Assignment of the backbone sup 1 H and sup 15 N NMR resonances of bacteriophage T4 lysozyme

• Published in: Biochemistry (Easton) Vol. 29:27
• Main Authors: McIntosh, L.P., Dahlquist, F.W., Wand, A.J., Lowry, D.F., Redfield, A.G.
• Format: Journal Article
• Language: English
• Published: United States 10.07.1990
• Subjects: 550601 - Medicine- Unsealed Radionuclides in Diagnostics; AMINO ACID SEQUENCE; AMMONIUM CHLORIDES; AMMONIUM COMPOUNDS; AMMONIUM HALIDES; BACTERIOPHAGES; BARYONS; CHEMICAL SHIFT; CHLORIDES; CHLORINE COMPOUNDS; ELEMENTARY PARTICLES; ENZYMES; FERMIONS; GLYCOSYL HYDROLASES; HADRONS; HALIDES; HALOGEN COMPOUNDS; HYDROLASES; ISOTOPES; LABELLED COMPOUNDS; LIGHT NUCLEI; LYSOZYME; MAGNETIC RESONANCE; MICROORGANISMS; MOLECULAR STRUCTURE; NITROGEN 15; NITROGEN ISOTOPES; NMR SPECTRA; NUCLEAR MAGNETIC RESONANCE; NUCLEI; NUCLEONS; O-GLYCOSYL HYDROLASES; ODD-EVEN NUCLEI; OVERHAUSER EFFECT; PARASITES; PROTONS; RADIOLOGY AND NUCLEAR MEDICINE; RESONANCE; SPECTRA; STABLE ISOTOPES; VIRUSES
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00479a003

The proton and nitrogen ({sup 15}NH-H{sup {alpha}}-H{sup {beta}}) resonances of bacteriophage T4 lysozyme were assigned by {sup 15}N-aided {sup 1}H NMR. The assignments were directed from the backbone amide {sup 1}H-{sup 15}N nuclei, with the heteronuclear single-multiple-quantum coherence (HSMQC) spectrum of uniformly {sup 15}N enriched protein serving as the master template for this work. The main-chain amide {sup 1}H-{sup 15}N resonances and H{sup {alpha}} resonances were resolved and classified into 18 amino acid types by using HMQC and {sup 15}N-edited COSY measurements, respectively, of T4 lysozymes selectively enriched with one or more of {alpha}-{sup 15}N-labeled Ala, Arg, Asn, Asp, Gly, Gln, Glu, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. The heteronuclear spectra were complemented by proton DQF-COSY and TOCSY spectra of unlabeled protein in H{sub 2}O and D{sub 2}O buffers, from which the H{sup {beta}} resonances of many residues were identified. The NOE cross peaks to almost every amide proton were resolved in {sup 15}N-edited NOESY spectra of the selectively {sup 15}N enriched protein samples. Residue specific assignments were determined by using NOE connectivities between protons in the {sup 15}NH-H{sup {alpha}}-H{sup {beta}} spin systems of known amino acid type. Additional assignments of the aromatic proton resonances were obtained from {sup 1}H NMR spectra of unlabeled and selectively deuterated protein samples. The secondary structure of T4 lysozyme indicated from a qualitative analysis of the NOESY data is consistent with the crystallographic model of the protein.