Further crystallographic studies on lysozyme

Abstract

This thesis is concerned with further structural studies-primarily using crystallographic techniques-on the well known enzyme lysozyme. The introductory chapter briefly surveys the available X ray results, which are extensive. A substantial part of these results pertains to hen egg white (HEW) lysozyme. The tertiary structure of the protein and the geometrical basis of its activity, in terms of the binding of the NAG–NAM copolymer to the cleft of the molecule, were established through pioneering studies on the tetragonal crystal form. Several other crystal forms of HEW lysozyme-one triclinic, another monoclinic, and two orthorhombic-have also been extensively examined. Other chicken type lysozymes studied crystallographically include those from humans, tortoise, and turkey. Studies on chicken type lysozymes have contributed not only to molecular enzymology but also to understanding the mobility and hydration of proteins. Lysozymes from Streptomyces erythraeus, bacteriophage T4, and goose egg white, which have been studied by X ray methods, are not homologous to chicken type lysozyme. However, interesting structural and evolutionary relationships have been established among chicken type, T4, and goose lysozymes. The explanation of lysozyme’s lytic activity derived from X ray results has been based entirely on binding of the polysaccharide component of peptidoglycan to the hexasaccharide cleft in the enzyme. However, the peptidoglycan is known to be a better substrate than the NAG–NAM copolymer, suggesting that the conventional explanation of lysozyme activity may be incomplete. Recently, it has been shown that the enzyme, when bound to dyes such as bromophenol red (BPR) and bromophenol blue (BPB), remains active against polysaccharide but not against the bacterial cell wall. It was therefore of interest to characterise the binding site of these dyes using X ray techniques. Spectroscopic and inhibition studies performed as a prelude to the crystallographic work are described in Chapter 2. These studies were carried out at pH 4.6, the pH used for crystallising tetragonal lysozyme. At this pH, BPR exists predominantly in the neutral form, while BPB exists in both ionic and neutral forms. Both dyes bind to lysozyme at this pH without appreciable change in their ionisation state and competitively inhibit the enzyme’s lytic activity. Attempts were made to crystallise BPR and BPB; good crystals were obtained only for BPB. Chapter 3 presents the X ray structure analysis of BPB crystals. BPB molecules in the crystal exist in the closed neutral form. The molecule consists essentially of three planar groupings: 1. the sulfonphthalein ring system, 2. two dibromophenol rings attached to the tetrahedral carbon of the five membered ring. The dibromophenol rings are inclined at 73° to each other and make angles of 85° and 68° with the sulfonphthalein system. There is no steric or electronic reason why the closed form of BPR should differ significantly from BPB; thus, the X ray analysis likely describes both structures. Attempts to prepare lysozyme–dye complexes via soaking were successful only with BPR. Chapter 4 describes the 5.5 Å resolution X ray analysis of the lysozyme–BPR complex. Data were collected diffractometrically from native and complex crystals. Native phase angles provided by Prof. D. C. Phillips were also used. Difference Fourier maps consistently revealed density corresponding to the closed form of BPR, with geometry similar to closed form BPB. The residues located in the immediate neighbourhood of the bound dye include Arg5, Lys33, Ala22, Trp123, and Arg125, with Phe34 and Phe38 nearby. Most residues here are invariant or conserved across lysozymes. The dye binding site, located outside the main cleft close to subsite F, is presumably involved in interaction with the peptide component of peptidoglycan in the bacterial cell wall. Hydration of lysozyme and the structural changes it induces have received significant attention, largely from studies on non crystalline samples. Chapter 5 reports analogous studies on single crystals. Various crystal forms were examined under controlled humidity (100–75% relative humidity). Tetragonal, orthorhombic, and monoclinic lysozyme exhibited reversible structural transformations around 90% r.h., shown by abrupt changes in diffraction pattern, unit cell dimensions, and solvent content. Triclinic lysozyme did not transform in this range. Pre soaking tetragonal crystals in MPD lowered the transformation humidity. These transformations likely involve crystal packing changes accompanied by conformational transitions, providing a powerful tool for studying hydration and conformational flexibility in proteins. ________________________________________ Publications Based on This Work 1. The crystal structure of 3,3',5,5'-tetrabromophenol sulfonphthalein (Bromophenol Blue), Acta Cryst., C40, 500–502 (1984). 2. Water-mediated structural transformations in a new crystal form of ribonuclease A and tetragonal lysozyme, Current Science, 53, 231–235 (1984). 3. X ray characterisation of an additional binding site in lysozyme, FEBS Letters, 1985 (in press). 4. Water mediated transformations in protein crystals, Acta Cryst., 1985 (submitted).