[en] The in vivo incorporation of deuterated amino acids into egg white proteins of Japanese quail is described. Using a synthetic diet, the level of incorporation of selectively deuterated tyrosine, tryptophan, histidine, and phenylalanine into lysozyme was greater than 80% as demonstrated by proton NMR. Load and load-chase experiments using [3H]phenylalanine or [3H]leucine monitored the fast uptake time (t 1/2 = 2 days) and confirmed the high levels of incorporation. The potential of this system for preparation of other proteins for NMR spectroscopy is discussed

[en] Complete main-chain (NH and αCH)¹H NMR assignments are reported for the 130 residues of human lysozyme, along with extensive assignments for side-chain protons. Analysis of 2-D NOESY experiments shows that the regions of secondary structure for human lysozyme in solution are essentially identical with those found previously in a similar study of hen lysozyme and are in close accord with the structure of the protein reported previously from x-ray diffraction studies in the crystalline state. Comparison of the chemical shifts, spin-spin coupling constants, and hydrogen exchange behavior are also consistent with closely similar structures for the two proteins in solution. In a number of cases specific differences in the NMR parameters between hen and human lysozymes can be correlated with specific differences observed in the crystal structures

[en] Assignments of ¹H NMR resonances of 121 of the 129 residues of hen egg white lysozyme have been obtained by sequence-specific methods. Spin systems were identified with phase-sensitive two-dimensional (2-D) correlated spectroscopy and single and double relayed coherence transfer spectroscopy. For key types of amino acid residues, particularly alanine, threonine, valine, and glycine, complete spin systems were identified. For other residues a less complete definition of the spin system was found to be adequate for the purpose of sequential assignment. Sequence-specific assignments were achieved by phase-sensitive 2-D nuclear Overhauser enhancement spectroscopy (NOESY). Exploitation of the wide range of hydrogen exchange rates found in lysozyme was a useful approach to overcoming the problem of spectral overlap. The sequential assignment was built up from 21 peptide segments ranging in length from 2 to 13 residues. The NOESY spectra were also used to provide information about the secondary structure of the protein in solution. Three helical regions and two regions of β-sheet were identified from the NOESY data; these regions are identical with those found in the X-ray structure of hen lysozyme. Slowly exchanging amides are generally correlated with hydrogen bonding identified in the X-ray structure; a number of exceptions to this general trend were, however, found. The results presented in this paper indicate that highly detailed information can be obtained from 2-D NMR spectra of a protein that is significantly larger than those studies previously

[en] Published in summary form only

[en] Human urinary-type plasminogen activator (urokinase) and proteolytic fragments corresponding to the kringle, EGF-kringle, and protease domains have been examined by ¹H NMR spectroscopy. The intact protein shows a very well-resolved spectrum for a molecule of this size (MW 54,000), with resonance line widths not greatly increased from those of the isolated domains. On increasing the temperature, the protein at pH values close to 4 was found to undergo two distinct and reversible conformational transitions. These were identified, by comparison with spectra of the proteolytic fragments, as the unfolding of the kringle (and EGF) domains (at ∼ 42 degree C) and of a segment of the protease domain (at ∼ 60 degree C). The remaining segment of the protease domain showed persistent structure to at least 85 degree C at pH 4; only at lower pH values could complete unfolding be achieved. The results indicate that the structures and stabilities of the isolated domains are closely similar to those in the intact protein and suggest that there is a degree of independent motion at least between the kringle and protease domains

[en] ³¹P CP/MAS spectra have been obtained from 2'-CMP bound to ribonuclease A in the crystalline state. The chemical shift value is closely similar to that found in solution NMR studies under similar conditions, and corresponds to that of the dianionic state of the free compound. It is suggested that the NMR approach may be of general applicability for the comparison of the binding properties of small molecules to proteins in crystals and solution. 21 refs.; 3 figs

[en] Published in summary form only

[en] W(PMe₃)₃H₆ (1), an archetypal nine-coordinate transition-metal complex, has been studied in the crystalline state by variable-temperature ¹³C and ³¹P CP/MAS NMR spectroscopy. The NMR spectra are consistent with a tricapped-trigonal-prismatic geometry for the complex, with two phosphine ligands in eclipsed prismatic sites and the third capping the prismatic face opposite the other two. The two prismatic phosphines are shown to be on inequivalent sites. At temperatures above 340 K, exchange broadening shows the occurrence of ligand functionality interchange between the different phosphine environments. Analysis of these line shapes and magnetization-transfer data, in the slow limit of exchange, for the both ¹³C and ³¹P nuclei shows activation parameters of E_a = 148.8 ± 15 kJ mol^-1 and A = 6.6 x 10²³ s^-1 for the ligand-functionality-interchange process. The most likely mechanism involves a double-rearrangement mechanism with polyhedral edge stretches through a monocapped-square-antiprismatic geometry to a tricapped-trigonal-prismatic intermediate in which all phosphines occupy capping positions. This allows complete scrambling of the ligand functionality, in a manner such that the spatial movement of the phosphine ligands within the crystalline frame need only be relatively small. An additional dynamic process, involving a tripod reorientation of the PMe₃rotors, has been detected by dipolar broadening of the ¹³C NMR spectra. 31 refs., 10 figs., 2 tabs

[en] Published in summary form only

[en] Published in summary form only