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Investigation of native state of plasticity of interleukin 1-beta

Melinda Roy

Appointment Period: 1996-1996 / Grant Year: [11]

We investigated the native state plasticity of the inflammatory cytokine interleukin-1B (Il-1B). This cytokine has been implicated in apoptosis (programmed cell death) because it controls the activation of NF-KB through the interleukin receptor kinase. Unfolded intermediates relative to the native structure may indicate a fluidity or plasticity in the conformation of soluble IL-1-beta, which may explain the number of IL-1-beta interactions and mechanisms.

An equilibrium unfolding intermediate induced by guanidine hydrochloride (gdn-HCl) was characterized by fluorescence and NMR. Relative to other proteins, IL-1-beta shows effects at relatively low levels of gdn-HCl. The physiological significance may be that the native structure can easily access other conformations.

The fluorescent intensity data of IL-1-beta with respect to gdn-HCl indicate the formation of a stable equilibrium intermediate at 0.8M gdn-HCl, with unfolding essentially complete at 2M. The chemical shifts of IL-1-beta in 2D 1H-15N heteronuclear NMR are also affected by addition of gdn-HCL, with the intermediate and native forms in fast exchange (on the NMR time scale) and the intermediate and unfolded forms in slow exchange. Controls done with NaCl indicate a salt effect on chemical shift perturbation, which is probably a combination of ionic and destabilizing effects. However, there are additional chemical shift changes seen with the addition of gdn-HCl, which should be exclusively the result of unfolding.

Preliminary analysis of 3D-NOESY data indicates that the conformation of the unfolding intermediate is similar to the native state, a beta-barrel with a cap composed of beta-strands. The beta-sheet structure is largely unaffected and hairpin turns are as tight as always. However, a few tertiary interactions have altered (e.g., the aromatic ring of Trp120, and the change of the turn at Lys63-Asn66 from type I to type II), and the protein's state appears to be more fluid or dynamic, as evidenced by a surprising increase in the number of cross-strand NOE interactions seen. Loops appear to be seeing more backbone interactions. Triple resonance NMR experiments provide 13C chemical shift data, which are very closely correlated to protein secondary structure. Analysis of the 13C chemical shift data of the native and the intermediate confirms the interpretation of the NOE data, insofar as the secondary structure is largely unchanged. There are individual cases where the phi, psi anges have altered considerably, primarily at the loops and at hairpin turn Lys63-Asn66. The largest change in carbon chemical shift is that of K92, which is in a loop region already implicated in unfolding by other studies in this lab.

PUBLICATIONS (resulting from this training, and some recent ones)

Newlon MG, Roy M, Hausken ZE, Scott JD, Jennings PA. (1997) The A-kinase anchoring domain of type IIalpha cAMP-dependent protein kinase is highly helical. J Biol Chem. 272:23637-44.

Heidary DK, Gross LA, Roy M, Jennings PA. (1997) Evidence for an obligatory intermediate in the folding of interleukin-1 beta. Nat Struct Biol. 4:725-31.

Jennings P, Roy M, Heidary D, Gross L. (1998) Folding pathway of interleukin-1 beta. Nat Struct Biol. 5:11.

Newlon MG, Roy M, Morikis D, Hausken ZE, Coghlan V, Scott JD, Jennings PA. (1999) The molecular basis for protein kinase A anchoring revealed by solution NMR. Nat Struct Biol. 6:222-7.

Finke JM, Roy M, Zimm BH, Jennings PA. (2000) Aggregation events occur prior to stable intermediate formation during refolding of interleukin 1beta. Biochemistry. 39:575-83.

Heidary DK, O'Neill JC Jr, Roy M, Jennings PA. (2000) An essential intermediate in the folding of dihydrofolate reductase. Proc Natl Acad Sci USA 97:5866-70.

Banky P, Newlon MG, Roy M, Garrod S, Taylor SS, Jennings PA. (2000) Isoform-specific differences between the type Ialpha and IIalpha cyclic AMP-dependent protein kinase anchoring domains revealed by solution NMR. J Biol Chem. 275:35146-52.

Covalt JC Jr, Roy M, Jennings PA. (2001) Core and surface mutations affect folding kinetics, stability and cooperativity in IL-1 beta: does alteration in buried water play a role? J Mol Biol. 307:657-69.

Newlon MG, Roy M, Morikis D, Carr DW, Westphal R, Scott JD, Jennings PA. (2001) A novel mechanism of PKA anchoring revealed by solution structures of anchoring complexes. EMBO J. 20:1651-62.

Morikis D, Roy M, Sahu A, Troganis A, Jennings PA, Tsokos GC, Lambris JD. (2002) The structural basis of compstatin activity examined by structure-function-based design of peptide analogs and NMR. J Biol Chem. 277:14942-53.

Morikis D, Roy M, Newlon MG, Scott JD, Jennings PA. (2002) Electrostatic properties of the structure of the docking and dimerization domain of protein kinase A IIalpha. Eur J Biochem. 269:2040-51.

Roy M, Jennings PA. (2003) Real-time NMR kinetic studies provide global and residue-specific information on the non-cooperative unfolding of the beta-trefoil protein, interleukin-1beta. J Mol Biol. 328:693-703.

Banky P, Roy M, Newlon MG, Morikis D, Haste NM, Taylor SS, Jennings PA. (2003) Related protein-protein interaction modules present drastically different surface topographies despite a conserved helical platform. J Mol Biol. 330:1117-29.

Jiang T, Olson ES, Nguyen QT, Roy M, Jennings PA, Tsien RY. (2004) Tumor imaging by means of proteolytic activation of cell-penetrating peptides. Proc Natl Acad Sci USA 101:17867-72.

Roy M, Chavez LL, Finke JM, Heidary DK, Onuchic JN, Jennings PA. (2005) The native energy landscape for interleukin-1beta. Modulation of the population ensemble through native-state topology. J Mol Biol. 348:335-47.

Heidary DK, Roy M, Daumy GO, Cong Y, Jennings PA. (2005) Long-range coupling between separate docking sites in interleukin-1beta. J Mol Biol. 353:1187-98.