INTRINSIC FLUORESCENCE PROPERTIES AND STRUCTURAL-ANALYSIS OF P13(SUC1) FROM SCHIZOSACCHAROMYCES-POMBE

p(13suc1) acts in the fission yeast cell division cycle as a component of p34(cdc2). I, the present work, structural information contained i n 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%. Th e same changes are not obtained with p(13suc1) at 56 degrees C or afte r incubation at 100 degrees C, and the protein appears to be substanti ally temperature-stable. The fluorescence decay of p(13suc1) is best d escribed by three discrete lifetimes of 0.6 ns (tau(1)), 2.9 ns (tau(2 )), and 6.1 ns (tau(3)), with amplitudes that are dependent on the nat ive or unfolded state of the protein. Under native conditions, the two predominant decay-associated spectra, DAS-tau(2), (lambda(max), = 332 nm) and DAS-tau(3) (lambda(max) = 340 nm), derive from two different excitation DAS. Moreover distinct quenching mechanisms and collisional accessibilities (k(q)(tau(2))much greater than k(q)(tau(3))) are reso lved for each lifetime. An interpretation in terms of specific tryptop han residue (or protein conformer)-lifetime as signments is presented. The decay of the fluorescence anisotropy of native p13(suc1) is best described by a double exponential decay. The longer correlation time r ecovered (9 ns less than or equal to phi(2) less than or equal to 15ns ) can be associated with the rotational motion of the protein as a who le and a Stokes radius of 21.2 Angstrom has been calculated for p13(su c1). Anisotropy measurements obtained as a function of temperature ind icate that, in solution, the protein exists exclusively as a prolate m onomer. In 1 mM zinc, changes of the anisotropy decay parameters are c ompatible with subunits oligomerization.