Complete assignment of the deoxyribose 5'/5" proton resonances of the EcoRI DNA sequence using isotropic mixing

Using two-dimensional isotropic mixing spectroscopy all 5'/5" proton resonances of the EcoRI restriction site DNA dodecamer [d(CGCGAATTCGCG)]2 have been assigned. This completes the previous assignments of 1'H to 4'H resonances of the deoxyribose spin systems (Hare et al., 1983). With mixing times of up to 500 ms, many of these resonances showed connectivities of 5'/5" protons in the two-dimensional isotropic mixing spectrum. Relying only on through-bond connectivities makes these assignments independent of assumptions about the conformation of the DNA oligonucleotide. The assignment of the 5'H/5"H resonances will allow the interpretation of intra- and interresidue NOEs to these protons, providing information about the DNA backbone conformation.     

Main-chain-directed strategy for the assignment of 1H NMR spectra of proteins

A strategy for assigning the resonances in two-dimensional (2D) NMR spectra of proteins is described. The method emphasizes the analysis of through-space relationships between protons by use of the two-dimensional nuclear Overhauser effect (NOE) experiment. NOE patterns used in the algorithm were derived from a statistical analysis of the combinations of short proton-proton distances observed in the high-resolution crystal structures of 21 proteins. One starts with a search for authentic main-chain NH-C alpha H-C beta H J-coupled units, which can be found with high reliability. The many main-chain units of a protein are then placed in their proper juxtaposition by recognition of predefined NOE connectivity patterns. To discover these connectivities, the 2D NOE spectrum is examined, in a prescribed order, for the distinct NOE patterns characteristic of helices, sheets, turns, and extended chain. Finally, the recognition of a few amino acid side-chain types places the discovered secondary structure elements within the polypeptide sequence. Unlike the sequential assignment approach, the main-chain-directed strategy does not rely on the difficult task of recognizing many side-chain spin systems in J-correlated spectra, the assignment process is not in general sequential with the polypeptide chain, and the prescribed connectivity patterns are cyclic rather than linear. The latter characteristic avoids ambiguous branch points in the analysis and imposes an internally confirmatory property on each forward step.     

Sequence-specific recognition of DNA: assignment of nonexchangeable proton resonances in the consensus Pribnow promoter DNA sequence by two-dimensional NMR

The resonances of most of the nonexchangeable protons of both + and - strands of the consensus Pribnow dodecamer d( CGTTATAATGCG ) have been assigned by two-dimensional nuclear magnetic resonance methods. Application of the two-dimensional nuclear Overhauser effect ( NOESY ) sequential connectivity method, combined with two-dimensional autocorrelated ( COSY ) spectra to reveal scalar-coupled protons, results in assignment of virtually all of the base and sugar protons, except the sugar C5 protons which are inadequately resolved. Analysis of the nuclear Overhauser data indicates that the helix assumes a fairly uniform B form conformation.     

Spectral editing of two-dimensional magic-angle-spinning solid-state NMR spectra for protein resonance assignment and structure determination

Several techniques for spectral editing of 2D ¹³C?C¹³C correlation NMR of proteins are introduced. They greatly reduce the spectral overlap for five common amino acid types, thus simplifying spectral assignment and conformational analysis. The carboxyl (COO) signals of glutamate and aspartate are selected by suppressing the overlapping amide N?CCO peaks through ¹³C?C¹⁵N dipolar dephasing. The sidechain methine (CH) signals of valine, lecuine, and isoleucine are separated from the overlapping methylene (CH₂) signals of long-chain amino acids using a multiple-quantum dipolar transfer technique. Both the COO and CH selection methods take advantage of improved dipolar dephasing by asymmetric rotational-echo double resonance (REDOR), where every other ??-pulse is shifted from the center of a rotor period t_r by about 0.15 t_r. This asymmetry produces a deeper minimum in the REDOR dephasing curve and enables complete suppression of the undesired signals of immobile segments. Residual signals of mobile sidechains are positively identified by dynamics editing using recoupled ¹³C?C¹H dipolar dephasing. In all three experiments, the signals of carbons within a three-bond distance from the selected carbons are detected in the second spectral dimension via ¹³C spin exchange. The efficiencies of these spectral editing techniques range from 60?% for the COO and dynamic selection experiments to 25?% for the CH selection experiment, and are demonstrated on well-characterized model proteins GB1 and ubiquitin.     

Mestranol: the assignment of its 1H and 13C NMR spectra by means of two-dimensional NMR spectroscopy, and its photochemical decomposition

The 1H- and 13C-nmr spectra of mestranol were assigned with the help of a 2 D-J-resolved, a 2D spin echo J-correlated (SECSY) and a 2D 1H-13C hetero-shift correlation experiment. The analysis of the spectra facilitated the identification of some of the photodecomposition products of mestranol. It was shown that, upon irradiation with UV-B light in water-ethanol (1:1, v/v), products are formed by oxidation of rings B and C of the steroid.     

Complete assignment of the imino protons of Escherichia coli valine transfer RNA: two-dimensional NMR studies in water

The imino proton spectrum of Escherichia coli valine tRNA has been studied by two-dimensional nuclear Overhauser effect spectroscopy (NOESY) in H2O solution. The small nuclear Overhauser effects from the imino proton of an internal base pair to the imino protons of each nearest neighbor can be observed as off-diagonal cross-peaks. In this way most of the sequential NOE connectivity trains for all the helices in this molecule can be determined in a single experiment. AU resonances can be distinguished from GC resonances by the AU imino NOE to the aromatic adenine C2-H, thus leading to specific base-pair assignments. In general, the NOESY spectrum alone is not capable of assigning every imino proton resonance even in well-resolved tRNA spectra. Multiple proton peaks exhibit more than two cross-peaks, resulting in ambiguous connectivities, and coupling between protons with similar chemical shifts produces cross-peaks that are incompletely resolved from the diagonal. The sequence of the particular tRNA determines the occurrence of the latter problem, which can often be solved by careful one-dimensional experiments. The complete imino proton assignments of E. coli valine tRNA are presented.     

Refinement of the main chain directed assignment strategy for the analysis of 1H NMR spectra of proteins.

The underlying basis of the main chain directed (MCD) resonance assignment strategy for the analysis of 1H NMR spectra of proteins is reexamined. The criteria used in the construction of the patterns used in the MCD method have been extended to increase the robustness of the approach to the presence of variable protein secondary structure and significant spectral degeneracy. These criteria have led to the development of several dozen patterns exclusively involving the short distance relationships between main chain amide NH-C alpha-H-C beta H (NAB) J-coupled subspin systems of the amino acid residues. The MCD patterns have been examined for fidelity and frequency of occurrence in a database composed of the high resolution crystal structures of 39 proteins. The analysis has identified several extremely robust patterns, suitable for initiating a hierarchical construction of units of secondary structure based upon a systematic analysis of two-dimensional nuclear Overhauser effect spectra. A formal procedure, suitable for the computer assisted application of the MCD strategy, is developed. This procedure, termed MCDPAT, has been applied to the analysis of the crystal structures of human ubiquitin, T4 lysozyme, and ribonuclease A. It has been found that the MCDPAT procedure is conservative producing no significant errors and is globally successful in correctly identifying the appropriate units of secondary structure contained in these three proteins.     

Sequential resonance assignments in 1H NMR spectra of oligonucleotides by two-dimensional NMR spectroscopy

A sequential assignment procedure is outlined, based on two-dimensional NOE ( NOESY ) and two-dimensional J-correlated spectroscopy ( COSY ), for assigning the nonexchangeable proton resonances in NMR spectra of oligonucleotides. As presented here the method is generally applicable to right-handed helical oligonucleotides of intermediate size. We applied it to a lac operator DNA fragment consisting of d( TGAGCGG ) and d( CCGCTCA ) and obtained complete assignments for the adenine H8, guanine H8, cytosine H6 and H5, thymine H6 and 5-methyl, and the deoxyribose H1', H2', H2", H3', and H4' resonances, as well as some H5', H5" (pairwise) assignments. These assignments are required for the analysis of two-dimensional NOE and J-coupling data in terms of the solution structure of oligonucleotides.     

A modified strategy for sequence specific assignment of protein NMR spectra based on amino acid type selective experiments

The determination of the three-dimensional structure of a protein or the study of protein–ligand interactions requires the assignment of all relevant nuclei as an initial step. This is nowadays almost exclusively performed using triple-resonance experiments. The conventional strategy utilizes one or more pairs of three dimensional spectra to obtain redundant information and thus reliable assignments. Here, a modified strategy for obtaining sequence specific assignments based on two dimensional amino acid type selective triple-resonance experiments is proposed. These experiments can be recorded with good resolution in a relatively short time. They provide very specific and redundant information, in particular on sequential connectivities, that drastically increases the ease and reliability of the assignment procedure, done either manually or in an automated fashion. The new strategy is demonstrated with the protein domain PB1 from yeast CDC24p. Dedicated to Rüdiger Winter ( 06.04.2004)     

Solution structural characteristics of cyanometmyoglobin: resonance assignment of heme cavity residues by two-dimensional NMR

Steady-state nuclear Overhauser effects (NOE), two-dimensional (2D) nuclear Overhauser effect spectroscopy (NOESY), and 2D spin correlation spectroscopy (COSY) have been applied to the fully paramagnetic low-spin, cyanide-ligated complex of sperm whale ferric myoglobin to assign the majority of the heme pocket side-chain proton signals and the remainder of the heme signals. It is shown that the 2D NOESY map reveals essentially all dipolar connectivities observed in ordinary 1D NOE experiments and expected on the basis of crystal coordinates, albeit often more weakly than in a diamagnetic analogue. For extremely broad (approximately 600-Hz) and rapidly relaxing (Tf1 approximately 3 ms) signals which show no NEOSY peaks, we demonstrate that conventional steady-state NOEs obtained under very rapid pulsing conditions still allow detection of the critical dipoar connectivities that allow unambiguous assignments. The COSY map was found to be generally less useful for the hyperfine-shifted residues, with cross peaks detected only for protons greater than 6 A from the iron. Nevertheless, numerous critical COSY cross peaks between strongly hyperfine-shifted peaks were resolved and assigned. In all, 95% (53 of 56 signals) of the total proton sets within approximately 7.5 A of the iron, the region experiencing the strongest hyperfine shifts and paramagnetic relaxation, are now unambiguously assigned. Hence it is clear that the 2D methods can be profitably applied to paramagnetic proteins. The scope and limitations of such application are discussed. The resulting hyperfine shift pattern for the heme confirmed expectations based on model compounds. In contrast, while exhibiting fortuitous 1H NMR spectral similarities, a major discrepancy was uncovered between the hyperfine shift pattern of the axially bound (F8 histidyl) imidazole in the protein and that of the imidazole in a relevant model compound [Chacko, V.P., & La Mar, G. N. (1982) J. Am. Chem. Soc. 104, 7002-7007], providing direct evidence for a protein-based deformation of axial bonding in the protein.     

Mass transfer characterization of an airlift probe for oxygenating and mixing cell suspensions in an NMR spectrometer

The oxygen transfer characteristics of a 20-mm O.D. airlift contactor fitted with an oxygen microelectrode were determined by steady-state sulfite oxidation measurements. The volumetric mass transfer coefficient k(L)a was proportional to sparging power input per unit volume raised to a power which varied from 0.41 in water (coalescing bubbles) to 0.76 in NaCl solutions (noncoalescing bubbles). The highest observed k(L)a value was 0.012 s(-1) which is sufficient to aerate Escherichia coli in an NMR spectrometer at moderate to high cell densities, depending on the physiological state of the cells.     

Ansig for Windows: An interactive computer program for semiautomatic assignment of protein NMR spectra

Assignment of NMR spectra is a prerequisite for structure determination of proteins using NMR. The time spent on the assignment is comparatively long compared to that spent on other parts in the protein structure determination process, but it can be shortened by using either interactive or fully automated computer programs. To benefit from the advantages of both types of program we have developed a version of the interactive assignment program ANSIG to include automatized, yet user-supervised, routines. The new program includes tools for (i) semiautomatic sequential assignment, (ii) plotting of distances from PDB structure files directly in NMR spectra and (iii) statistical analysis of distance restraint violations with the possibility to directly zoom to violated NOEs in NOESY spectra.     

The rate enhancement produced by the ribosome: an improved model

As a model for mechanistic comparison with peptidyl transfer within the ribosome, the reaction of aqueous glycinamide with N-formylphenylalanine trifluoroethyl ester (fPhe-TFE) represents an improvement over earlier model reactions involving Tris. The acidity of trifluoroethanol (pKa 12.4) resembles that of tRNA (12.98) more closely than do the acidities of model reactants described earlier, and the reactivity of the simple nucleophile glycinamide is free of potential complications that arise from alternative reaction pathways available to Tris. At 25 degrees C, the uncatalyzed reaction of glycinamide with fPhe-TFE proceeds with a second-order rate constant of 3 x 10(-5) M-1 s-1; DeltaH(++) = +7.8 kcal/mol; TDeltaS(++)= -15.7 kcal/mol. The ribosomal reaction of puromycin with fMet-tRNA proceeds 3 x 107-fold more rapidly, with a second-order rate constant (kcat/Km) of 1 x 10(3) M-1 s-1; DeltaH(++) = +16.0 kcal/mol; TDeltaS(++)= +2.0 kcal/mol. That rate enhancement, an order of magnitude larger than estimated earlier, is fully explained by the more favorable entropy of activation of the ribosomal reaction. Experiments involving ethylene glycol esters suggest that neighboring -OH group effects are negligible in the presence of solvent water, which itself acts as a general base catalyst. In the desolvated interior of the ribosome, the vicinal 2'-OH group of aminoacyl-tRNA probably replaces water as a general base catalyst. But the catalytic effect of the ribosome itself is overwhelmingly entropic in origin, suggesting that the ribosome achieves its effect by physical desolvation and/or juxtaposition of the reactants in a manner conducive to peptidyl transfer.     

NMR distance measurements in DNA duplexes: sugars and bases have the same correlation times

To evaluate whether the sugar moieties of short DNA duplexes exhibit local motion of sufficient amplitude to affect interproton distance measurements, we have carried out a series of time-dependent NOESY experiments at increasingly shorter mixing times on dodecamer DNA duplexes. By use of the cytosine H5-H6 vector as a known distance in the bases and the geminal 2'H-2'H vector as a known distance in the sugars, the corresponding apparent cross-relaxation rates were sampled at various mixing times. While the ratio of the inverse sixth power of these two fixed distances is in the range 6-7, when the system is sampled at 100 ms the apparent initial rate of growth of the 2'H-2'H NOESY crosspeak is only 1.9-2.0 times faster than that of the H5-H6 crosspeak--in agreement with the results of Clore and Gronenborn [Clore, G. M., & Gronenborn, A. M. (1984) FEBS Lett. 172, 219; (1984) FEBS Lett. 175, 117] and of Gronenborn and Clore [Gronenborn, A. M., & Clore, G. M. (1985) Prog. NMR Spectrosc. 17, 1]. This observation was interpreted to indicate the existence of internal mobility with a 3-fold shorter correlation time for the sugar moieties in DNA and led to the use of this shorter correlation time to estimate sugar-sugar proton distances and many sugar-base proton distances in subsequent DNA structure determination. We have examined 2'H-2"H cross-relaxation and H5-H6 cross-relaxation at 100, 90, 60, 30, and 15 ms in dodecamer DNA duplexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

MAP-XSII: an improved program for the automatic assignment of methyl resonances in large proteins

NMR studies of large proteins have gathered much interest in recent years, especially after methyl-transverse relaxation optimized spectroscopy was successfully applied to systems as large as ~1 MDa in molecular weight. However, to fully take advantage of these spectra, there is a need for convenient and robust methods for making resonance assignments rapidly. Here, we present an improved version of our program MAP-XS (methyl assignment prediction from X-ray structure) for the automatic assignment of methyl peaks, based on nuclear Overhauser effects (NOE) correlations and chemical shifts together with available structures. No manual analysis of the NOE data is needed in this new version, which helps to further accelerate the assignment process. A refined algorithm as well as more efficient sampling produces results from single runs of MAP-XSII using unanalyzed NOE data are comparable to those achieved by the old version using manually curated data with every NOE peak correctly attributed to the two related methyl peaks; in addition, checking the results from multiple parallel runs against each other provides an effective mechanism for getting rid of the wrong assignments while keeping the correct ones, which significantly improves the reliability of final assignments. The new program is tested against three different proteins and delivers ~95 % correct assignments; positive results are also achieved for tests using different cut-off distances for NOEs, structures of lower resolutions, and ambiguous residue types.     

Graph-theoretical assignment of secondary structure in multidimensional protein NMR spectra: Application to the lac repressor headpiece

Summary A novel procedure is presented for the automatic identification of secondary structures in proteins from their corresponding NOE data. The method uses a branch of mathematics known as graph theory to identify prescribed NOE connectivity patterns characteristic of the regular secondary structures. Resonance assignment is achieved by connecting these patterns of secondary structure together, thereby matching the connected spin systems to specific segments of the protein sequence. The method known as SERENDIPITY refers to a set of routines developed in a modular fashion, where each program has one or several well-defined tasks. NOE templates for several secondary structure motifs have been developed and the method has been successfully applied to data obtained from NOESY-type spectra. The present report describes the application of the SERENDIPITY protocol to a 3D NOESY-HMQC spectrum of the ¹⁵N-labelled lac repressor headpiece protein. The application demonstrates that, under favourable conditions, fully automated identification of secondary structures and semi-automated assignment are feasible.Abbreviations 2D, 3D two-, three-dimensional - NOESY nuclear Overhauser enhancement spectroscopy - HMQC heteronuclear multiple quantum coherence - SSE secondary structure element - SERENDIPITY SEcondary structuRE ideNtification in multiDImensional ProteIn specTra analYsis Supplementary Material available from the authors: Two tables containing the total number of mappings resulting from the graph search procedure for simulated and experimental NOE data.     

Aliphatic chain length by isotropic mixing (ALCHIM): determining composition of complex lipid samples by 1H NMR spectroscopy

Quantifying the amounts and types of lipids present in mixtures is important in fields as diverse as medicine, food science, and biochemistry. Nuclear magnetic resonance (NMR) spectroscopy can quantify the total amounts of saturated and unsaturated fatty acids in mixtures, but identifying the length of saturated fatty acid or the position of unsaturation by NMR is a daunting challenge. We have developed an NMR technique, aliphatic chain length by isotropic mixing, to address this problem. Using a selective total correlation spectroscopy technique to excite and transfer magnetization from a resolved resonance, we demonstrate that the time dependence of this transfer to another resolved site depends linearly on the number of aliphatic carbons separating the two sites. This technique is applied to complex natural mixtures allowing the identification and quantification of the constituent fatty acids. The method has been applied to whole adipocytes demonstrating that it will be of great use in studies of whole tissues.     

Complete assignment of the 500 MHz 1H-NMR spectra of bleomycin A2 in H2O and D2O solution by means of two-dimensional NMR spectroscopy

1H-NMR spectra of bleomycin A2 recorded at 500 MHz in D2O and H2O at 24 degrees C and 3 degrees C were investigated. Resonances of the individual spin systems were identified by using two-dimensional correlation spectroscopy (COSY), two-dimensional spin echo correlated spectroscopy (SECSY) and by the application of two-dimensional Nuclear Overhauser Effect spectroscopy (NOESY). Employment of these techniques allowed the assignment of 113 exchangeable and 59 non-exchangeable protons in the 1H NMR spectrum of bleomycin A2. By means of 2D NOE spectroscopy also interresidual connectivities could be observed. Comparison of the NOESY spectra at 3 degrees C and 24 degrees C suggest that at low temperatures the central party of the bleomycin A2 molecule tends to adopt an extended conformation.     

Dilute spin-exchange assignment of solid-state NMR spectra of oriented proteins: Acetylcholine M2 in bilayers

The assignment of amide resonances in the two-dimensional PISEMA (Polarization Inversion with Spin Exchange at the Magic Angle) spectrum of uniformly ¹⁵N labeled M2 peptide corresponding to the channel-lining segment of the acetylcholine receptor in oriented phospholipid bilayers is described. The majority of the resonances were assigned through comparisons with spectra from selectively ¹⁵N labeled recombinant peptides and specifically ¹⁵N labeled synthetic peptides. Some resonances were assigned to specific amino acid residues by means of homonuclear ¹⁵N spin-exchange spectroscopy. A modification to the conventional spin-exchange pulse sequence that significantly shortens the length of the experiments by combining the intervals for ¹⁵ N spin-exchange and ¹H magnetization recovery is described.