Jurnal Vimentin

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Volume 301, number 2 FEB.5 LETTERS May 1992nucleic acid solution (690-740 PM) were added to the vimcntin solu-tion. and emission spectra were measured using cxcitution wav-elenyhta or280 and 295 nm. Assuming that tyrosine is not excited at295 nm, the contribution of the tyrosine residues to the total Ruorcs-cence wjascalculated by subtraction of the vimentin emission obtainedat an excitation wavelength of 295 nm from that obtained at 280 nm,after normalizing the two spccrra above 380 nm (whcrc tyrosine emis-sion is practically zero [ 141).

Vimentin fluorescence excitation at 280 nm

Wavelength nm)

Vimentin fluorescence excitation at 295 nm

Wavelength nm)Fig. 1. F!uorescence emission spectra or vimentin (0.59 PM) withincreasing amounts or pdT (stock 740 PM): I, 0 yl; 2, 8,5~1; 3, 24.5yl; 4. 48.5 ~1; 5, 78.5 ~1; 6. 118.5pl; 7, 189.5 ~1. (A) Excitationwavelength 280 nm. (B) Excitation wavelength 295 nm. Spectra arecorrected for dilution, the starting volume is 2,000 ~1.

3. RESULTSFig. IA and B show the fluorescence emissionspectra of vimentin determined after the addition ofincreasing amounts of poly(dT). The vimentin emissionat an excitation wavelength of 280 nm (A) shows amaximum at 310 nm, suggesting a large contribution of

tyrosine fluorescence to the total signal. The intensityof the blue side of the spectrum (290-320 nm) is signif-icantly decreased, while the red side of the spectrum(360-450 nm) is hardly affected when poly(dT) is added,Upon excitation with 295 nm light, a peak with a max-imum at 346 nm due to tryptophan fluorescence is ob-served, The intensity as well as the maximum emissionwavelength are practically unaffected upon titrationwith poly(dT). When the pure tyrosine emission is cal-culated via subtraction of the tryptophan emission (seesection 2) one observes that the tyrosine signal isquenched upon addition of poly(dT) (Fig. 2). Whenrelatively large amounts of poly(dTj are added (Fig. 2),saturation occurs and the tyrosine fluorescence remainsat a constant level, about 48% of the level in the absenceof poly(dT).Assuming that the observed tyrosine fluorescencequenching is caused by binding of the nucleic acid to theprotein, the stochiometry of binding 11, which is thenumber of nucleotides covered by each vimentin tetra-mer in the complex, can be determined from a fluorcs-cence titration curve, in which the quenching of tyrosinefluorescence is plotted against the ratio of the addednucleotide to the protein tetramer concentration (Fig.3). This is accomplished by drawing two straight lines

Calculated vimentin tyrosine fluorescence

Fig. 2. Cdlcuhilcd tyrosine contribution to the Ruoresccnce emissionatIer excitation at 280 nm (set text). For cxplunation or the numbersnear the spectra: see Fig. I.178


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Volume 301, number 2 FEBS May 1992Titration curve for vimentin with poly(dT)

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[poly(dT)] [vimentin]Fig. 3, Fluorescence titration curve for the binding ofpdT tovimcntin.The Ruorcsccnce due to tyrosine measured at 307 nni is plotted againstthe ratio of nuclcotides and pl*otcin ictramcrs.

representing the initial and final slopes of the curve. Theratio at the intersection of these lines corresponds to n.For the vimentin-poly(dT) complex, n=50 f 4 is ob-tained. Assuming Scatchard binding to independentsites, the apporant binding constant (Q,) can beachieved from the titration curve using the expression[15]:K,,=W(l-W[&l]&,=apparent binding constant (M-l), 8=ratio be-tween protein fluorescence change at a particular ligandconcentration and fluorescence change at a saturatingligand concentration and [P&total protein concentra-tion (M). Because this approximation does not correctfor the accumulation of gaps (which are smaller thanthe binding site size) between stretches of contiguouslybound vimentin molecules, this binding constant is a

Table IValues of the nllmber of nucleotidcs covered, 11, he apparent bindingconstant, f& and the maximum tyrodnc quenching due to the inter-action of vimcntin tctramers with nucleic acids

II Max. quenching(%IpoMdT)poly(rA)poWA)

50 t- 4 2.5 ?: 0.5 x IO 48 2 244+4 5.0 2 3.0 x IO 32 ?r.4

-= no measurable tyrosinc fluorescence quenching

minimum estimate only [ 161.However, KllPp s a usefulparameter to compare binding constants of differentpolynucleotides. For the binding of vimentin topoly(dT), &,,=2.5 & 0.5 x 10 M-i was calculated.Similar binding experiments as those reported forpoly(dT) were also performed for vimentin binding topoly(dA) and to poly(rA). The parameters n, K,,rPandthe total tyrosine quenching of poly(dT) and poly(rA)are listed in Table 1. In case of poly(rA), n is found tobe similar to poly(dT), while KicpP f poly(rA) is slightlylarger. Interstingly, titration with polp(dA) did not re-sult in a measurable fluorescence quenching (data notshown).4. DISCUSSION

The specific quenching of tyrosine fluorescence in thetitration experiments with poly(dT) and poly(rA) is in-terpreted as an interaction between tyrosyl rings of theprotein and the heterocyclic bases of the nucleic acids.Since it has been shown that the N-terminus of vimentinis essential for its interaction with nucleic acids [6], it isobvious that some or all tyrosine residues located in thisregion are involved in this process. The interactioncould be caused by intercalation of aromatic residues ofamino acids between nucleotide bases, as has beenshown for other ssDNA binding proteins such as gene5 protein of fd bacteriophage and phage T4gp32 [9,17].Crosslinking experiments of vimentin with tetrani-tromethane (Traub, unpublished results), which showedprevention from nitration of the tyrosyl residues byrRNA or fd DNA also indicate a shielding of tyrosinedue to the nucleic acid. Shape and emission maximumof the tryptophan spectrum remain co;tstant upon theaddition of saturating amounts of nucleic acids, indicat-ing that no conformational changes in the tryptophanregion occur and that the tryptophan residue, which islocated in the central rod domain [8], does not play arole in the nucleic acid binding process either.The high affinity for poly(dT) and the absence ofbinding of poly(dA) are in reasonable agreement withthe results of investigations via a quantitative filterbinding assay 171. The advantage of the fluorescencetechnique is that only spec!.ficbinding in which tyrosineresidues are involved, is detected. Moreover, the bind-ing site size /I and the degree of fluorescence quenching,which yields information about the binding process, canbe determined. In this study, it is shown that the bindingsite size for poly(dT) and poly(rA) is approximately thesame, suggesting the involvement of the same numberof tyrosine residues in the binding process. The appar-ent binding constant is somewhat larger for poly(rA).At saturating amounts of poly(dT), however, the tyro-sine quenching is significantly larger than at saturatingconditions of poly(rA). This could be due to two differ-ent modes of these nucleic acids to the protein. Thestriking difference in binding properties of poly(rA) and

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Volume 302. number 2 FEBS LETTERS May 1992poly(dA) suggests that not only base composition butbackbone properties of the nucleic acids as well play arole in the process. The physiological meaning of nu-cleic acid-vimentin interaction, if existent, is poorly un-derstood. An active role for IF subunit proteins in mod-ulating DNA replication, recombination, and repair, aswell as in gene expression, has been postulated [18].Vimentin has been identified as a DNA attachment prc;-tein within nuclei of Chinese Hamster Ovary cells [19].However, more experiments are needed to fully charac-terize the nucleic acid binding properties of vimentinunder several conditions. The present study, whichshows a strong specific interaction between vimentinand some types of ssDNA and RNA, may be helpful inthe search for the (possible) physiological role of thebinding.A~k,to,~,/~~~~nr~ol~s.e thank the Department of Plant Physiology ofthe Free University Amsterdam for the use of the Aminco fluorome-ter. This work was supported by the Netherlands Oryanizition ofScientific Research (NWO), in part via the Foundation of Biophysicsand Biology,REFERENCES[I] Stcinert. P.M. and Roop, D,R. (1988) Annu. Rev. Biochem. 57.593-625.[2] Quax-Jcukcn, Y.E,F.M.. Qunx, W.J, and Bloemcndal, H. (1962)Proc. Natl. Acad. Sci. USA BO,3548-3552.

[3] Ip, W., Hartzer, M.K., Pang, S. and Robson, R.M. (1985) J. Mol.Biol. 183, 365-375,[4] Potschka, M. (1986) Biophys. J. 49, 129-130,[5] Truub. P., Nelson, W.J., Kuhn, S. and Vorgias, C.E. (1983) J.Biol. Chem. 258. 14561466.[6] Shoeman, R,L.. Wddk?. S., Schcrbarth, A. and Traub, P. (1988)J. Biol. Chem. 263, 18744-18749.[7] Shoeman, R,L, and Traub, P. (1990) J. Biol. Chem. 265, 90%9061.[8] Capetanuki. Y,G,, Kuisk. I., Rothblum, K.N. and Starnes, S.(1990) Oncogenc 5, 645-655.P-J1

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Prutorius, H,T.. Klein. M. and Day, L.A. (1975) J. Biol. Chcm.250. 9262-9269.Nelson, W-J,. Vorgius. C.E. and Traub, P. (1982) Biochem. Bio-phys. Rcs. Commun. 106. 1141-l 147.Van Amerongcn, H,, van Grondclle, R. and van dcr Vlict, PC.(1987) Biochemistry 26. 4646-4651.Peterson, G.L. (1917)Anal. Biochem. 83. 346-356.Bradford, M.M. (1976) Anal. Biochcm. 72. 248-254.Lakowicz, J.R. (1983) Principles of Fluorescence Spectroscopy,Plenum Press, New York.Kelly. R.C., Jcnscn, D.E. and von Hippel, P,N. (1976) 1. Biol,Chem. 251. 7240-7250.McGhce. J.D. and von Hippel. P.H. (1974) J. Mol. Biol. 86,469-489.Chase, J.W. and Williams, K.R. (19L6)Annu. Rev. Biochem. 55,103-136.Truub, P., Plagcns, U,. Kilhn, S. and Pcridcs, G. (1987) Forlschr,Zool. 34. 175-287.[I91 Cress, A.E. und Kurath, K.M. (1988) J. Biol. Chcm. 263. 19678-19683.

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