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Open Access Publications from the University of California

Conformational dynamics of interleukin-1beta and protein- membrane interactions

  • Author(s): Anderson, William David
  • et al.

Interleukin-1[beta] is a cytokine central in the immunological response to injury and infection. While the structure is well known, the i) modulating effects of point mutations upon function and the ii) process of post- translational export remain poorly understood. In the first part of the dissertation, the effects of several mutations important in protein folding and function, are examined. Water specific NMR experiments show the sites of hydrogen-bound water molecules in the [beta]-trefoil fold of IL-1[beta]. The structural hydration of several mutants with varying functional and folding propensities was found to be the same as wild-type, showing that the structural water interactions are robust. The conformational dynamics of the wild-type and two destabilized Thr-9 mutants were further explored using native state hydrogen exchange (NHX) NMR spectroscopy. The NHX experiments highlight the destabilizing effects of the T9Q and T9G mutations, yet the residue specific nature of NMR spectroscopy allowed the mapping of destabilizing, non-destabilizing, and hyper -stabilizing effects across the IL-1[beta] protein to sites distal from the mutation. These results are discussed in light of computational modeling of the T9G mutation. In the second part of the dissertation, protein- lipid interactions are explored, beginning with the novel finding of a weak but highly specific interaction between IL-1[beta] and phospholipid bicelles in solution. NHX NMR spectroscopy shows the destabilization of IL-1[beta] in the presence of bicelles. A combination of chemical shift analysis, heteronuclear ¹H-¹⁵N NOEs, and paramagnetic relaxation enhancement identified a contiguous surface on one side of the molecule as the putative site of interaction with bicelles. These results represent the first structural evidence of a membrane-protein interaction of IL-1[beta], and are discussed in light of the hypothesized non-classical secretion of IL-1[beta]. Finally, we have implemented transferred cross-saturation NMR experiments using the transmembrane Pf1 filamentous phage coat protein in isotropic bicelles. Protein residues can be localized to the core of the bicelle or the aqueous interface via selective pre-saturation of the [omega]- methyl or choline-methyl protons, respectively. Methyl- proton specific TCS in isotropic bicelles is an effective and simple method for characterizing the transmembrane region of proteins

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