Solution structure and dynamics of a serpin reactive site loop using interleukin 1 as a presentation scaffold

Human interleukin-1ß (IL1ß) was used as a presentationscaffold for the characterization of the reactive site loop(RSL) of the serpin 1-antitrypsin (A1AT), the physiologicalinhibitor of leukocyte elastase. A chimeric protein was generatedby replacement of residues 50–53 of IL1ß, correspondingto an exposed reverse turn in IL1ß, with the 10-residueP5-P5' sequence EAIPMSIPPE from A1AT. The chimera (antitrypsin-interleukin,AT-IL) inhibits elastase specifically and also binds the IL1ßreceptor. Multinuclear NMR characterization of AT-IL establishedthat, with the exception of the inserted sequence, the structureof the IL1ß scaffold is preserved in the chimera. Thestructure of the inserted RSL was analyzed relative to thatof the isolated 10-residue RSL peptide, which was shown to beessentially disordered in solution. The chimeric RSL was alsofound to be solvent exposed and conformationally mobile in comparisonwith the IL1ß scaffold, and there was no evidence of persistinginteractions with the scaffold outside of the N- and C-terminallinkages. However, AT-IL exhibits sigificant differences inchemical shift and NOE patterns relative to the isolated RSLthat are consistent with local features of non-random structure.The proximity of these features to the P1-P1' residues suggeststhat they may be responsible for the inhibitory activity ofthe chimera.     

Structural characterisation and comparison of the native and A-states of equine lysozyme

Native state 1H NMR resonance assignments for 125 of the 129 residues of equine lysozyme have enabled measurement of the hydrogen exchange kinetics for over 60 backbone amide and three tryptophan indole hydrogen atoms in the native state. Native holo equine lysozyme hydrogen exchange protection factors are as large as 10(6), the most protected residues being located in elements of secondary structure. High exchange protection in the domain interface correlates with the binding of Ca2+ in this region. Equine lysozyme differs from most non-Ca2+ binding lysozymes in forming a highly populated partially folded state at low pH. The protein in this A-state at pH 2.0 has been found to bind 1-anilino-naphthalene-8-sulphonate with the enhancement of fluorescent intensity and blue shift in the spectral maximum characteristic of molten globules. NMR spectra indicate that the A-state is globally much less ordered than native equine lysozyme but does not contain significant regions of random coil structure. The amides most protected against hydrogen exchange in the A-state (protection factors up to 10(2) at 5 degrees C) correspond to residues of three of the four alpha-helices of the native state; the side-chains of these residues form a hydrophobic cluster that includes five aromatic residues. Circular dichroism and tryptophan fluorescence indicate that these residues are substantially more constrained than similar residues in "classical" molten globules. Taken together, the data suggest a model for the A-state of equine lysozyme in which a more ordered core is surrounded by a less ordered but still compact polypeptide chain.