The Position of the Active Tryptophan Residue in Lysozyme*

The identification and complete assignment of the C-2 and N-1 proton nuclear magnetic resonances (NMR) of the six tryptophan residues of hen lysozyme are reported. Identification of the resonances required a detailed examination of the spectra of the protein in H20 and in 'HzO, and involved the application of spin-echo and Carr-Purcell-Meiboom-Gill pulse sequences. Assignment was achieved by observing the effects on the NMR spectra of performing specific chemical modifications, of binding paramagnetic species (lanthanide ions and spin labels), of binding inhibitors and protons and of carrying out solvent exchange experiments. The problems involved in completion of assignment are fully discussed. In the course of performing experiments to make assignments, several interesting aspects of the behaviour of the tryptophan residues in the protein structure were observed and are discussed.The application of nuclear magnetic resonance (NMR) spectroscopy to the study of proteins has made substantial progress over the last five years, and it is now possible to achieve well-resolved 'H and I3C spectra of small proteins [l -31. In our work on lysozyme, we are using NMR to investigate fundamental features of the structural and kinetic behavior of a protein in solution [4,5]. It is an essential feature of this work that we are able to study specific individual atoms or groups in the protein. It is therefore necessary that resonances of these individual groups are resolved and assigned to specific atoms in the protein. Indeed, the assignment process is an essential requirement in any detailed study of a molecule by NMR. In early studies of lysozyme by 'H NMR, assignments for a number of resolved resonances were proposed [6 -81. More recently we have been able to increase greatly the number of resolved resonances in the spectrum [l] and to apply rigorous techniques to assign resonances of a number of methyl groups and of several aromatic residues [5]. In the 13C spectrum of lysozyme, resonances of most non-protonated aromatic carbon atoms have been assigned [3]. Similar progress has been made in assigning the spectra of the pancreatic trypsin inhibitor protein [9,10] and of cytochromes c [ll], but the lack of detailed assignments of the resAbbreviafions. NMR, nuclear magnetic resonance; ppm, parts per million; Nbf-C1, 4-chloro-7-nitro-benzofurazan; GlcNAc, Nacetyl-glucosamine.Etzzyme. Lysozyme (EC 3.2.1.17).onances of other proteins reflects the difficuky of the required procedures. In this paper we describe the full assignment of the N-1 and (2-2 proton resonances of all the six tryptophdn residues of lysozyme. The strategy by which this assignment was achieved has been outlined in previous papers [1,4]. In essence, the process falls into two parts. The first stage involves assignment of a particular resonance to a nucleus of a type of amino acid residue. This stage makes use primarily of physical methods, such as determination of multiplet structure and coupling information. A number of different NMR techniques were used her...

Section: Preparation Of Modified Proteinsmentioning

confidence: 73%

“…Hen lysozyme specifically modified at Trp-62 was prepared as described by Hayashi et al [15]. In this modification N-bromo-succinimide (obtained from BDH) oxidises the tryptophan to the oxindole.…”

Section: Preparation Of Modified Proteinsmentioning

confidence: 99%

Study of the Tryptophan Residues of Lysozyme Using ¹H Nuclear Magnetic Resonance

Cassels

Dobson

Poulsen

et al. 1978

“…Since the primary and three dimensional structure of hen egg white lysozyme is now known in great detail, the studies on lysozyme are especially noteworthy [33,38]. Petterson [39] also has observed difference spectra when cellobiose was allowed to interact with the cellulase from Penicillium notatum.…”

Section: Discussionmentioning

confidence: 99%

Ultraviolet Difference Spectral Studies on Concanavalin A

Hassing

Goldstein

1970

The interaction of concanavalin A, the phytohemagglutinin of the jack bean, with a variety of glycosides has been studied by the technique of ultraviolet difference spectroscopy. Whereas methyl a-D-glUC0-and manno-pyranoside gave rise to relatively low intensity difference spectra, p-nitrophenyl a-D-mannopyranoside and a-D-glucopyranoside yielded large difference spectra upon interaction with concanavalin A. Using this technique as a measure of concanavalin A activity, it was demonstrated that the protein specifically binds low molecular weight carbohydrates at much lower pH values (e. g. pH 2.4) than previously believed. Although polysaccharides are also bound at these low pH values, they are not precipitated by concanavalin A. Molecular weight studies in acid media indicate that the protein does not dissociate and it is suggested that electrostatic repulsion of the protein molecules due to their high net positive charge prevents protein-polysaccharide lattice formation and hence failure of the complex to precipitate.The interaction of certain specific carbohydrates and carbohydrate-containing macromolecules with concanavalin A, the lectin present in jack bean, has been studied extensively. This protein has been shown t o interact to form it precipitate with various polysaccharides, e.g. glycogens, mannans, and dextrans, and with certain serum glycoproteins [I-121. Extensive inhibition studies, using mono-and oligosaccharides, have demonstrated that the specificity of the saccharide binding sites of concanavalin A are directed towards the unmodified hydroxyl groups at the (2-3, C-4 and C-6 positions of a-D-glucopyranosyl and a-D-mannopyranosyl residues [la-I?]. Equilibrium dialysis studies have indicated that one mole of monosaccharide (methyl a-D-ghcopyranoside or methyl a-D-mannopyranoside) is bound per 32000 to 34000 g of concanavalin A [IS, 191. Furthermore, the linearity of the Scatchard plots suggests a homogeneity of binding constants for a given ligand.Other studies have indicated the complexity of structure of concanavalin A. Olson and Liener [20] found that in 8 M urea at pH 7, concanavalin A dissociated into three fractions, with molecular weights corresponding to 16500, 42000 and in excess of 200000, each with the same peptide map, amino acid composition, and& N-terminal amino acid (alanine) . Recently Doyle et al.[30] demonstrated that when D-glUCOSe, an inhibitor of concanavalin A-polysaccharide precipitation, was added to concanavalin A, an ultraviolet difference spectrum was obtained. This report represents a detailed study of the carbohydrate-binding activity of concanavalin A, using the technique of ultraviolet difference spectroscopy. MATERIALS AND METHODSConcanavalin A, prepared as described previously [31] was stored in 1.0 M NaCl at 4". Heavy metalfree concanavalin A was prepared by dialysis of the native protein against 0.1 N HC1, followed by dialysis against 0.05M NaCl in the cold. The demetallized protein did not form a precipitate with dextran B-135543 [ll].All carbohydrates used wer...

“…Three tryptophans, Trp62, Trp63 and Trpl08, are located in the substrate-binding cleft and are believed to interact with sugar residues placed in subsites B-D. Of these, Trp62 is proposed to be involved in apolar interaction with a sugar ring bound to subsite B and hydrogen bonding to the hydroxyl oxygen atom at positon 6 of a sugar ring in subsite C (Blake et al, 1967;Cheetham et al, 1992). Trp62 is the residue most exposed to solvent and is very susceptible to chemical reagents (Hayashi et al, 1965). Selective chemical modifications of Trp62 produce marked changes in its enzymic activity.…”

mentioning

confidence: 99%

Effects of subsite alterations on substrate‐binding mode in the active site of hen egg‐white lysozyme

Koyanagi

Maenaka

Sunada

et al. 1993

The subsite structures in the active site of hen egg‐white lysozyme were altered by site‐directed mutagenesis. Replacement of Trp62, which is involved in apolar interaction with a sugar ring, and Asp101, which is hydrogen bonded to the same sugar ring in subsite B, led to a shift of the oligosaccharide‐binding mode in the active‐site cleft. Consequently, the double‐mutant lysozyme (Trp62His, Asp101Gly) exhibited a drastic change of substrate‐binding without any significant loss of enzymic activity. Conversion of Asn37, which is postulated to be involved in interaction with a sugar ring in subsite F, had a reverse effect on substrate binding. Nuclear magnetic resonance analysis of mutant lysozymes, in which Trp62 was replaced with Phe or His, suggested that these replacements not only altered the structure of the amino acid side chain at position 62 of the lysozyme, but also induced local structural changes around the residue at position 62.