Proton NMR measurements of bacteriophage T4 lysozyme aided by 15N isotopic labeling: structural and dynamic studies of larger proteins.

Abstract

A strategy for resolution and assignment of single proton resonances in proteins of molecular mass up to at least 40 kDa is presented. This approach is based on '5N (or "3C) labeling of selected residues in a protein. The resonances from protons directly bonded to labeled atoms are detected in a two-dimensional 'H-'5N (or '3C) spectrum. The nuclear Overhauser effects from isotopically tagged protons are selectively observed in one-dimensional isotope-directed measurements. Using this approach, we have observed approximately 160 resonances from '5N-bonded protons in the backbone and sidechains of uniformly 15N-labeled T4 lysozyme (molecular mass = 18.7 kDa). Partial proton-deuterium exchange can be used to simplify the 'H-15N spectrum of this protein. These resonances are identified by amino acid class using selective incorporation of 15N-labeled amino acids and are assigned to specific residues by mutational substitution, multiple 15N and 13C labeling, and isotope-directed nuclear Overhauser effect measurements. For example, using a phenyl[15N]alanine-labeled lysozyme variant containing two consecutive phenylalanine residues in an a-helical region, we observe an isotopedirected nuclear Overhauser effect from the amide proton of Phe-66 to that of Phe-67.The wild-type and numerous temperature-sensitive and stable mutants of bacteriophage T4 lysozyme have been extensively characterized by thermodynamic (1, 2, 4) and crystallographic studies (5-8). To obtain detailed information about the effects of point mutations on the structure and dynamics of this enzyme, we wished to complement these studies using nuclear magnetic resonance (NMR). Unfortunately, the 164-residue size (18.7 kDa) (9) of this protein is near the practical limits of modem two-dimensional (2D) proton-proton NMR methods (10). Therefore, T4 lysozyme also serves as an excellent system for development of new NMR methodologies for investigating larger biomolecules.The strategy that we and others have adopted is based on the selective observation of protons directly bonded to 13C or '5N nuclei biosynthetically incorporated in a protein (11-16).Proton-observe heteronucleus-perturb NMR (17, 18) is used to detect only the resonances from isotopically labeled protons in one-dimensional 1H or 2D 'H-15N or 13C spectra. The -100 Hz spin-spin coupling between a 13C or 15N nucleus and a directly bonded proton is central to these techniques. This scalar coupling is about ten times greater than that exerted between one proton and a vicinal proton. Such small proton-proton couplings are the basis for the identification of proton resonances using previously developed 2D-NMR. A fundamental barrier to extending these conventional proton-only methods to larger proteins is that the proton signal decays during the time required for development of coherences due to spin-spin interactions. In other words, the proton linewidths exceed the proton-proton spin coupling. A second limitation of proton-only 2D-NMR methods is that the spectra become increasingly complicat...

Mechanisms for Benzene Dissociation through the Excited State of T4 Lysozyme L99A Mutant

“…S5 and S6). These fluctuations are consistent with an array of experimental observables for these residues: low NMR order parameters (41), lower hydrogen exchange protection factors relative to WT (42,43), high crystallographic B-factors (44), and propensity to deform upon ligand binding to the nearby cavity (18,45). These excursions are unproductive in allowing benzene egress, and the residue transitions are not concerted as they are when benzene egresses (compare Fig.…”

Backbone dynamics of TFE‐induced native‐like fold of region 4 of Escherichia coli RNA polymerase σ70 subunit

“…Preparation of recombinant 107‐residue fragment of ECσ, rECσ, containing C‐terminal residues 528–613 of σ 70 comprising a C‐terminal fragment of region 3.2 and the whole domain 4 preceded by a 21‐amino acid segment MGSSHHHHHHSSGLVPRGSHM carrying (His) 6 ‐Tag followed by thrombin cleavage site, was described previously 18. Resonance assignmentswere done for 13 C and 15 N uniformly double‐labeled rECσ,28 whereas 15 N relaxation was measured for 15 N labeled protein. The protein samples were homogeneous on polyacrylamide gel electrophoresis and electrospray mass spectrometry tests.…”

Solution structure of the phosphocarrier protein HPr from Bacillus subtilis by two‐dimensional NMR spectroscopy

“…The HPr purified from this strain lacks the methionine residue at position 1 (Wittekind et al, 1990). For expression of uniformly labeled "N-enriched HPr protein, a minimal growth medium (McIntosh et al, 1987) was used with "NH4CI (99% isotopic enrichment, Cambridge Isotope Laboratories, Woburn, Massachusetts). Two samples of unlabeled HPr, one in D 2 0 and the other in 90%/10% H20/D20, were prepared for NMR analysis as previously described (Wittekind et al, 1990).…”