p13(sac1) 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 (τ1), 2.9 ns (τ2), and 6.1 ns (τ3), with amplitudes that are dependent on the native or unfolded state of the protein. Under native conditions, the two predominant decay-associated spectra, DAS-τ2 (λ(max) = 332 nm) and DAS- τ3 (λ(max) = 340 nm), derive from two different excitation DAS. Moreover distinct quenching mechanisms and collisional accessibilities (k(q)(τ2)>>k(q)(τ3)) 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 ≤ φ2 ≤ 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.

Intrinsic fluorescence properties and structural analysis of p13suc1 from Schizosaccharomyces pombe / Neyroz, P; Menna, C; Polverini, Eugenia; Masotti, L.. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - 271:44(1996), pp. 27249-27258. [10.1074/jbc.271.44.27249]

Intrinsic fluorescence properties and structural analysis of p13suc1 from Schizosaccharomyces pombe

POLVERINI, Eugenia;
1996-01-01

Abstract

p13(sac1) 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 (τ1), 2.9 ns (τ2), and 6.1 ns (τ3), with amplitudes that are dependent on the native or unfolded state of the protein. Under native conditions, the two predominant decay-associated spectra, DAS-τ2 (λ(max) = 332 nm) and DAS- τ3 (λ(max) = 340 nm), derive from two different excitation DAS. Moreover distinct quenching mechanisms and collisional accessibilities (k(q)(τ2)>>k(q)(τ3)) 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 ≤ φ2 ≤ 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.
1996
Intrinsic fluorescence properties and structural analysis of p13suc1 from Schizosaccharomyces pombe / Neyroz, P; Menna, C; Polverini, Eugenia; Masotti, L.. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - 271:44(1996), pp. 27249-27258. [10.1074/jbc.271.44.27249]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11381/1510142
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