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

Abstract

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...

Protein dynamics studied by NMR

“…We demonstrated the error this can lead to in interpretation in a very early NMR study of a chemical modification of a tryptophan residue (Cassels et al 1978). We demonstrated the error this can lead to in interpretation in a very early NMR study of a chemical modification of a tryptophan residue (Cassels et al 1978).…”

“…Relaxation of aromatics, fast flipping rates, > 105 s-1 is very common (Wtithrich 1986) as is oscillating behaviour of tryptophan (Cassels et al 1978). Life is a product of the existence of water.…”

Isolation and photo-oxidation of lysozyme fragments

“…By nuclear magnetic resonance studies (NMR) (Cassels et al 1978;Lenkinsky et al 1979) and optically detected magnetic resonance (ODMR) (Rousslang et al 1979) it has been possible to determine the degree of availability towards the solvent of Trp residues in lysozyme. In this way it has been shown that residue 62 is the most exposed one, followed by residues 123 and 63; the others are quite more shielded.…”

The blind watchmaker and rational protein engineering

“…Photo-CIDNP (chemically induced nuclear polarisation) is an interesting technique for the study of surface positioned aromatic residues in proteins (Broadhurst et al, 1991;Cassels et al, 1978;Hore and Kaptain, 1983;Scheffier et al, 1985). By introducing a dye and exciting it with a laser, it is possible to transfer magnetisation to aromatic residues, where it can be observed.…”