Abstract
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 degrees C or after incubation at 100 degrees 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 (tau1), 2.9 ns (tau2), and 6.1 ns (tau3), with amplitudes that are dependent on the native or unfolded state of the protein. Under native conditions, the two predominant decay-associated spectra, DAS-tau2 (lambdamax = 332 nm) and DAS-tau3 (lambdamax = 340 nm), derive from two different excitation DAS. Moreover distinct quenching mechanisms and collisional accessibilities (kq(tau2)>>kq(tau3)) 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 </= phi2 </= 15ns) can be associated with the rotational motion of the protein as a whole and a Stokes radius of 21.2 A 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.
Highlights
P13suc1 acts in the fission yeast cell division cycle both in G1 and G2 (Forsburg and Nurse, 1991; Reed, 1992)
Identified in Schizosaccharomyces pombe as extragenic suppressor of certain cdc2 temperature-sensitive mutations (Hayles et al, 1986), in the yeast lysates the product of the suc1 gene was found associated with the major cell cycle regulator, p34cdc2 (Brizuela et al, 1987; Draetta et al, 1987)
From that source p13suc1-Sepharose beads have been prepared, and their affinity binding to p34cdc2 has been widely used to purify p34cdc2 (Brizuela et al, 1989)
Summary
P13suc acts in the fission yeast cell division cycle both in G1 and G2 (Forsburg and Nurse, 1991; Reed, 1992). Identified in Schizosaccharomyces pombe as extragenic suppressor of certain cdc temperature-sensitive mutations (Hayles et al, 1986), in the yeast lysates the product of the suc gene was found associated with the major cell cycle regulator, p34cdc (Brizuela et al, 1987; Draetta et al, 1987). The human homologues of the p13suc1/p18CKS proteins, p9CKShs1/ p9CKShs, have been identified as the products of the genes CKShs and CKShs, respectively (Richardson et al, 1990) According to these findings p13suc and its homologous proteins appear to be as ubiquitous as the p34 family of kinases, suggesting the essential role of p13suc as components of the cell cycle control mechanisms. Fluorescence methods provide a useful tool to obtain dynamic and static information on the structure of proteins and macromolecular assemblies (Beechem and Brand, 1985; Eftink, 1991) These techniques can be used to investigate molecular interactions in the living cell. These information, combined with the recent characterization of the protein crystal structure (Endicott et al, 1995), will be useful for future studies on p13suc structure/function relationships
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