CSI 2.0: a significantly improved version of the Chemical Shift Index

Protein chemical shifts have long been used by NMR spectroscopists to assist with secondary structure assignment and to provide useful distance and torsion angle constraint data for structure determination. One of the most widely used methods for secondary structure identification is called the Chemical Shift Index (CSI). The CSI method uses a simple digital chemical shift filter to locate secondary structures along the protein chain using backbone ¹³C and ¹H chemical shifts. While the CSI method is simple to use and easy to implement, it is only about 75–80 % accurate. Here we describe a significantly improved version of the CSI (2.0) that uses machine-learning techniques to combine all six backbone chemical shifts (¹³C_α, ¹³C_β, ¹³C, ¹⁵N, ¹HN, ¹H_α) with sequence-derived features to perform far more accurate secondary structure identification. Our tests indicate that CSI 2.0 achieved an average identification accuracy (Q3) of 90.56 % for a training set of 181 proteins in a repeated tenfold cross-validation and 89.35 % for a test set of 59 proteins. This represents a significant improvement over other state-of-the-art chemical shift-based methods. In particular, the level of performance of CSI 2.0 is equal to that of standard methods, such as DSSP and STRIDE, used to identify secondary structures via 3D coordinate data. This suggests that CSI 2.0 could be used both in providing accurate NMR constraint data in the early stages of protein structure determination as well as in defining secondary structure locations in the final protein model(s). A CSI 2.0 web server (http://csi.wishartlab.com) is available for submitting the input queries for secondary structure identification.     

Probabilistic Approach to Determining Unbiased Random-coil Carbon-13 Chemical Shift Values from the Protein Chemical Shift Database

We describe a probabilistic model for deriving, from the database of assigned chemical shifts, a set of random coil chemical shift values that are “unbiased” insofar as contributions from detectable secondary structure have been minimized (RCCS_u). We have used this approach to derive a set of RCCS_u values for ¹³C^α and ¹³C^β for 17 of the 20 standard amino acid residue types by taking advantage of the known opposite conformational dependence of these parameters. We present a second probabilistic approach that utilizes the maximum entropy principle to analyze the database of ¹³C^α and ¹³C^β chemical shifts considered separately; this approach yielded a second set of random coil chemical shifts (RCCS). Both new approaches analyze the chemical shift database without reference to known structure. Prior approaches have used either the chemical shifts of small peptides assumed to model the random coil state (RCCS_peptide) or statistical analysis of chemical shifts associated with structure not in helical or strand conformation (RCCS). We show that the RCCS values are strikingly similar to published RCCS_peptide and RCCS values. By contrast, the RCCS_u values differ significantly from both published types of random coil chemical shift values. The differences (RCCS_peptide−RCCS_u) for individual residue types show a correlation with known intrinsic conformational propensities. These results suggest that random coil chemical shift values from both prior approaches are biased by conformational preferences. RCCS_u values appear to be consistent with the current concept of the “random coil” as the state in which the geometry of the polypeptide ensemble samples the allowed region of (ϕ,ψ)-space in the absence of any dominant stabilizing interactions and thus represent an improved basis for the detection of secondary structure. Coupled with the growing database of chemical shifts, this probabilistic approach makes it possible to refine relationships among chemical shifts, their conformational propensities, and their dependence on pH, temperature, or neighboring residue type.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Chemical speciation applied to bio-inorganic chemistry

The processes of converting new chemistry into new healthcare, environment-friendly, products are considerably facilitated when chemical speciation data are available. These identify causal links between speciation and biological responses based upon specific chemical species rather than total concentrations. The effective roles of bismuth for ulcer treatment, of Ca, Zn, Sn and F in dentifrices, and of zinc species and common cold symptoms are described. A new readily biodegradable ligand for minimising the environmental impact of washing powders, Al and Alzheimer's disease (and the absence of a causal link), and an audit of Cu and Zn flow from wound dressing through wound fluid to healing tissue, are all discussed in detail.     

Switched-angle spinning applied to bicelles containing phospholipid-associated peptides

In a model study, the proton NMR spectrum of the opioid pentapeptide leucine-enkephalin associated with bicelles is investigated. The spectral resolution for a static sample is limited due to the large number of anisotropic interactions, in particular strong proton–proton couplings, but resolution is greatly improved by magic-angle sample spinning. Here we present two-dimensional switched-angle spinning NMR experiments, which correlate the high-resolution spectrum of the membrane-bound peptide under magic-angle spinning with its anisotropic spectrum, leading to well-resolved spectra. The two-dimensional spectrum allows the exploitation of the high resolution of the isotropic spectrum, while retaining the structural information imparted by the anisotropic interactions in the static spectrum. Furthermore, switched-angle spinning techniques are demonstrated that allow one to record the proton spectrum of ordered bicellar phases as a function of the angle between the rotor axis and the magnetic field direction, thereby scaling the dipolar interactions by a predefined factor.     

Chemical modification of peptides by hydrazine.

High pressure ('performance') liquid chromatography on reverse-phase supports has been used to characterize the products arising from the hydrazine treatment of peptides. In addition to converting arginine residues into ornithine, the reaction was found to cleave predominately Gly-Xaa, Xaa-Gly, Asn-Xaa and Xaa-Ser peptide bonds. Peptide-bond cleavage and deguanidation was studied as a function of time of exposure to hydrazine, hydrazine concentration and temperature. The convenience of this method of chromatography for the rapid low-cost separation and isolation of peptides, as well as their reaction products, is illustrated at the level of material required for solid-phase microsequencing.     

Chemical conversion of aspartyl peptides to isoaspartyl peptides. A method for generating new methyl-accepting substrates for the erythrocyte D-aspartyl/L-isoaspartyl protein methyltransferase

Mammalian protein carboxyl methyltransferases have recently been proposed to recognize atypical configurations of aspartic acid and may possibly function in the metabolism of covalently altered cellular proteins. Consistent with this proposal, the tetrapeptide tetragastrin, containing a single "normal" L-aspartyl residue (L-Trp-L-Met-L-Asp-L-Phe-NH2) was found here not to be an in vitro substrate for erythrocyte carboxyl methyltransferase activity. However, chemical treatment of tetragastrin by methyl esterification and then de-esterification of the aspartic acid residue yielded a mixture of peptide products, the major one of which could now be enzymatically methylated. We show here that this new peptide species is the isomeric beta-aspartyl form of tetragastrin (L-iso-tetragastrin; L-Trp-L-Met-L-Asp-L-Phe-NH2), and it appears that isomerization proceeds via an intramolecular succinimide intermediate during the de-esterification procedure. L-iso-Tetragastrin is stoichiometrically methylated (up to 90% in these experiments) with a Km for the enzyme of 5.0 microM. Similar chemical treatment of several other L-aspartyl peptides also resulted in the formation of new methyltransferase substrates. This general method for converting normal aspartyl peptides to isoaspartyl peptides may have application in the reverse process as well.     

Random-breakage mapping method applied to human DNA sequences.

The random-breakage mapping method [Game et al. (1990) Nucleic Acids Res., 18, 4453-4461] was applied to DNA sequences in human fibroblasts. The methodology involves NotI restriction endonuclease digestion of DNA from irradiated calls, followed by pulsed-field gel electrophoresis, Southern blotting and hybridization with DNA probes recognizing the single copy sequences of interest. The Southern blots show a band for the unbroken restriction fragments and a smear below this band due to radiation induced random breaks. This smear pattern contains two discontinuities in intensity at positions that correspond to the distance of the hybridization site to each end of the restriction fragment. By analyzing the positions of those discontinuities we confirmed the previously mapped position of the probe DXS1327 within a NotI fragment on the X chromosome, thus demonstrating the validity of the technique. We were also able to position the probes D21S1 and D21S15 with respect to the ends of their corresponding NotI fragments on chromosome 21. A third chromosome 21 probe, D21S11, has previously been reported to be close to D21S1, although an uncertainty about a second possible location existed. Since both probes D21S1 and D21S11 hybridized to a single NotI fragment and yielded a similar smear pattern, this uncertainty is removed by the random-breakage mapping method.     

The Krylov-Bogoliubov-Mitropolskii method applied to models of population dynamics

In this study the possibility of applying the asymptotic method of Krylov-Bogoliubov-Mitropolskii to problems of population dynamics is shown. Especially a general Volterra-Gause-Witt type model for prey-predator interaction is investigated. A discussion on the results obtained is given for the general model and for a particular case as well.     

The characteristics method applied to the study of muscle dynamics

In two-state sliding filament models of muscle contraction a partial differential equation must be solved to find the cross-bridge distribution functionn(x, t). In this paper the analytical form of this function is obtained by integration along the characteristic line and special cases are presented in which the explicit expression forn(x, t) can be completely determined. These analytical solutions provide a direct mathematical connection between the microscopic contraction parameters contained in the kinetic theories and macroscopic muscle dynamics and are thus used to investigate what parameters influence the transient contractile tension in typical experimental conditions. The results of this investigation are consistent with relevant aspects of muscle physiology.     

The maximum likelihood identification method applied to insect morphometric data

To distinguish species or populations using morphometric data is generally processed through multivariate analyses,in particular the discriminant analysis.We explored another approach based on the maximum likelihood method.Simple statistics based on the assumption of normal distribution at a single variable allows to compute the chance of observing a particular data (or sample) in a given reference group.When data are described by more than one variable,the maximum likelihood (MLi) approach allows to combine these chances to fmd the best fit for the data.Such approach assumes independence between variables.The assumptions of normal distribution of variables and independence between them are frequently not met in morphometrics,but improvements may be obtained after some mathematical transformations.Provided there is strict anatomical correspondence of variables between unknown and reference data,the MLi classification produces consistent classification.We explored this approach using various input data,and compared validated classification scores with the ones obtained after the Mahalanobis distance-based classification.The simplicity of the method,its fast computation,performance and versatility,make it an interesting complement to other classification techniques.     

Chemical macrocyclization of peptides fused to antibody fc fragments

To extend the plasma half-life of a bicyclic peptide antagonist, we chose to link it to the Fc fragment of the long-lived serum protein IgG1. Instead of chemically conjugating the entire bicyclic peptide, we recombinantly expressed its peptide moiety as a fusion protein to an Fc fragment and subsequently cyclized the peptide by chemically reacting its three cysteine residues with tris-(bromomethyl)benzene. This reaction was efficient and selective, yielding completely modified peptide fusion protein and no side products. After optimization of the linker and the Fc fragment format, the bicyclic peptide was fully functional as an inhibitor (K(i) = 76 nM) and showed an extended terminal half-life of 1.5 days in mice. The unexpectedly clean reaction makes chemical macrocyclization of peptide-Fc fusion proteins an attractive synthetic approach. Its good compatibility with the Fc fragment may lend the bromomethylbenzene-based chemistry also for the generation of antibody-drug conjugates.     

A rapid population method for action spectra applied to Halobacterium halobium.

We have developed a simple and rapid technique for measuring the action spectra for phototaxis of populations of microorganisms and applied it to halobacteria. A microscope with a dark-field condenser was used to illuminate the cell suspension in a sealed chamber with light of wavelength greater than 750 nm; in this region of the spectrum, the halobacteria show no phototactic response. A 150-micron spot of light from a xenon arc lamp, whose wavelength and intensity can be varied, was projected through the objective lens into the center of the dark field. The objective lens imaged this measuring spot through a 780-nm cut-off filter on an aperture in front of a photomultiplier. The intensity of the scattered 750-nm light, and therefore the photomultiplier current, is proportional to the number of cells in the measuring spot. A third lamp provided background light of variable wavelength and intensity through the dark-field condenser. To minimize secondary effects due to large changes in cell density, we recorded the initial changes in the photomultiplier current over 1 min after the actinic light had been switched on. By plotting the rate of change against wavelength, we obtained action spectra after the proper corrections for changes in light intensity with wavelength were applied and saturation effects were avoided.     

Methionine anchoring applied to the solid-phase synthesis of lysine-containing 'head-to-tail' cyclic peptides

Lysine-containing 'head-to-tail' cyclic peptides can be prepared via a side-chain anchoring solid-phase synthesis strategy. A handle is prepared using a methionine residue, the C -carboxylof which forms an amide with the N -amine of lysine. Subsequently, the linear peptide sequence is assembled, appropriatedeblocking steps are carried out, and on-resin head-to-tail cyclizationfollows. Optionally, acid-labile protecting groups may be removed while the peptide remains resin-bound. The final cleavage step uses CNBr, and releases the free or protected cyclic peptide into solution.