FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2079362211> ?p ?o ?g. }

• W2079362211 endingPage "27258" @default.
• W2079362211 startingPage "27249" @default.
• W2079362211 abstract "p13suc1 acts in the fission yeast cell division cycle as a component of p34cdc2. In the present work, structural information contained in the intrinsic fluorescence of p13suc1 has been extracted by steady-state and time-resolved fluorescence techniques. In its native form, the steady-state emission spectrum of p13suc1 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 p13suc1 at 56°C or after incubation at 100°C, and the protein appears to be substantially temperature-stable. The fluorescence decay of p13suc1 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 (λmax = 332 nm) and DAS-τ3 (λmax = 340 nm), derive from two different excitation DAS. Moreover distinct quenching mechanisms and collisional accessibilities (kq(τ2)≫kq(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 p13suc1 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 p13suc1. 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. p13suc1 acts in the fission yeast cell division cycle as a component of p34cdc2. In the present work, structural information contained in the intrinsic fluorescence of p13suc1 has been extracted by steady-state and time-resolved fluorescence techniques. In its native form, the steady-state emission spectrum of p13suc1 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 p13suc1 at 56°C or after incubation at 100°C, and the protein appears to be substantially temperature-stable. The fluorescence decay of p13suc1 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 (λmax = 332 nm) and DAS-τ3 (λmax = 340 nm), derive from two different excitation DAS. Moreover distinct quenching mechanisms and collisional accessibilities (kq(τ2)≫kq(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 p13suc1 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 p13suc1. 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. INTRODUCTIONp13suc1 acts in the fission yeast cell division cycle both in G1 and G2 (Forsburg and Nurse, 27Forsburg S.L. Nurse P. Annu. Rev. Cell Biol. 1991; 7: 227-256Crossref PubMed Scopus (268) Google Scholar; Reed, 57Reed S.I. Annu. Rev. Cell Biol. 1992; 8: 529-561Crossref PubMed Scopus (266) Google Scholar). Originally identified in Schizosaccharomyces pombe as extragenic suppressor of certain cdc2 temperature-sensitive mutations (Hayles et al., 32Hayles J. Beach D. Durkacz B. Nurse P. Mol. & Gen. Genet. 1986; 202: 291-293Crossref PubMed Scopus (153) Google Scholar), in the yeast lysates the product of the suc1 gene was found associated with the major cell cycle regulator, p34cdc2 (Brizuela et al., 12Brizuela L. Draetta G. Beach D. EMBO J. 1987; 6: 3507-3514Crossref PubMed Scopus (259) Google Scholar; Draetta et al., 18Draetta G. Brizuela L. Potashkin J. Beach D. Cell. 1987; 50: 319-325Abstract Full Text PDF PubMed Scopus (219) Google Scholar). The addition of p13suc1 to the kinase assay in vitro was able to rescue the defect in the Cdc2 mutant kinase activity (Booher et al., 11Booher R.N. Alfa C.E. Hyams J.S. Beach D.H. Cell. 1989; 58: 485-497Abstract Full Text PDF PubMed Scopus (258) Google Scholar; Moreno et al., 51Moreno S. Hayles J. Nurse P. Cell. 1989; 58: 361-372Abstract Full Text PDF PubMed Scopus (401) Google Scholar), and the protein was proposed to act as a regulatory component of the p34cdc2 (Brizuela et al., 12Brizuela L. Draetta G. Beach D. EMBO J. 1987; 6: 3507-3514Crossref PubMed Scopus (259) Google Scholar; Draetta, 16Draetta G. Trends. Biochem. Sci. 1990; 15: 378-383Abstract Full Text PDF PubMed Scopus (269) Google Scholar).The nucleotide sequence of suc1 has been determined (Hindley et al., 34Hindley J. Phear G. Stein M. Beach D. Mol. Cell. Biol. 1987; 7: 504-511Crossref PubMed Scopus (119) Google Scholar), and p13suc1 has been expressed in Escherichia coli (Brizuela et al., 12Brizuela L. Draetta G. Beach D. EMBO J. 1987; 6: 3507-3514Crossref PubMed Scopus (259) Google Scholar). 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., 13Brizuela L. Draetta G. Beach D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4362-4366Crossref PubMed Scopus (93) Google Scholar). The discovery of p34cdc2 homologue in human suggested the universality of cell cycle control mechanisms (Lee and Nurse, 45Lee M.G. Nurse P. Nature. 1987; 237: 31-35Crossref Scopus (757) Google Scholar). In addition, concurrently with finding a p34cdc2 homologous protein kinase, in the different eucaryotic species investigated, suc1 gene-related homologues have also been found (Simanis and Nurse, 61Simanis V. Nurse P. Cell. 1986; 45: 261-268Abstract Full Text PDF PubMed Scopus (315) Google Scholar; Draetta et al., 18Draetta G. Brizuela L. Potashkin J. Beach D. Cell. 1987; 50: 319-325Abstract Full Text PDF PubMed Scopus (219) Google Scholar; Paris et al., 1990; Elledge and Spottswood, 25Elledge S.J. Spottswood M.R. EMBO J. 1991; 10: 2653-2659Crossref PubMed Scopus (196) Google Scholar; Hellmich et al., 33Hellmich M.R. Pant H.C. Wada E. Battey J.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10867-10871Crossref PubMed Scopus (242) Google Scholar; Lew et al., 47Lew J. Beaudette K. Litwin C.M.E. Wang J.H. J. Biol. Chem. 1992; 267: 13383-13390Abstract Full Text PDF PubMed Google Scholar). In the budding yeast Saccharomyces cerevisiae the CKS1 gene codes for a protein, p18CKS, suppressor of Cdc28 mutations (Reed, 57Reed S.I. Annu. Rev. Cell Biol. 1992; 8: 529-561Crossref PubMed Scopus (266) Google Scholar), while in the early studies on HeLa cells (Draetta et al., 18Draetta G. Brizuela L. Potashkin J. Beach D. Cell. 1987; 50: 319-325Abstract Full Text PDF PubMed Scopus (219) Google Scholar), Cdc2 protein kinase was found associated with a 13-kDa polypeptide. More recently, the human homologues of the p13suc1/p18CKS proteins, p9CKShs1/p9CKShs2, have been identified as the products of the genes CKShs1 and CKShs2, respectively (Richardson et al., 58Richardson H.E. Stueland C.S. Thomas J. Reed S.I. Genes Dev. 1990; 4: 1332-1344Crossref PubMed Scopus (144) Google Scholar). According to these findings p13suc1 and its homologous proteins appear to be as ubiquitous as the p34 family of kinases, thus suggesting the essential role of p13suc1 as components of the cell cycle control mechanisms.The relevance of the biological function of p13suc1 has been advanced by several intriguing observations. The p34cdc2-p13suc1-Sepharose complex is active as protein kinase (Brizuela et al., 13Brizuela L. Draetta G. Beach D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4362-4366Crossref PubMed Scopus (93) Google Scholar). The same matrix has been found to deplete M phase Xenopus extracts of the “M phase-promoting factor” (MPF) 1The abbreviations used are: MPFM phase-promoting factorDASdecay-associated spectraEDASexcitation decay-associated spectraGdnHClguanidine hydrochloride. (Dunphy et al., 22Dunphy W.G. Brizuela L. Beach D. Newport J. Cell. 1988; 54: 423-431Abstract Full Text PDF PubMed Scopus (554) Google Scholar), and this evidence has been confirmed among different cell species (Draetta and Beach, 17Draetta G. Beach D. Cell. 1988; 54: 17-26Abstract Full Text PDF PubMed Scopus (524) Google Scholar; Arion et al., 3Arion D. Meijer L. Brizuela L. Beach D. Cell. 1988; 55: 371-378Abstract Full Text PDF PubMed Scopus (352) Google Scholar; Draetta et al., 19Draetta G. Luca F. Westendorf J. Brizuela L. Ruderman J. Beach D. Cell. 1989; 56: 829-838Abstract Full Text PDF PubMed Scopus (438) Google Scholar; Labbé et al., 40Labbé J.C. Capony J.P. Caput D. Cavadore J.C. Derancourt J. Kaghad M. Lelias J.M. Picard A. Doree M. EMBO J. 1989; 8: 3053-3058Crossref PubMed Scopus (375) Google Scholar; Pondaven et al., 56Pondaven P. Meijer L. Beach D. Genes Dev. 1990; 4: 9-17Crossref PubMed Scopus (93) Google Scholar; Meijer et al., 49Meijer L. Arion D. Goldsteyn R. Pines J. Brizuela L. Hunt T. Beach D. EMBO J. 1989; 8: 2275-2282Crossref PubMed Scopus (194) Google Scholar, 50Meijer L. Azzi L. Wang J.Y.J. EMBO J. 1991; 10: 1545-1554Crossref PubMed Scopus (105) Google Scholar). In addition, p13suc1 inhibits the entry into mitosis in Xenopus extracts (Dunphy et al., 22Dunphy W.G. Brizuela L. Beach D. Newport J. Cell. 1988; 54: 423-431Abstract Full Text PDF PubMed Scopus (554) Google Scholar) and, microinjected in mammalian oocytes, inhibits the entry into meiosis (Gavin et al., 28Gavin A.C. Vassalli J.D. Cavadore J.C. Schorderet-Slatkine S. Mol. Reprod. Dev. 1992; 33: 287-296Crossref PubMed Scopus (23) Google Scholar). Moreover, in vitro activation of the inactivated precursor of MPF (pre-MPF) from Xenopus oocytes results in tyrosine dephosphorylation of the p34cdc2 protein. p13suc1 completely blocks p34cdc2 tyrosine 15 dephosphorylation and kinase activation (Dunphy and Newport, 21Dunphy W.G. Newport J.W. Cell. 1989; 58: 181-191Abstract Full Text PDF PubMed Scopus (201) Google Scholar). A model has been proposed in which a Cdc2-specific tyrosine kinase and phosphatase, as well as p13suc1, might interact to regulate the Cdc2 kinase (Dunphy and Newport, 21Dunphy W.G. Newport J.W. Cell. 1989; 58: 181-191Abstract Full Text PDF PubMed Scopus (201) Google Scholar).Although these data suggest that p13suc1 plays a significant role in the regulation of p34cdc2, there is no clear agreement about the involvement of the suc1 product and its homologues in the steps of p34cdc2 biochemistry (Dunphy, 20Dunphy W.G. Trends Cell Biol. 1994; 4: 202-207Abstract Full Text PDF PubMed Scopus (249) Google Scholar).Fluorescence methods provide a useful tool to obtain dynamic and static information on the structure of proteins and macromolecular assemblies (Beechem and Brand, 6Beechem J.M. Brand L. Annu. Rev. Biochem. 1985; 54: 43-71Crossref PubMed Scopus (664) Google Scholar; Eftink, 23Eftink M.R. Suelter C.H. Methods of Biochemical Analysis.Vol 35. John Wiley & Sons, Inc., New York1991: 127-205Google Scholar). In addition, these techniques can be used to investigate molecular interactions in the living cell. In this paper, we describe steady-state and time-resolved fluorescence studies on the intrinsic fluorescence of p13suc1. A characterization of its photophysics and hydrodynamic properties is presented. These information, combined with the recent characterization of the protein crystal structure (Endicott et al., 26Endicott J.A. Noble M.E. Garman E.F. Brown N. Rasmussen B. Nurse P. Johnson L.N. EMBO J. 1995; 14: 1004-1014Crossref PubMed Scopus (67) Google Scholar), will be useful for future studies on p13suc1 structure/function relationships.MATERIALS AND METHODSThe E. coli [BL21(DE3)]LysS strain expressing the suc1 gene product was kindly provided by Dr. Giulio Draetta (Mitotix, Cambridge, MA). Bacterial growth, induction by isopropyl-β-D-thiogalactopyranoside, and purification of the protein were performed following the procedure described by Brizuela et al. (12Brizuela L. Draetta G. Beach D. EMBO J. 1987; 6: 3507-3514Crossref PubMed Scopus (259) Google Scholar). A final gel filtration step on a Sephacryl S-100 HR column (80 × 2 cm, flow rate: 0.1 ml/min) eluted with a buffer containing 50 mM Tris-HCl, pH 8.0, and 2 mM EDTA was added to obtain full homogeneity of the purified material. Protein concentration was determined by the Lowry method (Lowry et al., 48Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) and by a bicinchoninic acid-based method (Smith et al., 62Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18442) Google Scholar) using bovine serum albumin as a standard. Routinely, purity of p13suc1 was evaluated by reverse phase high performance liquid chromatography on a Vydac 208TP column (25 × 1.0 cm) eluted in the presence of 0.1% trifluoroacetic acid at increasing acetonitrile concentrations (0-80% of a 70:30, acetonitrile:H2O solution). The protein had an apparent Mr of ∼13.100 on 13% SDS-polyacrylamide gel electrophoresis (Laemmli, 43Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205998) Google Scholar) as measured on a Bio-Rad model GS-670 videodensitometer and using the Molecular Analyst software package. Isoelectrofocusing was performed on LKBAmpholine® PAGplates with a LKB apparatus. The measured pI = 5.84 was in excellent agreement with the theoretical value of pI = 5.80 obtained from the SWISS-PROT data base (ExPASy.WWW) of the Geneva University. The NH2-terminal region of the protein, blotted onto Pro-Blot (Applied Biosystem), was sequenced by Edman degradation up to 43 residues in a pulsed liquid Applied Biosystem model 476A protein sequencer. The obtained, 2-43, 2The NH2-terminal methionine residue was not detected by NH2-terminal amino acid sequencing (C. Menna, personal observation) as confirmed by Endicott et al. (26Endicott J.A. Noble M.E. Garman E.F. Brown N. Rasmussen B. Nurse P. Johnson L.N. EMBO J. 1995; 14: 1004-1014Crossref PubMed Scopus (67) Google Scholar). primary structure shared 100% identity with the nucleotide sequence of suc1 as determined by Hindley et al. (34Hindley J. Phear G. Stein M. Beach D. Mol. Cell. Biol. 1987; 7: 504-511Crossref PubMed Scopus (119) Google Scholar).Bacto-Agar, Tryptone, and yeast extract were obtained from Difco. isopropyl-β-D-thiogalactopyranoside was obtained from Fluka. Soybean trypsin inhibitor, aprotinin, leupeptin, tosylphenylalanine chloromethyl ketone, and phenylmethylsulfonyl fluoride were from Sigma and stored following the instructions of the supplier.Sephadex G-25, G-50, Sepharose CL-6B, and Sephacryl S-100 HR were from Pharmacia Biotech Inc.Fluorescence Spectroscopy MeasurementsTechnical steady-state fluorescence excitation and emission spectra were obtained with a Jasko FP-550 spectrofluorometer using excitation and emission slit widths of 5 nm each. Fluorescence polarization measurements were performed using two Polacoat dichroic polarizers (Jasko FP-2010) installed in the excitation and the emission paths to record the relative intensities for the four combinations of vertically (v) and horizontally (h) polarized beams (Ivv, Ivh, Ihh, Ihv). The resulting steady-state emission anisotropy, <r>, was calculated as follows, 〈r〉=Ivv⋅G−IvhIvv⋅G+2Ivhwhere G = Ihh/Ihv is the grating correction factor introduced to normalize for the different sensitivity of the system to detect the horizontally and vertically polarized emission (Azumi and McGlynn, 4Azumi T. McGlynn S.P. J. Chem. Phys. 1962; 37: 2413-2420Crossref Scopus (421) Google Scholar; Paoletti and LePecq, 54Paoletti J. LePecq J.-B. Anal. Biochem. 1969; 31: 33-41Crossref PubMed Scopus (32) Google Scholar).Fluorescence quenching measurements of p13suc1 were performed using acrylamide and potassium iodide as quenchers. With acrylamide, protein samples at increasing concentrations of the quencher were prepared by adding small aliquots from a 8 M stock solution. At the excitation wavelength of 295 nm no corrections for the optical density of the samples were required. With KI, protein samples at increasing concentrations of the quencher were prepared at constant ionic strength using KCl as a counter-ion. Stock solutions of KI (4 M) were freshly prepared in the presence of ≈1 × 10−4M Na2S2O3 to avoid 3− formation (Lehrer, 46Lehrer S.S. Biochemistry. 1971; 10: 3254-3263Crossref PubMed Scopus (1662) Google Scholar). Fluorescence decay experiments were recorded at increasing quencher concentrations (0-0.42 M). Curves were collected at three emission wavelengths (330, 340, and 350 nm) for each concentration of the quencher, and the data were analyzed by the global procedure. Steady-state and time-resolved fluorescence results were analyzed according to the Stern-Volmer equation (Lehrer, 46Lehrer S.S. Biochemistry. 1971; 10: 3254-3263Crossref PubMed Scopus (1662) Google Scholar; Lakowicz, 42Lakowicz J.R. Principles of Fluorescence Spectroscopy. Plenum Press, New York1983Crossref Google Scholar), F0F=1+KSV[Q]=τ0τwhere F0 and F and τ0 and τ are the fluorescence intensity and the lifetime in the absence and the presence of the quencher Q. KSV is the Stern-Volmer constant and represents a measure of the quenching of the fluorescence intensity. In the case the total emitted fluorescence can be separated in discrete intensity contributions, f(i), from distinct species, the overall KSV is the weighted sum of the specific quenching constant/species, Σf(i)KSV(i). The expression for the collisional quenching ratio is, then, given by Equation 3 (Laws and Contino, 44Laws W.R. Contino P.B. Methods Enzymol. 1992; 210: 448-463Crossref PubMed Scopus (59) Google Scholar), F0F=∑i=1nf(i)1+KSV(i)[Q]−1=τ0τ(Eq. 3) while in the presence of a static contribution and considering the sphere of action model, the complete expression for the quenching ratio is given by Equation 4 (Laws and Contino, 44Laws W.R. Contino P.B. Methods Enzymol. 1992; 210: 448-463Crossref PubMed Scopus (59) Google Scholar), F0F=∑i=1nf(i)1+KSV(i)[Q]eV(i)(Q]−1(Eq. 4) where V(i) represent the volume of the species-associated interaction sphere.Moreover, KSV is equal to kqτ0; where kq is the apparent rate constant for the collisional quenching process and represents a measure of the overall accessibility of the fluorophores. Relative bimolecular collisional quenching constants, kq(τi), can be obtained for each fluorescence lifetime. In this case, a simple modified form of the Stern-Volmer plot of 1/τi versus [Q] directly provide the kq(τi) as the slope of the graph.Nanosecond time-resolved fluorescence measurements were obtained by the time-correlated single photon counting method (O'Connor and Philips, 53O'Connor D.V. Philips D. Time Correlated Single Photon Counting. Academic Press, London1984Google Scholar) using a model 5000U Fluorescence Lifetime Spectrometer (IBH Consultants Ltd., Glasgow, United Kingdom). Hydrogen (0.50 bar) was used as a filling gas of the thyratron-gated flash lamp. The instrument response function determined from a scattering solution of Ludox was typically 1.4 ns (full width at half-maximum) using a Hamamatzu R3235 photomultiplier. The channel width was 0.106 ns/channel, and data were collected in 1024 channels. The decay of the total fluorescence intensity (104 counts in the peak) was recorded under “magic angle” conditions (Badea and Brand, 5Badea M.G. Brand L. Methods Enzymol. 1979; 61: 378-425Crossref PubMed Scopus (233) Google Scholar), and the wavelength-dependent time shift of the photomultiplier (Wahl et al., 66Wahl Ph. Auchet J.C. Donzel B. Rev. Sci. Instrum. 1974; 45: 28-32Crossref Scopus (147) Google Scholar) was determined in a separate experiment using melatonin (Sigma) as a standard. To resolve the excitation (EDAS) and the emission (DAS) spectra associated with the individual decay constants (Knutson et al., 38Knutson J.R. Walbridge D.G. Brand L. Biochemistry. 1982; 21: 4671-4679Crossref PubMed Scopus (173) Google Scholar), experimental curves were collected for equal dwell times and by stepping the excitation and the emission monochromators in increments of 2-5 nm. Time-resolved parameters were normalized with respect to the relative excitation and emission steady-state spectra.The decay of the emission anisotropy of p13suc1 was measured as described previously (Badea and Brand, 5Badea M.G. Brand L. Methods Enzymol. 1979; 61: 378-425Crossref PubMed Scopus (233) Google Scholar), using a combination of two Polacoat dichroic polarizers parallel (vv) and crossed (vh) with respect to the excitation and the emission paths. A depolarizer DPU-15 (Optics for Research, Caldwell, NJ) placed in front of the emission monochromator slit was used to minimize “G-factor” corrections (G ≈ 1.007). Decay curves of the polarized components of the emitted fluorescence were separately collected within the same experimental time course by alternative collection of the “Ivv” and “Ivh” curves, plus the exciting function “lamp”.Fluorescence Data AnalysisFluorescence intensity decay. The decay data were analyzed by nonlinear least square method (Knight and Selinger, 1971; Grinvald and Steinberg, 30Grinvald A. Steinberg I.Z. Anal. Biochem. 1974; 59: 583-598Crossref PubMed Scopus (719) Google Scholar), and decay curves collected at multiple emission wavelengths were simultaneously analyzed according to the global procedure described by Knutson et al. (39Knutson J.R. Beechem J.M. Brand L. Chem. Phys. Lett. 1983; 102: 501-507Crossref Scopus (541) Google Scholar). When appropriate, the decay constants were linked across spectral regions. The experimental data (counts/channel > 0.5% of the total counts in the peak) were analyzed assuming that the fluorescence decay follows a multiexponential law, I(t)=∑i=1nαi⋅e−t/π(Eq. 5) where the relative amplitudes, αi, and the decay constants, τi, are the numerical parameters to be recovered. The best fit between the theoretical curve and the data was evaluated from the plot of residuals, the autocorrelation function of the residuals, and the reduced Chi-square (χ2) (Bevington, 9Bevington P.R. Data Reduction and Error Analysis for the Physical Science. McGraw-Hill Inc., New York1969Google Scholar). The DAS and the EDAS were obtained by the global procedure (Beechem et al., 7Beechem J.M. Ameloot M. Brand L. Anal. Instr. 1985; 14: 379-402Crossref Scopus (17) Google Scholar), and the fluorescence relative intensities at the various wavelengths were expressed as αi·τi products. Percent fractional contributions of each decay component to the total emitted fluorescence was, then, calculated as, αi·τi/Σαi·τi.Fluorescence Anisotropy DecayThe anisotropy decay can be described by a sum of discrete exponential terms as follows (Wahl, 65Wahl Ph. Biochim. Biophys. Acta. 1969; 175: 55-64Crossref PubMed Scopus (64) Google Scholar; Tao, 64Tao T. Biopolymers. 1969; 8: 609-632Crossref Scopus (469) Google Scholar), r(t)=∑i=1nβi⋅e−t/ϕi(Eq. 6) where the sum of the pre-exponential terms βi is the anisotropy in the absence of rotation, r0, and the Φi values are the rotational correlation times. For a globular protein that approximates the spherical symmetry, the anisotropy decay is reduced to a single exponential. Under this condition the correlation time can be related to the hydrated volume of the rotating protein, V, by the Einstein-Stokes relation, Φ = Vη/kT; where η is the solvent viscosity, k is the Boltzman constant, and T is the experimental temperature. Alternatively, complex anisotropy decays suggest deviation from simple spherical symmetry.The parameters for the decay of anisotropy, r(t), were recovered from the analysis of the experimental decays of the polarization components, Ivv(t) and Ivh(t), by the system analysis approach introduced by Gilbert (29Gilbert C.W. Cundall R.B. Dale R.E. Time-resolved Fluorescence Spectroscopy in Biochemistry and Biology. Plenum Press, New York1983: 605-606Google Scholar). According to this method, the fitting functions to obtain r(t) are the following (Ameloot et al., 1Ameloot M. Hendrickx H. Herreman W. Pottel H. Van Cauwelaert F. Van Der Meer W. Biophys. J. 1984; 46: 525-539Abstract Full Text PDF PubMed Scopus (115) Google Scholar; Cross and Fleming, 14Cross A.J. Fleming G.R. Biophys. J. 1984; 46: 45-56Abstract Full Text PDF PubMed Scopus (200) Google Scholar), Ivv(t)=1/3s(t)⋅(1+2r(t))(Eq. 7) Ivh(t)=1/3s(t)⋅(1−r(t))(Eq. 8) where s(t) represents the decay of the total fluorescence intensity and does not depends on molecular reorientation. Common parameters were linked, and η/T terms were introduced in the analysis of anisotropy decay curves obtained at multiple temperatures.The variability of the decay parameters was evaluated by determining the joint confidence intervals (Johnson, 35Johnson M.L. Biophys. J. 1983; 44: 101-106Abstract Full Text PDF PubMed Scopus (134) Google Scholar). All the steady-state and time-resolved fluorescence experiments were run at least twice using different preparations of p13suc1. The inter-experimental variability was less than 10′.Circular DichroismCircular dichroism spectra were recorded on a Jasco J-710 spectropolarimeter. Protein samples (0.15 mg/ml) in 50 mM phosphate buffer, pH 7.4, 2 mM EDTA were measured in a 1-mm path length cell at 0.5-nm interval. Eight spectra were accumulated and averaged to achieve appropriate signal-to-noise ratios. The fractional composition of the secondary structure of p13suc1 in terms of α-helix, β-sheet, and random coil was evaluated according to the procedure described by Andrade (1993). According to that procedure, neural network analysis of the protein topological map in the 200-230 nm wavelength range was performed using the k2d.PC software.RESULTSThe isolated protein resulted homogeneously pure as judged by the amino acids sequence of its 2-43 NH2-terminal region, by high performance liquid chromatography, by SDS-polyacrylamide gel electrophoresis, and by the measure of its pI (see “Materials and Methods”).Circular DichroismThe CD spectra of p13suc1 recorded at 20°C (curve 1), at 56°C (curve 2), and at 20°C after incubation at 100°C for 10 min (curve 3) is presented in Fig. 1. The data were recorded in the 180-300 nm spectral range. The protein was dialyzed overnight against 50 mM phosphate buffer, 2 mM EDTA, at pH 7.4, to remove Tris-HCl. From these data a secondary structure composition of 23%α-helix, 19%β-sheet, and 58% random coil was obtained for the native protein at 20°C. At 56°C, an expected loss of ordered secondary structure is observed. However, when the spectrum is recorded at 20°C, with a sample previously incubated at 100°C, a small fraction of secondary structure is recovered and the intensity of the aromatic band (235 nm) is completely restored to the native level.Steady-state Intrinsic Fluorescencep13suc1 contains two tryptophan (Trp-71 and Trp-82) and seven tyrosine residues (Tyr-27, Tyr-31, Tyr-36, Tyr-38, Tyr-55, Tyr-85, and Tyr-103). The steady-state emission spectra of the intrinsic fluorescence of p13suc1 recorded under native and denaturing conditions are presented in Fig. 2. Under native conditions, emission spectra were obtained exciting the protein in both the tryptophan, 295 nm (curve 1), and the tyrosine, 275 nm (not shown), absorption bands. No differences of the steady-state emission spectra profiles were observed, suggesting a very poor contribution of the tyrosine residues. The maximum of the fluorescence intensity was centered at 336 nm, as expected for chromophores partially buried inside the protein. In 4.0 M GdnHCl, curve 2, the emission spectrum of p13suc1 was shifted to 355-360 nm, and its intensity was decreased of approximately 70%. Higher guanidinium concentrations (6.0 M) did not cause further effects on the fluorescence spectrum. Emission spectra were also recorded in the absence of GdnHCl, at 56°C, curve 3; at 20°C, by cooling the same sample, curve 4; and finally at 20°C, with a sample previously incubated at 100°C for 10 min, curve 5. These data suggest that, up to 56°C, the structural transitions induced on p13suc1 by heat are mostly reversible and even more severe temperature treatments do not denature the protein completely.Fig. 2Steady-state emission spectra of the intrinsic fluorescence of p13suc1. The native protein (0.1 mg/ml) was dissolved in 50 mM Tris-HCl, pH 8.0, 2 mM EDTA, at 20°C, in the absence (solid line 1) and the presence of 4.0 M GdnHCl (solid line 2). Emission spectra were also recorded in the absence of GdnHCl: at 56°C (dotted line 3), at 20°C by cooling the same sample (dotted line 4), and at 20°C with a sample incubated at 100°C for 10 min (dotted line 5). The excitation wavelength was 295 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Time-resolved Intrinsic FluorescenceTo obtain dynamic information on p13suc1, the intrinsic fluorescence decay of the protein has been resolved in the nanosecond time scale. The decay of the fluorescence intensity was measured exciting the samples at 295 nm. A ty" @default.
• W2079362211 created "2016-06-24" @default.
• W2079362211 creator A5051194757 @default.
• W2079362211 creator A5057459234 @default.
• W2079362211 creator A5070902719 @default.
• W2079362211 creator A5080363205 @default.
• W2079362211 date "1996-11-01" @default.
• W2079362211 modified "2023-10-17" @default.
• W2079362211 title "Intrinsic Fluorescence Properties and Structural Analysis of p13 from Schizosaccharomyces pombe" @default.
• W2079362211 cites W1246035719 @default.
• W2079362211 cites W124931762 @default.
• W2079362211 cites W129634405 @default.
• W2079362211 cites W1548138662 @default.
• W2079362211 cites W1566217027 @default.
• W2079362211 cites W1578554939 @default.
• W2079362211 cites W1643898426 @default.
• W2079362211 cites W1723327061 @default.
• W2079362211 cites W1775749144 @default.
• W2079362211 cites W1969338076 @default.
• W2079362211 cites W1969350379 @default.
• W2079362211 cites W1973435955 @default.
• W2079362211 cites W1975849939 @default.
• W2079362211 cites W1977646528 @default.
• W2079362211 cites W1980585800 @default.
• W2079362211 cites W1983185345 @default.
• W2079362211 cites W1985232413 @default.
• W2079362211 cites W1991217128 @default.
• W2079362211 cites W1994088804 @default.
• W2079362211 cites W1995725157 @default.
• W2079362211 cites W1997360761 @default.
• W2079362211 cites W2008066024 @default.
• W2079362211 cites W2009837683 @default.
• W2079362211 cites W2012185830 @default.
• W2079362211 cites W2015188954 @default.
• W2079362211 cites W2016634429 @default.
• W2079362211 cites W2017798184 @default.
• W2079362211 cites W2024197567 @default.
• W2079362211 cites W2024990576 @default.
• W2079362211 cites W2037774606 @default.
• W2079362211 cites W2047799300 @default.
• W2079362211 cites W2051184492 @default.
• W2079362211 cites W2056104143 @default.
• W2079362211 cites W2064262345 @default.
• W2079362211 cites W2068034418 @default.
• W2079362211 cites W2069249321 @default.
• W2079362211 cites W2070125667 @default.
• W2079362211 cites W2070278756 @default.
• W2079362211 cites W2072463712 @default.
• W2079362211 cites W2074177074 @default.
• W2079362211 cites W2078528113 @default.
• W2079362211 cites W2080160898 @default.
• W2079362211 cites W2080263723 @default.
• W2079362211 cites W2086247286 @default.
• W2079362211 cites W2089414651 @default.
• W2079362211 cites W2100238612 @default.
• W2079362211 cites W2100837269 @default.
• W2079362211 cites W2103675400 @default.
• W2079362211 cites W2117939247 @default.
• W2079362211 cites W2120626070 @default.
• W2079362211 cites W2136640235 @default.
• W2079362211 cites W2162279862 @default.
• W2079362211 cites W2175640000 @default.
• W2079362211 cites W2205702781 @default.
• W2079362211 cites W2302488076 @default.
• W2079362211 cites W2333124839 @default.
• W2079362211 cites W2339158758 @default.
• W2079362211 cites W4232534952 @default.
• W2079362211 cites W58458559 @default.
• W2079362211 doi "https://doi.org/10.1074/jbc.271.44.27249" @default.
• W2079362211 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/8910298" @default.
• W2079362211 hasPublicationYear "1996" @default.
• W2079362211 type Work @default.
• W2079362211 sameAs 2079362211 @default.
• W2079362211 citedByCount "13" @default.
• W2079362211 countsByYear W20793622112013 @default.
• W2079362211 countsByYear W20793622112016 @default.
• W2079362211 countsByYear W20793622112023 @default.
• W2079362211 crossrefType "journal-article" @default.
• W2079362211 hasAuthorship W2079362211A5051194757 @default.
• W2079362211 hasAuthorship W2079362211A5057459234 @default.
• W2079362211 hasAuthorship W2079362211A5070902719 @default.
• W2079362211 hasAuthorship W2079362211A5080363205 @default.
• W2079362211 hasBestOaLocation W20793622111 @default.
• W2079362211 hasConcept C120665830 @default.
• W2079362211 hasConcept C121332964 @default.
• W2079362211 hasConcept C12554922 @default.
• W2079362211 hasConcept C185592680 @default.
• W2079362211 hasConcept C2776519988 @default.
• W2079362211 hasConcept C2777576037 @default.
• W2079362211 hasConcept C2779222958 @default.
• W2079362211 hasConcept C2779429346 @default.
• W2079362211 hasConcept C55493867 @default.
• W2079362211 hasConcept C70721500 @default.
• W2079362211 hasConcept C86803240 @default.
• W2079362211 hasConcept C91881484 @default.
• W2079362211 hasConceptScore W2079362211C120665830 @default.
• W2079362211 hasConceptScore W2079362211C121332964 @default.
• W2079362211 hasConceptScore W2079362211C12554922 @default.

• next