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Volume 271, Number 44, Issue of November 1, 1996 pp. 27249-27258
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Intrinsic Fluorescence Properties and Structural Analysis of p13^suc1 from Schizosaccharomyces pombe

(Received for publication, May 30, 1996, and in revised form, August 9, 1996)

From the Dipartimento di Biochimica ``G. Moruzzi,'' Sezione di Biochimica Farmaceutica, Università di Bologna, 40127 Bologna, Italy

p13^suc1 acts in the fission yeast cell division cycle as a component of p34^cdc2. In the present work, structural information contained in the intrinsic fluorescence of p13^suc1 has been extracted by steady-state and time-resolved fluorescence techniques. In its native form, the steady-state emission spectrum of p13^suc1 is centered at 336 nm. Upon denaturation by guanidine HCl (4.0 M), the emission spectrum is shifted to 355-360 nm and the fluorescence intensity decreases 70%. The same changes are not obtained with p13^suc1 at 56 °C or after incubation at 100 °C, and the protein appears to be substantially temperature-stable. The fluorescence decay of p13^suc1 is best described by three discrete lifetimes of 0.6 ns (₁), 2.9 ns (₂), and 6.1 ns (₃), with amplitudes that are dependent on the native or unfolded state of the protein. Under native conditions, the two predominant decay-associated spectra, DAS-₂ (_max = 332 nm) and DAS-₃ (_max = 340 nm), derive from two different excitation DAS. Moreover distinct quenching mechanisms and collisional accessibilities (k_q(₂)k_q(₃)) are resolved for each lifetime. An interpretation in terms of specific tryptophan residue (or protein conformer)-lifetime assignments is presented. The decay of the fluorescence anisotropy of native p13^suc1 is best described by a double exponential decay. The longer correlation time recovered (9 ns  ₂  15ns) can be associated with the rotational motion of the protein as a whole and a Stokes radius of 21.2 Å has been calculated for p13^suc1. Anisotropy measurements obtained as a function of temperature indicate that, in solution, the protein exists exclusively as a prolate monomer. In 1 mM zinc, changes of the anisotropy decay parameters are compatible with subunits oligomerization.

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