Biomedical subjects

M R Wormald

Publications and source records attributed to M R Wormald.

42 records · Page 3Linked to original sources

PubMed1991

The conformational effects of N-glycosylation on the tailpiece from serum IgM.

1H-NMR spectroscopy has been used to study the conformation and dynamics of the isolated tailpiece from human serum immunoglobulin M, a 22-residue peptide containing a single asparagine glycosylation site. The peptide is isolated as a set of glycoforms, varying only in the sequence of the oligosaccharide attached at the glycosylation site. The oligosaccharides present have the general formula (Man)n(GlcNAc)2, with 45% having n = 6, 45% having n = 8 and 10% having n = 7 and/or 9. They have been identified and their NMR parameters compared to those found for the isolated oligosaccharides in free solution. The conformation and dynamics of the peptide component have also been studied, using NOE data and hydrogen-exchange experiments, and the results compared to those obtained from the aglycosyl peptide of the same sequence. The presence of the peptide is found to have no measurable effect on the conformation of the oligosaccharides. However, the presence of oligosaccharide causes a decrease in the conformational mobility of the backbone and sidechains of the peptide in the region of the glycosylation site. This is proposed to result from interactions between the oligosaccharide core and the amino acid side chains. Further, the conformation of the N-glycosidic linkage has been shown to be both rigid and planar. Thus, the conformational space available to an N-linked oligosaccharide in a glycoprotein relative to the protein may depend to a large extent upon the flexibility of the asparagine side chain. Various roles for the different glycoforms of the tail peptide are discussed.

Amino Acid Sequence↗

PubMed1991

Some structural features of the iron-uptake regulation protein.

An extensive proton nuclear magnetic resonance study of the iron-uptake regulation protein (Fur) from Escherichia coli has been made. Considerable difficulties were experienced in the NMR experiments in 1H2O which may be due unfavourable proton exchange rates in the pH range greater than 6.2, where the protein is soluble. Even in 2H2O, the two-dimensional NMR spectra were not easily interpreted due to widely differing line widths, as a result of the protein side-chains having very differing mobilities. Despite these problems, virtually all the 20 aromatic amino acids have been assigned. Small regions of the protein core were assigned by taking advantage of the approximately 20 non-exchanging peptide-NH resonances in 2H2O. Using two-dimensional J-correlated, homonuclear Hartmann-Hahn and NOE spectroscopies, we have been able to give some assignments in which there is considerable confidence for about one third of the amino acids. Taking advantages of two series of probe experiments, using Mn(II) and a spin label, together with longer range NOE data and result from structure predictions and CD data, we have put forward a tentative fold for the protein which is seen to have a relatively rigid series of interior strands and more flexible exterior strands, many of which are likely to be helical. The Mn(II) probe experiments have also allowed us to define the Fe(II) binding site.

Amino Acid Sequence↗

PubMed1990

Structure and function of X-Pro dipeptide repeats in the TonB proteins of Salmonella typhimurium and Escherichia coli.

The TonB protein is required for several outer membrane transport processes in bacteria. A short 33-residue peptide segment of TonB has been studied by 1H and 13C nuclear magnetic resonance spectroscopy. The sequence of this peptide segment contains multiple Glu-Pro and Lys-Pro dipeptide repeats that maintain rigid, elongated structures and flank a short connecting segment that adopts a beta-strand configuration. This TonB peptide is shown to interact specifically with the FhuA protein, the outer membrane receptor for ferrichrome-iron, providing the first direct evidence that the TonB protein interacts with outer membrane receptors. Interaction with the FhuA protein involves the extended structural element containing positively charged Lys-Pro repeats, and suggests a functional role for this segment of the TonB protein. As TonB is anchored in the cytoplasmic membrane the protein must, uniquely, span the periplasm. These data, together with studies described in the accompanying paper, suggest a model by which TonB serves to transduce conformational information over extended distances, from the cytoplasmic membrane to the outer membrane.

Amino Acid Sequence↗

PubMed1990

Solution NMR studies of colicin E1 C-terminal thermolytic peptide. Structural comparison with colicin A and the effects of pH changes.

The aqueous solution structure of the C-terminal thermolytic peptide of colicin E1 has been investigated using both one- and two-dimensional NMR techniques. The NMR data are consistent with a fold for the peptide very similar to that reported for the colicin A C-terminal peptide in the crystalline state, although some differences have been noted. The one-dimensional NMR spectrum of the peptide has been used to follow changes in both the structure and dynamics of the peptide on changing pH. The in vitro functionally competent form of the peptide (present in solution only below pH 6) does not differ in structure significantly from the higher pH form. However, small local conformational changes are observed together with an increase in mobility in some of the more hydrophilic regions. This suggests that the effect of lower pH is to change the ease with which the major conformational changes during insertion into a membrane can occur.

Amino Acid Sequence↗

PubMed1989

Identification and description of beta-structure in horse muscle acylphosphatase by nuclear magnetic resonance spectroscopy.

Nuclear magnetic resonance spectra of acylphosphatase were searched for signs of beta-structure, i.e. characteristic nuclear Overhauser enhancement patterns displayed in the two-dimensional spectra, typical chemical shifts, coupling constants and slow 2H-H exchange. The results provided identification of the main-chain resonances of amino acid residues involved in the beta-structure. The full sequential assignment of this region was gained by identification of some amino acid spin systems and their alignment with the primary sequence. The assignment of the side-chains was virtually completed subsequently and a list produced of nuclear magnetic resonance (n.m.r.) constraints derived from the spectra. The beta-structure consists of a beta-sheet with four antiparallel chains, one attached parallel chain, three tight turns and a beta-bulge. The conformation of the beta-sheet was determined by distance geometry calculation using the n.m.r. constraints (174 intraresidual, 107 sequential and 226 long-range distances, 32 torsion angles, phi, and 28 hydrogen bonds) as input. Observation of some interactions between the sheet and previously identified alpha-helical regions made it possible to give an outline of the three-dimensional structure of the enzyme.

Acid Anhydride Hydrolases↗

PubMedDate not supplied

Mapping space by NMR using susceptibility changes at phase boundaries.

A technique for differentiating high-resolution NMR signals from different regions of small objects is outlined and some initial results on model systems are given. This method uses inorganic paramagnetic or diamagnetic ions to create magnetic field gradients at phase boundaries.

Magnetic Resonance Spectroscopy↗