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Vacuum ultraviolet circular dichroism of fibronectin dominant tyrosine effects
J. Mol. Biol. (1987) 197, 743-745
Vacuum Ultraviolet
Circular Dichroism
of Fibronectin
Dominant Tyrosine Effects The spectroscopic authenticity of a very intense negative band at about 183 nm reported previously from conventional circular dichroism (c.d.) studies of bovine plasma fibronectin has now been confirmed by vacuum ultraviolet c.d. measurements on two prototype spectrometers, one using a conventional light source and the other using synchrotron radiation. Closely similar spectra were obtained from both instruments, and from both solid films and solutions. The spectra show no obvious parentage in the known c.d. of the peptide backbone, but have marked similarities to the c.d. of N-acetyltyrosineamide, both in the strong band at 183 nm and in a characteristic positive band at 230 nm, It is concluded that the c.d. of fibronectin is dominated by contributions from tyrosine side-chains and that, as suggested previously, these may provide a sensitive probe for molecular organization and interactions.
The fibronectin molecule has been shown to incorporate a series of discrete functional domains with specific affinities for cell surface receptors and various matrix components including collagen, fibrin and heparan sulphate (e.g. see Yamada, 1983). Much circumstantial evidence argues for folded tertiary conformations in these binding This includes protease resistance, domains. fluorescence studies indicating “buried” tryptophan residues that are exposed to the external aqueous environment only after denaturation, extensive disulphide bridging within certain domains, and the variations observed in specificity for different surface receptors when the cell-binding fragment (RGDS) is flanked by different peptide sequences (Ruoslahti & Pierschbacher, 1986). However studies by infrared spectroscopy indicate t,hat ordered secondary structure is limited to some /?-sheet (Venyaminov el al., 1983), and the unusually low intensity of fibronectin c.d.t over the wavelength range diagnostic of peptide conformation is also indicative of little conventional repeating structure. repeating structure. At low wavelength, however, a very intense negative cd. band centred at about 183 nm has been reported (Welsh et al., 1983). This band, which had not been observed previously, has no obvious parentage in known transitions of the peptide backbone (Brahms & Brahms, 1980) but, in common with another anomalous positive band at 230 nm, corresponds to a transition observed in the model compound N-acetyltyrosineamide, and was therefore attributed to optical activity of tyrosine side-chains. On the commercial equipment used in the previous study, however, the 183 nm band was
t Abbreviations vacuum ultraviolet’
used: c.d.. circular dichroism: circular dichroism.
0022-2836/87/2(H)743-03
$03.00/O
close to the transmission limit of the instrument, raising the obvious possibility that it was due to an out-of-light artefact, rather than of genuine spectroscopic origin. In the present work we have recorded the c.d. of fibronectin to about 175nm, both in solutions and solid films, on two different prototype vacuum ultraviolet circular dichroism (v.u.c.d.) spectrometers, one using a conventional light source (Pysh, 1976) and the other linked to the synchrotron light source at Brookhaven National Laboratory (Sutherland, 1981; Stevens et al., 1985). Our results confirm the spectroscopic authenticity of the transition, but show a substantially lower ellipticity than in the earlier investigation. Fibronectin was prepared from titrated bovine containing 2 mM-benzamidine, 1 mMplasma phenylmethylsulphonyl fluoride and 10 mM-EDTA under non-denaturing conditions according to the method of Vuento & Vaheri (1979). The protein was finally eluted from a gelatin-Sepharose column with 2 M-arginine hydrochloride (pH 7*5), and dialysed against 50 mM-Tris * HCI (pH 7.5). A 400-m] sample of plasma typically yielded about 70 mg of fibronectin. The purity was determined using SDS/ polyacrylamide gel electrophoresis, and a single band of molecular weight 440 x IO3 was seen under non-reducing conditions. A commercial sainple of N-acetyltyrosineamide (Aldrich) was used as supplied. The prototype v.u.c.d. spectrometers used have been described previously (Pysh, 1976; Sutherland, 1981). Solution v.u.c.d. spectra were recorded at ambient temperature (-25°C) using cells of pathlength 0.1 or 0.05 mm. Film spectra were obt’ained using the same fibronectin solutions evaported to dryness on calcium fluoride discs. A direct tracing of a typical fibronectin v.u.c.d. spectrum from the instrument at SUNY, Binghamton, using a conventional deuterium discharge Hinteregger light source, is shown in
v.u.c.d..
743
0 1987 Academic Press Limited
-161 180
220
200
240
260
X(“rn)
Figure 1. r.u.c.d. of fibronectin (1Zimg ml-‘; 25°C’; 0.1 mm pathlength). Direct tracing of spectrum obtained at SONY, Ringhamton. Molar ellipticity values were calculated using a mean residue weight of 116.
obtained at the National Figure 1. Spectra Synchrotron Light Source, Brookhaven were closely similar, apart from some improvement in signal-tonoise ratio. On both instruments the spectral features shown in Figure 1, including particularly the strong negative band at 183 nm, were also present in the c.d. of fibronectin films. Figure 2 shows the v.u.c.d. of N-acetyltyrosineamide at, neutral pH in distilled water. The spectrum has the same general form as reported previously from conventional c.d. measurements (Welsh et aZ., 1983), but the intensity of the shortwavelength negative band is about 60% greater (both in abosolute terms, and relative to the major positive band at N 200 nm), suggesting some peakflattening due to loss of transmission in the conventional spectrometer. This interpretation is further supported by the somewhat longer wavelength of the apparent minimum in the previous study (N 185 nm, in comparison with N 182 nm in the present investigation), as would be expected from progressive loss of signal towards the transmission limit of the instrument’. The close agreement between fibronectin v.u.c.d. spectra obtained on two separate instruments, and between solution spectra and those recorded using solid films (where transmission is enhanced by elimination of absorption of light by the solvent) provides strong evidence for the validity of the spectrum shown in Figure 1, and in particular for the authenticity of the strong negative band at 183 nm. The striking similarities between the v.u.c.d. of fibronectin (Fig. 1) and of N-acetyltyrosineamide differences from the (Fig. 5% and the major reported v.u.c.d. behaviour of globular proteins (Brahms & Brahms, 1980), reinforce the conclusion (Venyaminov et al., 1983; Welsh et al., 1983) that there is little conventional repeating secondary structure (a-helix; p-sheet) within bhe fibronectin
I
I
I
8
180
200
220
240
J
Xfnm)
Figure 2. v.u.c.d. of A-acetyltyrosineamide (A5’C’: pH 7). The spectrum above 210nm was rrcorded in a 0.1 mm cell at a roncentrat,ion of 5 mg ml *: at short)er wavelengths the concentration was 1 mg ml-i and the pathlength 0.05 mm. Molar ellipticities wcrc calculated using the formula molecular weight of 222.
molecule, and that its c.d. behaviour is dominated by spectral contributions from aromatic side-chains (part’icularly tyrosine). We have not yet, identified the specific domains from which these contributions arise. However, investigations by conventional c.d. (Odermatt et al.. 1982) show that the characteristic positive band at, 230 nm is particularly strong in the X-terminal heparin-binding domain (M, 29 x 103), whose primarv sequence incorporates five homologous “fingers” with tyrosine residues in conserved locations (Garcia-Pardo et al., 1983). It is not yet, clear whether the very much higher intensity reported by Welsh et al. (1983) for t’he negative band at 183 nm was due to a transmission artefact on the conventional spectrometer used, or to genuine sample-to-sample variation bet’ween fibronectin preparations. This will be investigated further in fut’ure studies of fibronectins from different biological sources, extracted by different isolation procedures. For the moment, however. our present results lend confidence to the continued use of circular dichroism, and specifically the strong transition at 183 nm, to probe the conformation and functional interactions of fibronectin. We thank Dr ,J. C. Sutherland for generous access t,o his v.u.c.d. facilities at Hrookhaven National Laborator)-. This work was supported by NIH grant
Letters to the Editor
References
E. S. Stevens Department of Chemistry State University of New York Binghamton, NY 13901, U.S.A. E. R. Morris? Department of Food Research & Technology Cranfield Institute of Technology Silsoe College, Silsoe Bedford MK45 4DT, England J. A. Charlton D. A. Rees National Institute for Medical Research Mill Hill, London NW7 IAA, England Received 12 June 1987
t Author to whom all correspondence
745
should be addressed.
Brahms, S. & Brahms, J. (1980). J. Mol. Biol. 138, 149178. Garcia-Pardo, A., Pearlstein, E. & Frangione, B. (1983). J. Biol. Chem. 258, 12670-12674. Odermatt, E., Engel, J., Richter, H. & Hormann, H. (1982). J. Mol. Biol. 159, 109-123. Pysh (Stevens). E. S. (1976). Annu. Rev. Biophys. Bioeny. 5, 63375. Ruoslahti, E. & Pierschbacher. M. D. (1986). Cell, 44, 517-518. Stevens, E. S., Morris, E. R., Rees, D. A. & Sutherland, J. C. (1985). J. Amer. Chem. Sot. 107, 2982-2983. Sutherland, J. C. (1981). BioScience, 31, 587492. Venyaminov. S. Y., Metsis, M. L., Chernousov, M. A. & Koteliansky, V. E. (1983). Eur. J. Biochem. 135, 485489. Vuento, M. & Vaheri. A. (1979). Biochem. J. 183, 331337. Welsh, E. J., Frangou, S. A., Morris, E. R., Rees, D. A. & Chavin, S. I. (1983). BiopoEymers, 22, 821-831. Yamada, K. M. (1983). Annu. Rev. Biochem. 52, 761-799.
Edited by M. F. Moody
Vacuum Ultraviolet
Circular Dichroism
of Fibronectin
Dominant Tyrosine Effects The spectroscopic authenticity of a very intense negative band at about 183 nm reported previously from conventional circular dichroism (c.d.) studies of bovine plasma fibronectin has now been confirmed by vacuum ultraviolet c.d. measurements on two prototype spectrometers, one using a conventional light source and the other using synchrotron radiation. Closely similar spectra were obtained from both instruments, and from both solid films and solutions. The spectra show no obvious parentage in the known c.d. of the peptide backbone, but have marked similarities to the c.d. of N-acetyltyrosineamide, both in the strong band at 183 nm and in a characteristic positive band at 230 nm, It is concluded that the c.d. of fibronectin is dominated by contributions from tyrosine side-chains and that, as suggested previously, these may provide a sensitive probe for molecular organization and interactions.
The fibronectin molecule has been shown to incorporate a series of discrete functional domains with specific affinities for cell surface receptors and various matrix components including collagen, fibrin and heparan sulphate (e.g. see Yamada, 1983). Much circumstantial evidence argues for folded tertiary conformations in these binding This includes protease resistance, domains. fluorescence studies indicating “buried” tryptophan residues that are exposed to the external aqueous environment only after denaturation, extensive disulphide bridging within certain domains, and the variations observed in specificity for different surface receptors when the cell-binding fragment (RGDS) is flanked by different peptide sequences (Ruoslahti & Pierschbacher, 1986). However studies by infrared spectroscopy indicate t,hat ordered secondary structure is limited to some /?-sheet (Venyaminov el al., 1983), and the unusually low intensity of fibronectin c.d.t over the wavelength range diagnostic of peptide conformation is also indicative of little conventional repeating structure. repeating structure. At low wavelength, however, a very intense negative cd. band centred at about 183 nm has been reported (Welsh et al., 1983). This band, which had not been observed previously, has no obvious parentage in known transitions of the peptide backbone (Brahms & Brahms, 1980) but, in common with another anomalous positive band at 230 nm, corresponds to a transition observed in the model compound N-acetyltyrosineamide, and was therefore attributed to optical activity of tyrosine side-chains. On the commercial equipment used in the previous study, however, the 183 nm band was
t Abbreviations vacuum ultraviolet’
used: c.d.. circular dichroism: circular dichroism.
0022-2836/87/2(H)743-03
$03.00/O
close to the transmission limit of the instrument, raising the obvious possibility that it was due to an out-of-light artefact, rather than of genuine spectroscopic origin. In the present work we have recorded the c.d. of fibronectin to about 175nm, both in solutions and solid films, on two different prototype vacuum ultraviolet circular dichroism (v.u.c.d.) spectrometers, one using a conventional light source (Pysh, 1976) and the other linked to the synchrotron light source at Brookhaven National Laboratory (Sutherland, 1981; Stevens et al., 1985). Our results confirm the spectroscopic authenticity of the transition, but show a substantially lower ellipticity than in the earlier investigation. Fibronectin was prepared from titrated bovine containing 2 mM-benzamidine, 1 mMplasma phenylmethylsulphonyl fluoride and 10 mM-EDTA under non-denaturing conditions according to the method of Vuento & Vaheri (1979). The protein was finally eluted from a gelatin-Sepharose column with 2 M-arginine hydrochloride (pH 7*5), and dialysed against 50 mM-Tris * HCI (pH 7.5). A 400-m] sample of plasma typically yielded about 70 mg of fibronectin. The purity was determined using SDS/ polyacrylamide gel electrophoresis, and a single band of molecular weight 440 x IO3 was seen under non-reducing conditions. A commercial sainple of N-acetyltyrosineamide (Aldrich) was used as supplied. The prototype v.u.c.d. spectrometers used have been described previously (Pysh, 1976; Sutherland, 1981). Solution v.u.c.d. spectra were recorded at ambient temperature (-25°C) using cells of pathlength 0.1 or 0.05 mm. Film spectra were obt’ained using the same fibronectin solutions evaported to dryness on calcium fluoride discs. A direct tracing of a typical fibronectin v.u.c.d. spectrum from the instrument at SUNY, Binghamton, using a conventional deuterium discharge Hinteregger light source, is shown in
v.u.c.d..
743
0 1987 Academic Press Limited
-161 180
220
200
240
260
X(“rn)
Figure 1. r.u.c.d. of fibronectin (1Zimg ml-‘; 25°C’; 0.1 mm pathlength). Direct tracing of spectrum obtained at SONY, Ringhamton. Molar ellipticity values were calculated using a mean residue weight of 116.
obtained at the National Figure 1. Spectra Synchrotron Light Source, Brookhaven were closely similar, apart from some improvement in signal-tonoise ratio. On both instruments the spectral features shown in Figure 1, including particularly the strong negative band at 183 nm, were also present in the c.d. of fibronectin films. Figure 2 shows the v.u.c.d. of N-acetyltyrosineamide at, neutral pH in distilled water. The spectrum has the same general form as reported previously from conventional c.d. measurements (Welsh et aZ., 1983), but the intensity of the shortwavelength negative band is about 60% greater (both in abosolute terms, and relative to the major positive band at N 200 nm), suggesting some peakflattening due to loss of transmission in the conventional spectrometer. This interpretation is further supported by the somewhat longer wavelength of the apparent minimum in the previous study (N 185 nm, in comparison with N 182 nm in the present investigation), as would be expected from progressive loss of signal towards the transmission limit of the instrument’. The close agreement between fibronectin v.u.c.d. spectra obtained on two separate instruments, and between solution spectra and those recorded using solid films (where transmission is enhanced by elimination of absorption of light by the solvent) provides strong evidence for the validity of the spectrum shown in Figure 1, and in particular for the authenticity of the strong negative band at 183 nm. The striking similarities between the v.u.c.d. of fibronectin (Fig. 1) and of N-acetyltyrosineamide differences from the (Fig. 5% and the major reported v.u.c.d. behaviour of globular proteins (Brahms & Brahms, 1980), reinforce the conclusion (Venyaminov et al., 1983; Welsh et al., 1983) that there is little conventional repeating secondary structure (a-helix; p-sheet) within bhe fibronectin
I
I
I
8
180
200
220
240
J
Xfnm)
Figure 2. v.u.c.d. of A-acetyltyrosineamide (A5’C’: pH 7). The spectrum above 210nm was rrcorded in a 0.1 mm cell at a roncentrat,ion of 5 mg ml *: at short)er wavelengths the concentration was 1 mg ml-i and the pathlength 0.05 mm. Molar ellipticities wcrc calculated using the formula molecular weight of 222.
molecule, and that its c.d. behaviour is dominated by spectral contributions from aromatic side-chains (part’icularly tyrosine). We have not yet, identified the specific domains from which these contributions arise. However, investigations by conventional c.d. (Odermatt et al.. 1982) show that the characteristic positive band at, 230 nm is particularly strong in the X-terminal heparin-binding domain (M, 29 x 103), whose primarv sequence incorporates five homologous “fingers” with tyrosine residues in conserved locations (Garcia-Pardo et al., 1983). It is not yet, clear whether the very much higher intensity reported by Welsh et al. (1983) for t’he negative band at 183 nm was due to a transmission artefact on the conventional spectrometer used, or to genuine sample-to-sample variation bet’ween fibronectin preparations. This will be investigated further in fut’ure studies of fibronectins from different biological sources, extracted by different isolation procedures. For the moment, however. our present results lend confidence to the continued use of circular dichroism, and specifically the strong transition at 183 nm, to probe the conformation and functional interactions of fibronectin. We thank Dr ,J. C. Sutherland for generous access t,o his v.u.c.d. facilities at Hrookhaven National Laborator)-. This work was supported by NIH grant
Letters to the Editor
References
E. S. Stevens Department of Chemistry State University of New York Binghamton, NY 13901, U.S.A. E. R. Morris? Department of Food Research & Technology Cranfield Institute of Technology Silsoe College, Silsoe Bedford MK45 4DT, England J. A. Charlton D. A. Rees National Institute for Medical Research Mill Hill, London NW7 IAA, England Received 12 June 1987
t Author to whom all correspondence
745
should be addressed.
Brahms, S. & Brahms, J. (1980). J. Mol. Biol. 138, 149178. Garcia-Pardo, A., Pearlstein, E. & Frangione, B. (1983). J. Biol. Chem. 258, 12670-12674. Odermatt, E., Engel, J., Richter, H. & Hormann, H. (1982). J. Mol. Biol. 159, 109-123. Pysh (Stevens). E. S. (1976). Annu. Rev. Biophys. Bioeny. 5, 63375. Ruoslahti, E. & Pierschbacher. M. D. (1986). Cell, 44, 517-518. Stevens, E. S., Morris, E. R., Rees, D. A. & Sutherland, J. C. (1985). J. Amer. Chem. Sot. 107, 2982-2983. Sutherland, J. C. (1981). BioScience, 31, 587492. Venyaminov. S. Y., Metsis, M. L., Chernousov, M. A. & Koteliansky, V. E. (1983). Eur. J. Biochem. 135, 485489. Vuento, M. & Vaheri. A. (1979). Biochem. J. 183, 331337. Welsh, E. J., Frangou, S. A., Morris, E. R., Rees, D. A. & Chavin, S. I. (1983). BiopoEymers, 22, 821-831. Yamada, K. M. (1983). Annu. Rev. Biochem. 52, 761-799.
Edited by M. F. Moody