Secondary structure of human interleukin 2 from 3D heteronuclear NMR experiments.

Recombinant 15N-labeled human interleukin 2 (IL-2) has been studied by 2D and 3D NMR using uniformly 15N-labeled protein. Assignment of the backbone resonances has enabled the secondary structure of the protein to be defined. The secondary structure was found to consist of four alpha-helical regions and a short section of antiparallel beta-sheet. This structure is more similar to recent published structures of interleukin 4 and granulocyte-macrophage colony-stimulating factor than to a structure of IL-2 previously obtained from low-resolution X-ray diffraction data.     

NMR studies on metal complexes of DNA oligomers

A short overview of NMR spectroscopic applications for the study of metal ion complexes of DNA oligomers is presented. One typical example is given to illustrate the scope of the methods: the NMR structure of a trans-DDP interstrand cross-linked duplex, d(CTCCTG*TGTCTC) x d(GAGATA*AGGAG). The solution structure of this double-stranded DNA oligonucleotide, containing a trans-diammineplatinum(II) interstrand cross-link, was determined using two-dimensional nuclear magnetic resonance (2D NMR) and NOE-restrained molecular dynamics and energy minimization refinement. The duplex is a non-palindromic 12/11-mer with a missing central residue in the lower strand and in addition it contains a GT mismatched base pair. The analysis indicated that an interstrand cross-link is established between G6-N7 of the upper strand and A18-N1 of the lower strand.     

Assignment of the protonated 13C resonances of apo-neocarzinostatin by 2D heteronuclear NMR spectroscopy at natural abundance

Summary Nearly complete assignment of the protonated carbon resonances of apo-neocarzinostatin, 113-amino acid antitumor antibiotic carrier protein, has been achieved at natural ¹³C abundance using heteronuclear 2D experiments. Most of the cross peaks in the proton-carbon correlation map were identified by the combined use of HMQC, HMQC-RELAY and HMQC-NOESY spectra, using already published proton chemical shifts. However, double-DEPT and triple-quantum experiments had to be performed for the edition of CH and CH₂ side-chain groups, respectively, which were hardly visible on HMQC-type maps. The triple-quantum pulse sequence was adapted from its original scheme to be applicable to a natural abundance sample. The correlation between carbon chemical shifts and the apo-neocarzinostatin structure is discussed. In particular, ¹³C alpha secondary shifts correlate well with the backbone conformation. These shifts also yield information about the main-chain flexibility of the protein. Assignments reported herein will be used further for interpretation of carbon relaxation times in a study of the internal dynamics of apo-neocarzinostatin.     

Identification of the DNA binding surface of H-NS protein from Escherichia coli by heteronuclear NMR spectroscopy.

The DNA binding domain of H-NS protein was studied with various N-terminal deletion mutant proteins and identified by gel retardation assay and heteronuclear 2D- and 3D-NMR spectroscopies. It was shown from gel retardation assay that DNA binding affinity of the mutant proteins relative to that of native H-NS falls in the range from 1/6 to 1/25 for H-NS(60-137), H-NS(70-137) and H-NS(80-137), whereas it was much weaker for H-NS(91-137). Thus, the DNA binding domain was defined to be the region from residue A80 to the C-terminus. Sequential nuclear Overhauser effect (NOE) connectivities and those of medium ranges revealed that the region of residues Q60-R93 in mutant protein H-NS(60-137) forms a long stretch of disordered, flexible chain, and also showed that the structure of the C-terminal region (residues A95-Q137) in mutant H-NS(60-137) was nearly identical to that of H-NS(91-137). 1H and 15N chemical shift perturbations induced by complex formation of H-NS(60-137) with an oligonucleotide duplex 14-mer demonstrated that two loop regions, i.e. residues A80-K96 and T110-A117, play an essential role in DNA binding.     

A complete set of novel 2D correlation NMR experiments based on heteronuclear J-cross polarization

A suite of multiple-purpose sensitivity-enhanced 2D correlation NMR experiments based on heteronuclear J-cross polarization (HCP) techniques are introduced for isotropic liquid samples. Several pulse sequences using an adaptable heteronuclear TOCSY mixing building block are proposed for different types of effective coherence-order-selective (COS) heteronuclear coherence-transfer mechanisms. They are based on the anisotropic behaviour of the involved HCP process that is easily described and analysed in terms of cartesian product-operator formalism. A number of different versions are given for in-phase to in-phase (II-COS: S (-) \\ to I (-)), in-phase to anti-phase (IA-COS: S (-) \\ to 2 I (-) S (z)), in-phase to spin-state-selective (IS(3)-COS: S (-) \\ to 2 I (-) S (alpha /beta)), anti-phase to in-phase (AI-COS: 2 I (z) S (-) \\ to I (-)), anti-phase to anti-phase (AA-COS: 2 I (z) S (-) \\ to 2 I (-) S (z)), anti-phase to spin-state-selective (AS(3)-COS: 2 I (z) S (-) \\ to 2 I (-) S (alpha /beta)) and spin-state-selective to spin-state-selective (S(3)S(3)-COS: 2 I (alpha /beta) S (-) \\ to 2 I (-) S (alpha /beta )) coherence transfers. The combination of the echo/anti-echo approach, heteronuclear gradient echoes and the preservation of equivalent pathways (PEP) methodology affords a general approach to obtain sensitivity-enhanced pure-absorption 2D spectra that can be used as interesting alternatives to conventional pulse-interrupted free-precession INEPT-based pulse schemes, such as HSQC-type and TROSY-type experiments.     

Prediction of 1H NMR chemical shifts of DNA oligomers

A set of parameters, devised for the prediction of 1H NMR chemical shifts of heterobase and anomeric protons in the high temperature (greater than 70 degrees C) spectra of RNA oligomers has been found to be applicable to the corresponding DNA oligomers. Fifteen examples of DNA oligomers that have had high temperature spectra recorded and assigned show a mean absolute difference between predicted and assigned shifts of 0.045 ppm. The parameters for uridine H-5 are applied to the calculation of thymidine methyl group shifts and give excellent agreement with experimental assigned shifts. The RNA parameter set is a practical means of assigning heterobase and anomeric protons in DNA oligomers. A programme using the RNA parameter set has been written which enables the sequence of short DNA oligomers to be predicted from their 1H NMR spectra.     

Identification of the Archaeoglobus fulgidus endonuclease III DNA interaction surface using heteronuclear NMR methods.

BACKGROUND: Endonuclease III is the prototype for a family of DNA-repair enzymes that recognize and remove damaged and mismatched bases from DNA via cleavage of the N-glycosidic bond. Crystal structures for endonuclease III, which removes damaged pyrimidines, and MutY, which removes mismatched adenines, show a highly conserved structure. Although there are several models for DNA binding by this family of enzymes, no experimental structures with bound DNA exist for any member of the family. RESULTS: Nuclear magnetic resonance (NMR) spectroscopy chemical-shift perturbation of backbone nuclei (1H, 15N, 13CO) has been used to map the DNA-binding site on Archaeoglobus fulgidus endonuclease III. The experimentally determined interaction surface includes five structural elements: the helix-hairpin-helix (HhH) motif, the iron-sulfur cluster loop (FCL) motif, the pseudo helix-hairpin-helix motif, the helix B-helix C loop, and helix H. The elements form a continuous surface that spans the active site of the enzyme. CONCLUSIONS: The enzyme-DNA interaction surface for endonuclease III contains five elements of the protein structure and suggests that DNA damage recognition may require several specific interactions between the enzyme and the DNA substrate. Because the target DNA used in this study contained a generic apurinic/apyrimidinic (AP) site, the binding interactions we observed for A. fulgidus endonuclease III should apply to all members of the endonuclease III family and several interactions could apply to the endonuclease III/AlkA (3-methyladenine DNA glycosylase) superfamily.     

Novel DNA superstructures formed by telomere-like oligomers.

DNA oligomers containing three or more contiguous guanines form tetrastranded parallel complexes, G4-DNA, in the presence of alkali cations. However, oligomers that have a single multi-guanine motif at their 3' or 5' end, with a guanine as the terminal base, also form higher order products. Thus, the oligomer T8G3T forms a unique G4-DNA product at neutral pH in the presence of Na+, K+, or Rb+; however, its isomeric counterpart T9G3 in K+ or Rb+ generates an additional ladder of products of substantially lower gel mobility. We show that these larger complexes contain, respectively, 8, 12, or 16 distinct strands of oligomer. The octamer structure formed by T9G3 assembles in moderate salt at room temperature and melts around 60 degrees C in 100 mM KCl. Methylation protection experiments suggest a nested head-to-tail superstructure containing two tetraplexes bonded front-to-back via G quartets formed by out-of-register guanines. Naturally occurring chromosomal telomeres, which all have guanines at their 3' termini, may be able to form these superstructures.     

Rapid screening for structural integrity of expressed proteins by heteronuclear NMR spectroscopy.

A simple and rapid method based on 15N labeling and 1H-15N heteronuclear single quantum coherence spectroscopy is presented to directly assess the structural integrity of overexpressed proteins in crude Escherichia coli extracts without the need for any purification. The method is demonstrated using two different expression systems and two different proteins, the B1 immunoglobulin-binding domain of streptococcal protein G (56 residues) and human interleukin-1 beta (153 residues). It is shown that high quality 1H-15N correlation spectra, recorded in as little as 15 min and displaying only cross-peaks arising from the overexpressed protein of interest, can be obtained from crude E. coli extracts.     

A new method to determine DNA sugar conformation from the joint use of 2D and 3D NMR data

The use of sugar restraints has been proven essential for assessing DNAstructures through molecular modeling studies. We present a new methodcombining 2D (COSY and NOESY) and 3D (NOESY-NOESY) experiments, whereconstraints on either the phase angles or the difference between phase anglesof two residues are obtained from comparison of 2D NOE H1-H4intensities and 3D NOE intensities containing the H1-H4transfer. All experiments lead to restraints that match, proving the validityof the method.     

Chemical shift assignments and secondary structure of the Grb2 SH2 domain by heteronuclear NMR spectroscopy

Summary The growth factor receptor-bound protein-2 (Grb2) is an adaptor protein that mediates signal transduction pathways. Chemical shift assignments were obtained for the SH2 domain of Grb2 by heteronuclear NMR spectroscopy, employing the uniformly ¹³C-/¹⁵N-enriched protein as well as the protein containing selectively ¹⁵N-enriched amino acids. Using the Chemical Shift Index (CSI) method, the chemical shift indices of four nuclei, ¹H, ¹³C, ¹³C and ¹³CO, were used to derive the secondary structure of the protein. Nuclear Overhauser enhancements (NOEs) were then employed to confirm the secondary structure. The CSI results were compared to the secondary structural elements predicted for the Grb2 SH2 domain from a sequence alignment [Lee et al. (1994) Structure, 2, 423–438]. The core structure of the SH2 domain contains an antiparallel -sheet and two -helices. In general, the secondary structural elements determined from the CSI method agree well with those predicted from the sequence alignment.Abbreviations crk viral p47^gag-crk - EGF epidermal growth factor - GAP GTPase-activating protein - PI3K phosphatidylinositol-3-kinase - PLC- phospholipase-C-, shc, src homologous and collagen - src sarcoma family of nonreceptor tyrosine kinase     

Self-association reaction of denatured staphylococcal nuclease fragments characterized by heteronuclear NMR

The self-association reaction of denatured staphylococcal nuclease fragments, urea-denatured G88W110, containing residues 1-110 and mutation G88W, and physiologically denatured 131-residue Delta 131 Delta, have been characterized by NMR at close to neutral pH. The two fragments differ in the extent and degree of association due to the different sequence and experimental conditions. Residues 13-39, which show significant exchange line broadening, constitute the main association interface in both fragments. A second weak association region was identified involving residues 79-105 only in the case of urea-denatured G88W110. For residues involved in the association reaction, significant suppression of the line broadening and small but systematic chemical shift variation of the amide protons were observed as the protein concentration decreased. The direction of chemical shift change suggests that the associated state adopts mainly beta-sheet-like conformation, and the beta-hairpin formed by strands beta 2 and beta 3 is native-like. The apparent molecular size obtained by diffusion coefficient measurements shows a weak degree of association for Delta 131 Delta below 0.4 mM protein concentration and for G88W110 in 4 M urea. In both cases the fragments are predominantly in the monomeric state. However, the weak association reaction can significantly influence the transverse relaxation of residues involved in the association reaction. The degree of association abruptly increases for Delta 131 Delta above 0.4 mM concentration, and it is estimated to form a 4 to 8 mer at 2 mM. It is proposed that the main region involved in association forms the core structure, with the remainder of residues largely disordered in the associated state. Despite the obvious influence of the association reaction on the slow motion of the backbone, the restricted mobility on the nanosecond timescale around the region of strand beta 5 is essentially unaffected by the association reaction and degree of denaturation.     

Recent progress in heteronuclear long-range NMR of complex carbohydrates: 3D H2BC and clean HMBC

The new NMR experiments 3D H2BC and clean HMBC are explored for challenging applications to a complex carbohydrate at natural abundance of ¹³C. The 3D H2BC experiment is crucial for sequential assignment as it yields heteronuclear one- and two-bond together with COSY correlations for the ¹H spins, all in a single spectrum with good resolution and non-informative diagonal-type peaks suppressed. Clean HMBC is a remedy for the ubiquitous problem of strong coupling induced one-bond correlation artifacts in HMBC spectra of carbohydrates. Both experiments work well for one of the largest carbohydrates whose structure has been determined by NMR, not least due to the enhanced resolution offered by the third dimension in 3D H2BC and the improved spectral quality due to artifact suppression in clean HMBC. Hence these new experiments set the scene to take advantage of the sensitivity boost achieved by the latest generation of cold probes for NMR structure determination of even larger and more complex carbohydrates in solution.     

Characterization of autocatalytic conversion of precursor BACE1 by heteronuclear NMR spectroscopy

Accumulation of the cytotoxic 40- to 42-residue beta-amyloid peptide represents the primary pathological process in Alzheimer's disease (AD). BACE1 (beta-site APP cleaving enzyme 1) is responsible for the initial required step in the neuronal amyloidogenic processing of beta-amyloid precursor protein and is a major drug target for the therapeutic intervention of AD. In the present study, BACE1 is initially synthesized as an immature precursor protein containing part of the pre domain and the entire pro domain, and undergoes autocatalytic conversion to yield the well-folded mature BACE1 enzyme. To understand the mechanism of the conversion and the role of the pro domain, we monitored the autocatalytic conversion of BACE1 by heteronuclear NMR spectroscopy and used chemical shift perturbations as a probe to study the structural changes accompanying the autocatalytic conversion. NMR data revealed local conformational changes from a partially disordered to a well-folded conformation associated with the conversion. The conformational changes are largely concentrated in the NH(2)-terminal lobe. Conversely, the active site conformations are conserved during the autocatalytic conversion. The precursor and mature BACE1 proteins were further characterized for their ability to interact with a substrate-based transition state BACE1 peptide inhibitor. The precursor BACE1 rapidly adopted the bound conformation in the presence of the inhibitor, which is identical to the bound conformation of the mature protein. The interaction of the inhibitor with both the precursor BACE1 and the fully processed BACE1 is in slow exchange on the NMR time scale, indicating a tight binding interaction. Overall, the NMR data demonstrated that the pro domain does not hinder inhibitor binding and may assist in the proper folding of the protein. The fully processed BACE1 represents a high quality well-folded protein which is highly stable over a long period of time, and is suitable for evaluation of inhibitor binding by NMR for drug intervention.     

Sensitivity improvement in 2D and 3D HCCH spectroscopy using heteronuclear cross-polarization

Summary A new method, which employs a sequence of heteronuclear-homonuclear-heteronuclear Hartmann-Hahn (HEHOHEHAHA) cross-polarization steps for obtaining through-bond H-C-C-H correlations in larger proteins (M_r > 15 kDa), is presented. The method has significantly higher sensitivity compared to INEPTHOHAHA-INEPT-based techniques. An additional feature of this experiment is that well-phaseable spectra may be obtained with a minimal (4-step) phase cycle and, consequently, experimental time can be utilized towards obtaining high resolution in indirect dimensions. Results from 2D and 3D HEHOHEHAHA experiments on T4-lysozyme are presented.     

Linear prediction enhancement of 2D heteronuclear correlated spectra of proteins

Summary Linear prediction has been used to extrapolate the t₁ domain of natural abundance¹H–¹³C correlated two-dimensional (2D) FIDs of insulin. The FIDs were obtained by two different heteronuclear correlation experiments, one that utilizes heteronuclear multiple-quantum coherence during t₁, and one that utilizes¹³C single-quantum coherence. It is shown that the enhancement of the resolution and sensitivity in the F₁ dimension of the Fourier transform spectrum that results from the linear prediction extrapolation allows the t₁ domain to be confined to a relatively short time period where the signal intensity is at maximum. In particular, it is found that the enhancement thus obtained is sufficiently good to allow an observation of the difference between the F₁ line widths in the single-quantum and double-quantum coherence spectra.     

Structure of a DNA intercalation complex as determined by NMR using a paramagnetic probe.

Longitudinal relaxation rates of the protons of the 3,8-dimethyl-N-methyl-phenanthrolinium (DMP) cation in solutions containing DNA are strongly affected by the addition of the paramagnetic manganese (II) ions due to the electron-nuclear dipolar interaction in the ternary Mn-DNA-DMP Complex. Two possible models for the DMP-DNA intercalation complex are examined and one of them is unequivocally discriminated on the basis of the proton relaxation data. It is concluded that in the intercalation complex the long axis of the DMP molecule is almost perpendicular to the hydrogen bonds of the DNA base-pairs.     

Sequence-specific resonance assignment and conformational analysis of subtilin by 2D NMR.

Subtilin, a 32-amino acid peptide with potent antimicrobial activity, has been isolated from Bacillus subtilis ATCC6633. The chemical structure has been confirmed by the unambiguous sequence-specific assignment of its 1H NMR spectrum. Detailed NMR analysis revealed that subtilin is a rather flexible molecule; the only observed conformational contraints were those imposed by the cyclic structures created by the lanthionine and 3-methyllanthionine residues. These results suggest that in aqueous solution subtilin and the homologous peptide nisin have similar conformations.