The fast internal dynamics of human ubiquitin have been studied by the analysis of 15N relaxation of backbone amide nitrogens. The amide 15N resonances have been assigned by use of heteronuclear multiple-quantum spectroscopy. Spin lattice relaxation times at 60.8 and 30.4 MHz and the steady-state nuclear Overhauser effect at 60.8 MHz have been determined for 67 amide 15N sites in the protein using two-dimensional spectroscopy. These data have been analyzed in terms of the model free treatment of Lipari and Szabo [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559]. The global motion of the protein is shown to be isotropic and is characterized by a correlation time of 4.1 ns rad-1. The generalized order parameters (S2) of backbone amide N-H vectors in the globular region of the protein range from 0.5 to 0.95. No apparent correlation between secondary structure and generalized order parameters is observed. There is, however, a strong correlation between the magnitude of the generalized order parameters of a given N-H vector and the presence of hydrogen bonding of the amide hydrogen or its peptide bond associated carbonyl. Using a chemical shift tensor breadth of 160 ppm, the N-H vectors of peptide linkages participating in one or more hydrogen bonds to the main chain show an average generalized order parameter of 0.80 (SD 0.06), while those amide NH of peptide linkages free of hydrogen-bonding interactions with the main chain show an average order parameter of 0.69 (SD 0.06).(ABSTRACT TRUNCATED AT 250 WORDS)
ABSTRACrThe extremely chemically resistant component of the cell wall of spores, polens, and some microorganisms, sporopolenin, is generaly accepted to be derived from carotenoids or carotenoid esters. However, we report here that '3C NMR analyses of sporopoUenin from several sources shows that this widely held view is incorrect, with one possible exception. Sporopollenin is not a unique substance but rather a series of related biopolymers derived from largely saturated precursors such as fatty adds. The biopolymers contain widely varying amounts of oxygen in the form of ether, hydroxyl, carboxylic acid, ester, and ketone groups.The outer cell wall, or exine, of many spores, pollens, and certain microorganisms is made of sporopollenin. The
The interaction between the peptide corresponding to the calmodulin-binding domain of the smooth muscle myosin light-chain kinase and (Ca2+)4-calmodulin has been studied by multinuclear and multidimensional nuclear magnetic resonance methods. The study was facilitated by the use of 15N-labeled peptide in conjunction with 15N-edited and 15N-correlated 1H spectroscopy. The peptide forms a 1:1 complex with calcium-saturated calmodulin which is in slow exchange with free peptide. The 1H and 15N resonances of the bound have been assigned. An extensive set of structural constraints for the bound peptide has been assembled from the analysis of nuclear Overhauser effects and three-bond coupling constants. The backbone conformation of the bound peptide has been determined using these constraints by use of distance geometry and related computational methods. The backbone conformation of the peptide has been determined to high precision and is generally indicative of helical secondary structure. Nonhelical backbone conformations are seen in the middle and at the C-terminal end of the bound peptide. These studies provide the first direct confirmation of the amphiphilic helix model for the structure of peptides bound to calcium-saturated calmodulin.
The interaction between calcium-saturated chicken calmodulin and a peptide corresponding to the calmodulin-binding domain of the chicken smooth muscle myosin light chain kinase has been studied by multinuclear and multidimensional nuclear magnetic resonance methods. Extensive 1H and 15N resonance assignments of calmodulin in the complex have been obtained from the analysis of two- and three-dimensional nuclear magnetic resonance spectra. The assignment of calmodulin in the complex was facilitated by the use of selective labeling of the protein with alpha-15N-labeled valine, alanine, lysine, leucine, and glycine. These provided reference points during the main-chain-directed analysis of three-dimensional spectra of complexes prepared with uniformly 15N-labeled calmodulin. The pattern of nuclear Overhauser effects (NOE) seen among main-chain amide NH, C alpha H, and C beta H hydrogens indicates that the secondary structure of the globular domains of calmodulin in the complex closely corresponds to that observed in the calcium-saturated state of the protein in the absence of bound peptide. However, the backbone conformation of residues 76-84 adopts an extended chain conformation upon binding of the peptide in contrast to its helical conformation in the absence of peptide. A sufficient number of NOEs between the globular domains of calmodulin and the bound peptide have been found to indicate that the N- and C-terminal regions of the peptide interact with the C- and N-terminal domains of calmodulin, respectively. The significance of these results are discussed in terms of recently proposed models for the structure of calmodulin-peptide complexes.
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