 detoxification enzymes and antioxidant proteins is induced at the transcriptional level by Nrf2 and negatively regulated at the posttranslational level by Keap1 through protein-protein interactions with and subsequent proteolysis of Nrf2. We found that the Neh2 domain of Nrf2 is an intrinsically disordered but biologically active regulatory domain containing a 33-residue central alpha-helix followed by a mini antiparallel beta-sheet. Isothermal calorimetry analysis indicated that one Neh2 molecule interacts with two molecules of Keap1 via two binding sites, the stronger binding ETGE motif and the weaker binding DLG motif. Nuclear magnetic resonance titration study showed that these two motifs of the Neh2 domain bind to an overlapping site on the bottom surface of the beta-propeller structure of Keap1. In contrast, the central alpha-helix of the Neh2 domain does not have any observable affinity to Keap1, suggesting that this region may serve as a bridge connecting the two motifs for the association with the two beta-propeller structures of a dimer of Keap1. Based on these observations, we propose that Keap1 recruits Nrf2 by the ETGE motif and that the DLG motif of the Neh2 domain locks its lysine-rich central alpha-helix in a correct position to benefit ubiquitin signaling.

Keap1 recruits Neh2 through binding to ETGE and DLG motifs: characterization of the two-site molecular recognition model.

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.

https://www.ifm.liu.se/edu/coursescms/TFKE40/literature/GardnerKay_AnnuRevBiophysBiomolStr_98.pdf

The use of 2H, 13C, 15N multidimensional NMR to study the structure and dynamics of proteins

To test whether distances derived from paramagnetic broadening of 15 N heteronuclear single quantum coherence (HSQC) resonances could be used to determine the global fold of a large, perdeuterated protein, we used site-directed spin-labeling of 5 amino acids on the surface of 15 N-labeled eukaryotic translation initiation factor 4E (eIF4E). eIF4E is a 25 kDa translation initiation protein, whose solution structure was previously solved in a 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate hydrate (CHAPS) micelle of total molecular mass 45-50 kDa. Distance-dependent line broadening consistent with the three-dimensional structure of eIF4E was observed for all spin-label substitutions. The paramagnetic broadening effects (PBEs) were converted into distances for modeling by a simple method comparing peak heights in 15 N-HSQC spectra before and after reduction of the nitroxide spin label with ascorbic acid. The PBEs, in combination with HN-HN nuclear Overhauser effects (NOEs) and chemical shift index (CSI) angle restraints, correctly determined the global fold of eIF4E with a backbone precision of 2.3 A (1.7 A for secondary structure elements). The global fold was not correctly determined with the HN-HN NOEs and CSI angles alone. The combination of PBEs with simulated restraints from another nuclear magnetic resonance (NMR) method for global fold determination of large proteins (methyl- protonated, highly deuterated samples) improved the quality of calculated structures. In addition, the combination of the two methods simulated from a crystal structure of an all R-helical protein (40 kDa farnesyl diphoshphate synthase) correctly determined the global fold where neither method individually was successful. These results show the potential feasibility of obtaining medium-resolution structures for proteins in the 40-100 kDa range via NMR.

/pdf/utilization-of-site-directed-spin-labeling-and-high-2xudhtd88j.pdf

Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear overhauser effect data.

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.

A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-, 13C-, 2H-labeled proteins

Use of 1HN-1HN NOEs to Determine Protein Global Folds in Perdeuterated Proteins

Characterization of the three-dimensional solution structure of human profilin: proton, carbon-13, and nitrogen-15 NMR assignments and global folding pattern

Calculations with a metric matrix distance geometry algorithm were performed that show that the standard implementation of the algorithm generally samples a very limited region of conformational space. This problem is most severe when only a small amount of distance information is used as input for the algorithm. Control calculations were performed on linear peptides, disulfide-linked peptides, and a double-stranded DNA decamer where only distances defining the covalent structures of the molecules (as well as the hydrogen bonds for the base pairs in the DNA) were included as input. Since the distance geometry algorithm is commonly used to generate structures of biopolymers from distance data obtained from NMR experiments, simulations were performed on the small globular protein basic pancreatic trypsin inhibitor (BPTI) that mimic calculations performed with actual NMR data. The results on BPTI and on the control peptides indicate that the standard implementation of the algorithm has two main problems: first, that it generates extended structures; second, that it has a tendency to consistently produce similar structures instead of sampling all structures consistent with the input distance information. These results also show that use of a simple root-mean-square deviation for evaluating the quality of the structures generated from NMR data may not be generally appropriate. The main sources of these problems are identified, and our results indicate that the problems are not a fundamental property of the distance geometry algorithm but arise from the implementations presently used to generate structures from NMR data. Several possible methods for alleviating these problems are discussed.

Limited sampling of conformational space by the distance geometry algorithm: implications for structures generated from NMR data

Determining local conformational variations in DNA: Nuclear magnetic resonance structures of the DNA duplexes d(CGCCTAATCG) and d(CGTCACGCGC) generated using back-calculation of the nuclear overhauser effect spectra, a distance geometry algorithm and constrained molecular dynamics

The experiments here show that chemically synthesized DNA containing fluorine at selected sites can be used to test specific predictions of a model for cro repressor--operator interaction. This is done by observation of the perturbation to the fluorine-19 NMR spectra of analogues of OR3 synthesized with 2'-deoxy-5-fluorouracil at specific positions in the DNA helix. Although the three-dimensional structure of the cro repressor from phage lambda has been determined by Matthews and co-workers [Anderson, W., Ohlendorf, D., Takeda, Y., & Matthews, B. (1981) Nature (London) 290, 754-758], direct structural observations on the complex of the protein with its specific DNA recognition sequence, OR3, are limited. From that structure of the protein, alone, a model of its complex to DNA was built by fitting B-form DNA, with some distortion [Ohlendorf, D., Anderson, W., Fisher, R., Takeda, Y., & Matthews, B. (1982) Nature (London) 298, 718-723]. That model proposes that the cro repressor contacts only one side of this DNA double helix and a number of specific protein--DNA contacts. To test the model, 2'-deoxy-5-fluorouracil was used to place the fluorine-19 nuclear spin-label on the side of the DNA contacting the cro repressor and on the opposite side facing away from the cro repressor. The results presented here are consistent with the prediction that lambda phage cro repressor contacts only one side of the DNA double helix.

William J. Metzler

Papers

Use of 1HN-1HN NOEs to Determine Protein Global Folds in Perdeuterated Proteins

Characterization of the three-dimensional solution structure of human profilin: proton, carbon-13, and nitrogen-15 NMR assignments and global folding pattern

Limited sampling of conformational space by the distance geometry algorithm: implications for structures generated from NMR data

Determining local conformational variations in DNA: Nuclear magnetic resonance structures of the DNA duplexes d(CGCCTAATCG) and d(CGTCACGCGC) generated using back-calculation of the nuclear overhauser effect spectra, a distance geometry algorithm and constrained molecular dynamics

Lambda phage cro repressor interaction with its operator DNA: 2'-deoxy-5-fluorouracil OR3 analogues.