John Glushka

The conformational properties of methyl alpha-(2,8)-di/trisialosides and their N-acyl analogues: implications for anti-Neisseria meningitidis B vaccine design.

The conformational properties of di- and trisaccharide fragments of the polysialic acid O-antigen capsular polysaccharide (CPS) of Neisseria meningitidis B (NmB) have been investigated by a combination of solution phase NMR spectroscopy and explicit-solvent molecular dynamics (MD) simulations. Simulations employing 100 ns of conventional MD, as well as 160 ns of replica exchange MD (REMD), with the GLYCAM06 force field were shown to be in agreement with experimental NMR scalar J-coupling and NOE values. The presence of conformational families has been determined by monitoring interglycosidic torsion angles, by comparing structural superimpositions, as well as via a Bayesian statistical analysis of the torsional data. Attempts to augment the immunogenicity of NmB CPS often involve chemical modifications of the N-acetyl moiety. Here the effects of these chemical group modifications on the conformational properties of the trisialoside have been probed via REMD simulations of the N-glycolyl, N-propionyl, N-propyl and N-butanoyl analogues. Although there were conformational families unique to each non-native analogue, the chemical modifications resulted in largely equivalent overall conformational phase-spaces compared to the native trisialoside. On the basis of the conformational distributions, these shared conformational properties suggest that a recurrent global conformational epitope may be present in both the native and chemically modified CPS fragments. Explanations are therefore provided for monoclonal antibody cross-reactivity, in terms of recognition of a shared global CPS conformation, as well as for lack of cross-reactivity, in terms of fine structural differences associated with the N-acyl groups, which may be dominant in highly matured antibody responses.

Synthesis and NMR characterization of the trypanosomatid metabolite, N1,N8-bis(glutathionyl)spermidine disulphide (trypanothione disulphide)

An optimized chemical synthesis of the novel trypanosomatid metabolite,N1,N8-bis(glutathionyl)-spermidine(trypanothione disulphide) is described, and its solution structure has been investigated by NMR spectroscopy. The 1H, 13C, and 15N chemical shifts, as well as proton NOE data are compatible with two similar extended peptide segments connected by a flexible spermidine chain. The data do not support a predominant β sheet structure but indicate considerable flexibility in aqueous solution.

The binding of ATP and AMP to Escherichia coli adenylate kinase investigated by 1H and 15N NMR spectroscopy

[N6 15N]ATP and [N6 15N]AMP, complexed with E.coli adenylate kinase (AKe), were observed with 15N isotope-filtered NMR pulse sequences and 1H[15N] heterocorrelated experiments to determine differences between binding sites based on chemical shifts and competition by substrate analogs. The chemical shifts of the N6 amino proton and nitrogen signals changed significantly after mixing with adenylate kinase. Differences in chemical shifts between the bound ATP and AMP signals are slight. The response of these shifts to further addition of other substrates or Mg2+ supports the view that the unchelated nucleotides can bind to both the sites, whereas the metal complexed species are restricted to the MgATP/MgADP binding site.

Observation of arginyl-deoxyoligonucleotide interactions in TaqI endonuclease by detection of specific 1H NMR signals from 140kD [Nη1, Nη2, 15N Arg]TaqI/oligomer complexes

Proton and nitrogen signals of the guanidinium amines in [N eta 1, N eta 2 15N Arg]Taq I endonuclease were observed using isotope filtered experiments and proton detected 1H[15N] heterocorrelated two dimensional NMR spectroscopy. These rapidly exchanging protons could be detected in the free enzyme only at pH 4.5; at pH 8.5, no signals were measured after extensive signal averaging. Addition of deoxyribonucleotide oligomers resulted in the appearance of two groups of signals at about 6.8 and 7.5 ppm. Since these signals are independent of the presence of cognate sequence or Mg2+, it is assumed they represent nonspecific arginyl-DNA interactions. This labeling/NMR approach provides a new method for investigating the role of arginine in protein-DNA interactions.

Parametric Estimation in 1-D, 2-D, and 3-D NMR

The increased power of nmr as an analytical and structure determining method arises from the development of two-dimensional methods in which spectral maps reflect through-bond and through-space connectivities between atoms. The interpretation of these maps is made complex by many factors, including the large number of such connectivities in many applications, the multiplicity of the spectral elements, and the limited signal-to-noise ratio. As one element of solving these problems, we have developed methods to simplify the cataloging of positions and intensities of peaks, and for examining and selecting among them. Linear Predictive Singular Value Decomposition is one numerical method used for the extraction of peak characteristics. Simple tools have been developed to select among spectral characteristics, to reduce multiplets to single peaks, and to incorporate pre-existing chemical information into the analysis. Characteristics of this approach of parametric estimation permit somewhat different approaches to experimental design. This may be particularly valuable in the determination of multiple vicinal coupling constants about single bonds.