Estimation of protein secondary structure content directly from NMR spectra using an improved empirical correlation with averaged chemical shift

We have recently shown that the averaged chemical shift (ACS) of a nucleus in the protein backbone correlates well empirically to its secondary structure content (SSC). This allows the estimation of SSC directly from the NMR spectrum without the time intensive process of chemical shift assignment. Here, we present an empirical correlation that accounts both for contributions to the relevant protein and chemical shift databases made subsequent to the original analysis, and for missing or inconsistently referenced resonances. Our results affirm that this method provides a significant tool for initial structural prediction from NMR data prior to complete chemical shift assignment.     

An evaluation of chemical shift index-based secondary structure determination in proteins: Influence of random coil chemical shifts

Random coil chemical shifts are commonly used to detect protein secondary structural elements in chemical shift index (CSI) calculations. Though this technique is widely used and seems reliable for folded proteins, the choice of reference random coil chemical shift values can significantly alter the outcome of secondary structure estimation. In order to evaluate these effects, we present a comparison of secondary structure content calculated using CSI, based on five different reference random coil chemical shift value sets, to that derived from three-dimensional structures.Our results show that none of the reference random coil data sets chosen for evaluation fully reproduces the actual secondary structures. Among the reference values generally available to date, most tend to be good estimators only of helices. Based on our evaluation, we recommend the experimental values measured by Schwarzinger et al.(2000), and statistical values obtained by Lukin et al. (1997), as good estimators of both helical and sheet content.     

Influence of the completeness of chemical shift assignments on NMR structures obtained with automated NOE assignment

Reliable automated NOE assignment and structure calculation on the basis of a largely complete, assigned input chemical shift list and a list of unassigned NOESY cross peaks has recently become feasible for routine NMR protein structure calculation and has been shown to yield results that are equivalent to those of the conventional, manual approach. However, these algorithms rely on the availability of a virtually complete list of the chemical shifts. This paper investigates the influence of incomplete chemical shift assignments on the reliability of NMR structures obtained with automated NOESY cross peak assignment. The program CYANA was used for combined automated NOESY assignment with the CANDID algorithm and structure calculations with torsion angle dynamics at various degrees of completeness of the chemical shift assignment which was simulated by random omission of entries in the experimental ¹H chemical shift lists that had been used for the earlier, conventional structure determinations of two proteins. Sets of structure calculations were performed choosing the omitted chemical shifts randomly among all assigned hydrogen atoms, or among aromatic hydrogen atoms. For comparison, automated NOESY assignment and structure calculations were performed with the complete experimental chemical shift but under random omission of NOESY cross peaks. When heteronuclear-resolved three-dimensional NOESY spectra are available the current CANDID algorithm yields in the absence of up to about 10% of the experimental ¹H chemical shifts reliable NOE assignments and three-dimensional structures that deviate by less than 2 Å from the reference structure obtained using all experimental chemical shift assignments. In contrast, the algorithm can accommodate the omission of up to 50% of the cross peaks in heteronuclear- resolved NOESY spectra without producing structures with a RMSD of more than 2 Å to the reference structure. When only homonuclear NOESY spectra are available, the algorithm is slightly more susceptible to missing data and can tolerate the absence of up to about 7% of the experimental ¹H chemical shifts or of up to 30% of the NOESY peaks.Abbreviations: BmPBP^A – Bombyx mori pheromone binding protein form A; CYANA – combined assignment and dynamics algorithm for NMR applications; NMR – nuclear magnetic resonance; NOE – nuclear Overhauser effect; NOESY – NOE spectroscopy; RMSD – root-mean-square deviation; WmKT – Williopsis mrakii killer toxin     

Relaxation data in NMR structure determination: model calculations for the lysozyme-Gd3+ complex

The effect of including paramagnetic relaxation data as additional restraints in the determination of protein tertiary structures from NMR data has been explored by a systematic series of model calculations. The system used for testing the method was the 2.0 A resolution tetragonal crystal structure of hen egg white lysozyme (129 amino acid residues) and structures were generated using a version of the hybrid "distance geometry-dynamic simulated annealing" procedure. A limited set of 769 NOEs was used as restraints in all the calculations; the strengths of these were categorized into three classes on the basis of distances observed in the crystal structure. The values of 50 phi angles were also restrained on the basis of amide-alpha coupling constants calculated from the X-ray structure. Five sets of 12 structures were determined using differing sets of paramagnetic relaxation data as restraints additional to those involving the NOE and coupling constant data. The paramagnetic relaxation data were modeled on the basis of the distances of defined protons from the crystallographic binding site of Gd3+ in lysozyme. Analysis of the results showed that the relaxation data significantly improved the correspondence between the set of generated structures and the crystal structure, and that the more well defined the relaxation data, the more significant the improvement in the quality of the structures. The results suggest that the inclusion of paramagnetic relaxation restraints could be of significant value for the experimental determination of protein structures from NMR data.     

Principal components analysis of protein structure ensembles calculated using NMR data

One important problem when calculating structures of biomolecules from NMR data is distinguishing converged structures from outlier structures. This paper describes how Principal Components Analysis (PCA) has the potential to classify calculated structures automatically, according to correlated structural variation across the population. PCA analysis has the additional advantage that it highlights regions of proteins which are varying across the population. To apply PCA, protein structures have to be reduced in complexity and this paper describes two different representations of protein structures which achieve this. The calculated structures of a 28 amino acid peptide are used to demonstrate the methods. The two different representations of protein structure are shown to give equivalent results, and correct results are obtained even though the ensemble of structures used as an example contains two different protein conformations. The PCA analysis also correctly identifies the structural differences between the two conformations.     

Analysis of 1H chemical shifts in DNA: Assessment of the reliability of 1H chemical shift calculations for use in structure refinement

The reliability of ¹H chemical shift calculations for DNA is assessed by comparing the experimentally and calculated chemical shifts of a reasonably large number of independently determined DNA structures. The calculated chemical shifts are based on semiempirical relations derived by Giessner-Prettre and Pullman [(1987) Q. Rev. Biophys., 20, 113–172]. The standard deviation between calculated and observed chemical shifts is found to be quite small, i.e. 0.17 ppm. This high accuracy, which is achieved without parameter adjustment, makes it possible to analyze the structural dependencies of chemical shifts in a reliable fashion. The conformation-dependent ¹H chemical shift is mainly determined by the ring current effect and the local magnetic anisotropy, while the third possible effect, that of the electric field, is surprisingly small. It was further found that for a double helical environment, the chemical shift of the sugar protons, H2 to H5, is mainly affected by the ring current and magnetic anisotropy of their own base. Consequently, the chemical shift of these sugar protons is determined by two factors, namely the type of base to which the sugar ring is attached, C, T, A, or G, and secondly by the -angle. In particular, the H2 shift varies strongly with the -angle, and strong upfield H2 shifts directly indicate that the -angle is in the syn domain. The H1 shift is not only strongly affected by its own base, but also by its 3-neighboring base. On the other hand, base protons, in particular H5 of cytosine and methyl protons of thymine, are affected mainly by the 5-neighboring bases, although some effect (0.2 ppm) stems from the 3-neighboring base. The H2 protons are mainly affected by the 3-neighboring base. As a result of these findings a simple scheme is proposed for sequential assignment of resonances from B-helices based on chemical shifts.     

Improved efficiency of protein structure calculations from NMR data using the program DIANA with redundant dihedral angle constraints

Summary A new strategy for NMR structure calculations of proteins with the variable target function method (Braun, W. and Go, N. (1985)J. Mol. Biol.,186, 611) is described, which makes use of redundant dihedral angle constraints (REDAC) derived from preliminary calculations of the complete structure. The REDAC approach reduces the computation time for obtaining a group of acceptable conformers with the program DIANA 5-100-fold, depending on the complexity of the protein structure, and retains good sampling of conformation space.Dedicated to the memory of Professor V.F. Bystrov     

An NMR method for the determination of protein binding interfaces using TEMPOL-induced chemical shift perturbations

Background

The determination of protein–protein interfaces is of crucial importance to understand protein function and to guide the design of compounds. To identify protein–protein interface by NMR spectroscopy, ¹³C NMR paramagnetic shifts induced by freely diffusing 4-hydroxy-2, 2, 6, 6-tetramethyl-piperidine-1-oxyl (TEMPOL) are promising, because TEMPOL affects distinct ¹³C NMR chemical shifts of the solvent accessible nuclei belonging to proteins of interest, while ¹³C nuclei within the interior of the proteins may be distinguished by a lack of such shifts.

Method

We measured the ¹³C NMR paramagnetic shifts induced by TEMPOL by recording ¹³C–¹³C TOCSY spectra for ubiquitin in the free state and the complex state with yeast ubiquitin hydrolase1 (YUH1).

Results

Upon complexation of ubiquitin with YUH1, ¹³C NMR paramagnetic shifts associated with the protein binding interface were reduced by 0.05 ppm or more. The identified interfacial atoms agreed with the prior X-ray crystallographic data.

Conclusions

The TEMPOL-induced ¹³C chemical shift perturbation is useful to determine precise protein–protein interfaces.

General significance

The present method is a useful method to determine protein–protein interface by NMR, because it has advantages in easy sample preparations, simple data analyses, and wide applicabilities.     

Prediction algorithm for amino acid types with their secondary structure in proteins (PLATON) using chemical shifts

The algorithm PLATON is able to assign sets of chemical shifts derived from a single residue to amino acid types with its secondary structure (amino acid species). A subsequent ranking procedure using optionally two different penalty functions yields predictions for possible amino acid species for the given set of chemical shifts. This was demonstrated in the case of the -spectrin SH3 domain and applied to 9 further protein data sets taken from the BioMagRes database. A database consisting of reference chemical shift patterns (reference CSPs) was generated from assigned chemical shifts of proteins with known 3D-structure. This reference CSP database is used in our approach for extracting distributions of amino acid types with their most likely secondary structure elements (namely -helix, -sheet, and coil) for single amino acids by comparison with query CSPs. Results obtained for the 10 investigated proteins indicates that the percentage of correct amino acid species in the first three positions in the ranking list, ranges from 71.4% to 93.2% for the more favorable penalty function. Where only the top result of the ranking list for these 10 proteins is considered, 36.5% to 83.1% of the amino acid species are correctly predicted. The main advantage of our approach, over other methods that rely on average chemical shift values is the ability to increase database content by incorporating newly derived CSPs, and therefore to improve PLATON's performance over time.     

Determination of the three-dimensional structure of oligosaccharides in the solid state from experimental 13C NMR data and ab initio chemical shift surfaces

A novel method for the determination of the three-dimensional (3D) structure of oligosaccharides in the solid state using experimental 13C NMR data is presented. The approach employs this information, combined with 13C chemical shift surfaces (CSSs) for the glycosidic bond carbons in the generation of NMR pseudopotential energy functions suitable for use as constraints in molecular modeling simulations. Application of the method to trehalose, cellobiose, and cellotetraose produces 3D models that agree remarkably well with the reported X-ray structures, with phi and psi dihedral angles that are within 10 degrees from the ones observed in the crystals. The usefulness of the approach is further demonstrated in the determination of the 3D structure of the cellohexaose, an hexasaccharide for which no X-ray data has been reported, as well as in the generation of accurate structural models for cellulose II and amylose V6.     

Direct structure refinement of high molecular weight proteins against residual dipolar couplings and carbonyl chemical shift changes upon alignment: an application to maltose binding protein

The global fold of maltose binding protein in complex with -cyclodextrin has been determined using a CNS-based torsion angle molecular dynamics protocol involving direct refinement against dipolar couplings and carbonyl chemical shift changes that occur upon alignment. The shift changes have been included as structural restraints using a new module, CANI, that has been incorporated into CNS. Force constants and timesteps have been determined that are particularly effective in structure refinement applications involving high molecular weight proteins with small to moderate numbers of NOE restraints. Solution structures of the N- and C-domains of MBP calculated with this new protocol are within 2 Å of the X-ray conformation.     

The 13C Chemical-Shift Index: A simple method for the identification of protein secondary structure using 13C chemical-shift data

Summary 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 ¹H chemical shifts. By extending the Chemical-Shift Index to include ¹³C, ¹³C 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.Supplementary material is available in the form of a 10-page table (Table S1) describing the exact location of secondary structures in all 20 proteins as determined using the methods described in this paper. Requests for Table S1 should be directed to the authors.     

Metropolis Monte Carlo calculations of DNA structure using internal coordinates and NMR distance restraints: An alternative method for generating a high-resolution solution structure

Summary A new method, a restrained Monte Carlo (rMC) calculation, is demonstrated for generating high-resolution structures of DNA oligonucleotides in solution from interproton distance restraints and bounds derived from complete relaxation matrix analysis of two-dimensional nuclear Overhauser effect (NOE) spectral peak intensities. As in the case of restrained molecular dynamics (rMD) refinement of structures, the experimental distance restraints and bounds are incorporated as a pseudo-energy term (or penalty function) into the mathematical expression for the molecular energy. However, the use of generalized helical parameters, rather than Cartesian coordinates, to define DNA conformation increases efficiency by decreasing by an order of magnitude the number of parameters needed to describe a conformation and by simplifying the potential energy profile. The Metropolis Monte Carlo method is employed to simulate an annealing process. The rMC method was applied to experimental 2D NOE data from the octamer duplex d(GTA-TAATG)·d(CATTATAC). Using starting structures from different locations in conformational space (e.g. A-DNA and B-DNA), the rMC calculations readily converged, with a root-mean-square deviation (RMSD) of <0.3 Å between structures generated using different protocols and starting structures. Theoretical 2D NOE peak intensities were calculated for the rMC-generated structures using the complete relaxation matrix program CORMA, enabling a comparison with experimental intensities via residual indices. Simulation of the vicinal proton coupling constants was carried out for the structures generated, enabling a comparison with the experimental deoxyribose ring coupling constants, which were not utilized in the structure determination in the case of the rMC simulations. Agreement with experimental 2D NOE and scalar coupling data was good in all cases. The rMC structures are quite similar to that refined by a traditional restrained MD approach (RMSD<0.5 Å) despite the different force fields used and despite the fact that MD refinement was conducted with additional restraints imposed on the endocyclic torsion angles of deoxyriboses. The computational time required for the rMC and rMD calculations is about the same. A comparison of structural parameters is made and some limitations of both methods are discussed with regard to the average nature of the experimental restraints used in the refinement.Abbreviations MC Monte Carlo - rMC restrained Monte Carlo - MD molecular dynamics - rMD restrained molecular dynamics - DG distance geometry - EM energy minimization - 2D NOE two-dimensional nuclear Overhauser effect - DQF-COSY double-quantum-filtered correlation spectroscopy - RMSD root-mean-square deviationTo whom correspondence should be addressed.     

Determination of the torsion angles of alanine and glycine residues of model compounds of spider silk (AGG)(10) using solid-state NMR methods

Spiders synthesize several kinds of silk fibers. In the primary structure of spider silk, one of the major ampullate (dragline, frame) silks, spidroin 1, and flagelliform silk (core fibers of adhesive spiral), there are common repeated X-Gly-Gly (X = Ala, Leu, Pro, Tyr, Glu, and Arg) sequences, which are considered to be related to the elastic character of these fibers. In this paper, two dimensional spin diffusion solid-state NMR under off magic angle spinning (OMAS), ¹³C chemical shift contour plots, and Rotational Echo DOuble Resonance (REDOR) were applied to determine the torsion angles of one Ala and two kinds of Gly residues in the Ala-Gly-Gly sequence of ¹³C=O isotope-labeled (Ala-Gly-Gly)₁₀. The torsion angles were determined to be (, ) = (–90°, 150° ) within an experimental error of ±10° for each residue. This conformation is characterized as 3₁ helix which is in agreement with the structure proposed from the X-ray powder diffraction pattern of poly(Ala-Gly-Gly). The 3₁ helix of (Ala-Gly-Gly)₁₀ does not change by formic acid treatment although (Ala-Gly)₁₅ easily changes from the silk I conformation (the structure of Bombyx mori silk fibroin before spinning in the solid state) to silk II conformation (the structure of the silk fiber after spinning) by such treatment. Thus, the 3₁ helix conformation of (Ala-Gly-Gly)₁₀ is considered very stable. Furthermore, the torsion angles of the 16th Leu residue of (Leu-Gly-Gly)₁₀ were also determined as (, ) = (–90°, 150° ) and this peptide is also considered to take 3₁ helix conformation.     

Conformational isomerism of the binuclear N,N-pentamethylenedithiocarbamate cadmium(II) complex, [Cd2{S2CN(CH2)5}4] on multinuclear (N, Cd) CP/MAS NMR and single-crystal X-ray diffraction data

Crystalline N,N-cyclo-pentamethylenedithiocarbamate (PmDtc) cadmium(II) complex was prepared and studied by means of ¹⁵N, ¹¹³Cd CP/MAS NMR spectroscopy and single-crystal X-ray diffraction. The unit cell of the cadmium(II) compound comprises two centrosymmetric isomeric binuclear molecules [Cd₂{S₂CN(CH₂)₅}₄], which display structural inequivalence in both ¹⁵N and ¹¹³Cd NMR and XRD data. There are pairs of the dithiocarbamate ligands exhibiting different structural functions in both isomeric molecules. Each of the terminal ligands is bidentately coordinated to the cadmium atom and forms a planar four-membered chelate ring [CdS₂C]; whereas pairs of the tridentate bridging ligands combine two neighbouring cadmium atoms forming an extended eight-membered tricyclic moieties [Cd₂S₄C₂], whose geometry can be approximated by a ‘chair’ conformation. The structural states of cadmium atoms were characterised by almost axially symmetric ¹¹³Cd chemical shift tensors. All experimental ¹⁵N resonance lines were assigned to the nitrogen structural sites in both isomeric binuclear molecules.     

Nonmyristoylated Matrix Protein from the Mason-Pfizer Monkey Virus Forms Oligomers

We studied the oligomeric properties of betaretroviral nonmyristoylated matrix protein (MA) and its R55F mutant from the Mason-Pfizer monkey virus in solution by means of chemical crosslinking and NMR spectroscopy. By analyzing crosslinked products and using concentration-dependent NMR chemical shift mapping, we have proven that the wild-type (WT) MA forms oligomers in solution. Conversely, no oligomerization was observed for the R55F mutant. Structural comparison of MAs explained their different behaviors in solution, concluding that the key residues involved in intermonomeric interaction are exposed in the WT MA but buried in the mutant, preventing the oligomerization of R55F. The final model of oligomerization of the WT MA was derived by concerted use of chemical shift mapping and diffusion-ordered spectroscopy measured on a set of protein samples with varying concentrations. We found that the Mason-Pfizer monkey virus WT MA exists in a monomer-dimer-trimer equilibrium in solution, with the corresponding dissociation constants of 2.3  and 0.24 mM, respectively. Structures of the oligomers calculated with HADDOCK software are closely related to the structures of other retroviral MA trimers.