Robert Joseph Paton Williams

Proton Magnetic Resonance Studies of the Tyrosine Residues of Hen Lysozyme-Assignment and Detection of Conformational Mobility

This paper reports the detection of all the proton magnetic resonances from the aromatic rings of the three tyrosine residues of hen egg white lysozyme. Procedures, including spin decoupling, for the full assignment of these residues are described. To explain the appearance and behaviour of these resonances it is postulated that the tyrosine rings are able to flip between equivalent conformations at rates exceeding 104s-1.

Nuclear Magnetic Resonance Studies on the Structure of Lysozyme in Solution

Forty resonances in the proton magnetic resonance (p.m.r.) spectrum of hen egg-white lysozyme have now been assigned to specific amino acid residues by using methods outlined in the preceding paper (Campbell, Dobson & Williams 1975a). The analysis of both the broadening and shift perturbations of these resonances by lanthanide cations bound at a single site provides definite evidence th at there is a close similarity between the p.m.r. solution and X-ray crystal structures. More detailed quantitative structural correspondence cannot be established readily for the p.m.r. study has demonstrated th at certain sidechain groups are mobile, and the solution structure is sensitive to the precise ionic size of the lanthanide cation. The assigned resonances have been used to detect and investigate local conformational changes in the active site of the protein brought about through binding of protons, metal ions and inhibitors. Kinetic as well as thermodynamic parameters can be extracted from the measurements.

Structure, Morphology, Composition and Organization of Biogenic Minerals in Limpet Teeth

The structure, morphology, composition, and organization of inorganic solids in the radula teeth of the limpet Patella vulgata have been studied by electron microscopy, electron diffraction, and e. d. X. a. of fractured, acid-treated, and sectioned tissue. Minerals first appear in the tooth base and comprise: amorphous and poorly crystalline granular, particulate, and sheet-like phases of variable composition (Fe, Si, P, Ca); irregular laths of crystalline goethite; and single crystals of prismatic goethite. The presence of localized Si and P may inhibit goethite crystallization in many regions of the tooth base. Mineralization of the tooth cusp begins with goethite impregnation of the posterior region. Crystals are deposited in the form of thin fibrous strands (15–20 nm width) with the [001] crystallographic axis initially parallel to the posterior tooth wall. Mineralization proceeds by an increase in the number and thickness of the crystals within the posterior region. In contrast, the anterior zone is only partly impregnated with crystals aligned parallel to the long axis of the cusp. The mature crystals are well ordered, acicular in morphology but with extensive growth distortions, and organized along regularly interspaced (30–50 nm) electron-dense filaments within the cusp. Removal of iron reveals the presence of silica-impregnated fibres, folded sheets, and tubular structures (often 30–60 nm in diameter) within essentially intact teeth. We propose that goethite crystallization and organization is regulated, in part, by spatial constraints established by an ordered filamentous organic matrix and that silica impregnates the matrix components at a later stage in mineralization thus maintaining the structural integrity of the organic tissue.

The Ultrastructure of the Calcium Carbonate Balance Organs of the Inner Ear: An Ultra-High Resolution Electron Microscopy Study

The balance organs of the inner ear of vertebrates, found as single, large growths of aragonite (‘otoliths’) in fish and small clumped masses (‘otoconia’) of either aragonite (amphibians) or calcite (mammals), have long been regarded as polycrystalline and single crystals respectively. The use of ultra-high resolution electron microscopy and electron diffraction to study comparatively crushed samples of these biominerals and samples of geological calcium carbonates, as examples of pure inorganic crystals, reveals that the biological structures are composed of microcrystals joined together by organic matrices to form composite crystals. Such structures either grow to a finite, controlled size (otoconia) or have daily growth patterns (otoliths). Mechanisms of growth are proposed to link these seemingly different patterns varying only in the number of nucleation sites and the degree of biological as against chemical control over the growth.

The Bakerian Lecture, 1981: Natural Selection of the Chemical Elements

Biochemistry is the study of an intricate interwoven ‘designed’ use of many elements in cells. It can only be fully appreciated in terms of the patterns of flow of chemicals, of ionic and electronic charge, and of energy directed in space. This requires a knowledge of the selection of the elements not only in analytical terms of uptake and chemical combination but also in terms of their spatial separation and functional specification. Starting from the abundance and availability of the elements an attempt is made here to analyse the roles of the elements, showing that much of the ‘chosen’ chemistry is an inevitable consequence of atomic properties. Selection has played upon this chemistry, extracting the utmost value from it, as seen in the refinement of functions of individual elements so that each element plays a quite separate and distinct role. Unique qualities dominate comparative similarities through the use of evolved specific small molecule and protein ligands. Proteins provide the evolutionary media for the development of function. It was the recognition and separation of each element in their specific sites (proteins) that allowed elements to be positioned in space. In turn the spatial organization generates, through feedback, the flow of other elements. Biological chemistry is only understandable in terms of the symbiotic use of some 25 elements and should not be related to so-called organic rather than to so-called inorganic chemistry.

Studies of exchangeable hydrogens in lysozyme by means of Fourier transform proton magnetic resonance

Fourier transform proton magnetic resonance techniques have been used to observe the solvent exchangeable hydrogens of hen and human lysozymes. Spectra of samples in both D2O and H2O were obtained, the spectra in H2O being recorded while selectively saturating the H2O resonance with an applied radio frequency field. Difference spectroscopy has shown that there are no detectable differences in conformation between the proteins in D2O and H2O. Difference spectroscopy has also been used to follow the exchange of hydrogens with solvent (D2O) and to observe the resonances of the exchangeable (NH) hydrogens separately from those of the non-exchangeable (CH) ones. The resonances from the indole C2 and N1 protons of a tryptophan, residue 108, have been firmly assigned in the spectra of both hen and human lysozyme by using lanthanide ion shift and relaxation probes in conjunction with crystallographic information. These assignments have been confirmed by studying the effects of pH on the resonances. This study has revealed an interesting interaction between the residues tryptophan 108 and glutamic acid 35. The structures of the active sites of the two lysozymes examined in solution are very similar to each other and to their crystal structures.

Biological applications of the Oxford scanning proton microprobe

Abstract The use of a scanning proton microprobe for the analysis of elements in biological hard and soft materials is discussed with examples. The full potential of the method is likely to be achieved shortly with improvement in resolution to the subcellular level.

Structural and Analytical Studies of the Silicified Macrohairs from the Lemma of the Grass Phalaris canariensis L.

Silicified macrohairs from mature and immature lemmas of the grass Phalaris canariensis L. have been studied by scanning (s.e.m.) and transmission electron microscopy (t.e.m.) and energy dispersive X-ray analysis (e.d.X.a.) at various times after emergence of the inflorescence. Within the macrohairs a variety of morphologies of silica particles was observed. E.d.X.a. revealed the time-dependent concentrations in the macrohairs of the elements K, Cl, P, and S, in addition to Si. Much lower levels of these elements were found in highly silicified mature macrohairs than in the immature macrohairs. It is proposed that the hairs are silicified under strict cellular control.

The internal structure of the chromaffin granule

The intact chromaffin granule has been studied by high resolution proton and phosphorus magnetic resonance spectroscopy. In the granule the proton peaks of adrenaline and ATP are shifted upfield differentially with respect to their positions in free solution. All lines in both proton and phosphorus magnetic resonance spectra were considerably broadened. The n.m.r. spectrum of the chromogranin protein in the vesicles was also well resolved. A structure of the vesicle is proposed.

Iron Oxide Biomineralization in the Radula Teeth of the Limpet Patella vulgata; Mossbauer Spectroscopy and High Resolution Transmission Electron Microscopy Studies

Iron oxide biomineralization in the radula teeth of the common limpet (Patella vulgata) has been studied by Mössbauer spectroscopy and high resolution transmission electron microscopy. The results indicate that the teeth comprise two iron-containing phases: (i) a superparamagnetic, microcrystalline and poorly ordered goethite-like component located essentially within the tooth bases; and (ii) stoichiometric well-ordered goethite crystals of acicular morphology sited within the tooth cusps. The goethite crystals are initially deposited in the form of long thin fibrous single crystals elongated along the [001] direction and with extensive irregularities in crystal thickness. Mature crystals often show morphological distortions that are not associated with localized structural imperfections or domain boundaries. The implications of these results in terms of tooth development are discussed.