Vol. 85, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages
December 29,1978
Conformational
Study of Calf Thymus lMGl4 Nonhistone
1385-1391
Protein
Kashayar Javaherian and Shohreh Amini Institute of Biochemistry and Biophysics University of Tehran, P.O. Box 314-1700, Tehran, IRAN Received
October
18, 1978
Physicochemical study of nonhistone protein HMG14 from calf thymus has been undertaken. The protein has a random structure with a molecular weight of approximately 10,000. On interaction with DNA, it behaves like histones and nonhistone protein HMG17. Both circular dichroism and melting absorption technics show that the protein has an ionic interaction with DNA without causing significant changes in DNA structure. In conrast to HMGl and HMG2 which reduce linking number of circular DNA, nonhistone protein HMG14 and HMG17 do not introduce any changes in topological winding number of DNA. Introduction The present model of chromatin structure provides us with a framework to investigate the properties of chromosomal macromolecules. In particular, we are now in a better position to learn about the structural roles of some of the nonhistone proteins. A group of such proteins were isolated from calf thymus chromatin at 0.35M Nacl by Johns and his collaborators(l). The proteins were termed HMG (High Mobility Group). Calf thymus HMG consists mainly of four proteins designated HMGl, HMG2, HMG14 and HMG17 (2). HMG proteins have been isolated from a number of higher organisms and they are believed to be present in all tissues (3,4,5,6). HMGl and HMG2 proteins are shown to be very similar to each other in structure and sequence (for a review on this subject see ref. 7).Recently it was demonstrated that these two proteins reduce the linking number of circular DNA pointing to unwinding or supercoiling of DNA molecule by HMGl and HMG2 proteins (8). Some physicochemical study of HMG17 has been undertaken before and was found out that it behaved very much like histones (9,lO). We now report our observations on conformational aspects of HMG14 and its interactions with DNA. From our results we conclude that HMG14 demonstrates a great deal of similarity to HMG17 nonhistone protein. Methods HMG14 was prepared according to the method of Sanders (11). 0l-Sephadex C 25 was used for final separation of different HMG proteins. Slab gel electrophoresis was performed according to Laemmli (12) using lo-15% acrylamide gradient. Molecular 0006-291X/78/0854-1385$01.00/0 1385
Copyright 0 I978 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 85, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
weight of the protein was determined by using Beckman Model E analytical ultracentrifuge equipped with an optical scanner. To reduce the time of equilibrium, short columns were employed. Measurements were made at 23Onm. Calf thymus DNA was purchased from Sigma. T7 DNA was a gift from Dr. T. Kovacic, Harvard University Protein concentration was determined by weighing the dry powder. For DNA concentration E260mg/ml = 20 was used. Circular dichroism measurements were carried out at room temperature using Jobin Yvon Mark III dichrograph. Ellipticity calculations were done in terms of mean residue weight (DNA=340, HMG14 =llO). Thermal denaturation results were obtained on a Gilford spectrophotometer 2400-2 equipped with a thermoprogrammer. The unit was connected to a computer for calculating and plotting the derivative curves. Temperature scanning rate was 1 C/min. To determine whether HMG14 and HMG17 reduce the linking number of DNA, we employed a procedure described elsewhere (8). Briefly, 0.5pg of nicked PM2 DNA was used in each reaction mixture. After adding appropriate amounts of HMG14 and HMG17 to DNA, the circular DNA was closed enzymatically by E. coli ligase. The products of the reaction were examined on 0.7% agarose at 4'C, 40 volts using 5mM magnesium acetate, 40mM Tris-Hcl, pH=8.0, lmM EDNA as electrophoresis buffer. RESULTS and DISCUSSION Electrophoresis of HMG14 is shown in Fig. 1. Slots (a) and (b) contain HMGl and HMG2 respectively. Slot (c) is HMG complex which includes all four HMG proteins. As one can see from slot (d), HMG14 is contaminated with upper bands, very likely HMG2. However, the impurity does not affect the results presented here. Amino acid analysis of purified HMG14 was in complete agreement with earlier results (ref.(ll), data not shown). Both HMG14 and HMG17 contain no aromatic residues. We would like to point out that SDS gel electrophoresis of HMG14 and HMG17 show pink color in the presence of Coomassie Blue when the gel is allowed to remain at room temperature. The reason for this color change is not clear. Both HMG14 and HMG17 show anomalous positions on SDS gels (13). Sedimentation equilibrium investigation resulted in the molecular weight calculation of 8500-10500 for HMG14. The buffer was 0.1 M Tris, pH=8 and the time of run was 22 hours at 20°C. The protein does not show aggregation. With regard to the calculated molecular weight, it should be pointed out that sedimentation equilibrium result for HMG17 was 7500-8500 (9) which is below the value determined from amino acid sequence (9200). In view of the fact that HMG14 has a higher molecular weight than HMG17, we believe the upper limit 10500 determined for HMG14 is closer to the true molecular weight of the protein. DNA-protein precipitation curve is shown in Fig. 2. Increasing ionic strength from 0.1 to 0.3M Tris reduces the DNA-protein interaction. The data is consis-
1386
Vol. 85, No. 4, 1978
BIOCHEMICAL
8
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
b
C
d
Fig. 1. Sodium dodecyl sulfate slab gel electrophoresis the intense band is HMGl4.
of HMG14. In slot
(d)
Fig. 2. Precipitation curve of DNA-HMG14 complexes. Approximately lml of calf thymus DNAwas taken ( A26O=2) and HMG14was added. After centrifugation at 32OOpMfor 1 hour the absorption of supernatant was measured. 0.1 M Tris, pH=8. (-------) 0.3 M Tris, pH=8. ( ------)
1387
Vol. 85, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0.01 hi Hcl
-
pH=8.3
---
0.2 MNacl,pH=8.3.-.-
Fig.
3. Circular complex.
dichroism of HMG14. (a) far ultraviolet spectrum (b) DNA-W14 The buffer was 0.1 M Tris, pH=8.0. All ratios are by weights.
tent with the hypothesis that HMG14 interaction with DNA is mainly ionic. The general shape of the curve is similar to W17 with DNA(g). However it seems that more HI%14 is required, in comparison with HMG17, to neutralize DNA. Circular dichroism results are presented in Fig. 3. Far ultraviolet data reveal that HMG14 has a random structure under different ionic conditions. Circular dichroism measurements of DNA-protein complex in near ultraviolet show that the HMG14 protein introduces some minor changes in DNA spectrum. Clearly, the stru cture of DNA bases is not changed significantly upon complex formation with HMG14. T7 DNA melting derivative curves are similar to HMG17 and histone proteins(Fig.4). Addition of HN.24 gives rise to a biphasic profile. The first transition is due to DNA and the second one is DNA-HMG14 complex. The presence of protein stabilizes DNA against heat denaturation. Finally in Fig. 5, the agarose gel electrophoresis of FM2 DNA in the presence of HMG14 and HMG17 is shown. Each band differs by&=1 from its neighboring band(&s linking number). The results show that HMG14 and HMG17,in contrast to HMGl and HMGZ, do not change the topological winding number of DNA. The observed slight change ofO( for HMG14 (slots b and c) is due to For both HMG14 and HMG17 when the ratio contamination from other proteins.
1388
Vol. 85, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DNA Protein/DNA=+ Protein/DNA 210
220
230
240 Circular
250 dichroism
Figure
50
55
60
65
260 h(nml
270
200
290
----
= 1 -300
310
320
of DNA.HMG,&
3 (continued)
7o T@‘)
75
60
65
90
I
95
Fig. 4. Absorption melting curves of T7 DNA-HMG14.Buffer was 2.5 x 10e7 M FDTA, pH=8.0. (
) DNA, ( - - - - - ) +&4
1389
= $, ( ------
) HMG17=1. --Dir
Vol. 85, No. 4, 1978
8lOCHEMlCAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
abcdefghi
Fig. 5. Agarose gel electrophoresis
of covalently
HMG14and HMG17. (a) control
closed PM? in the presence of
DNA. b-e are DNA-HMG14 at different
weight ratios,(b)l,(c)Z,(d)3(e)4.f-i
are for y$
HMG14 DNA weight ratios, (f)l,
k)2,01)3,(il4.
of protein
to DNAexceeds 2, precipitation
occurs which explains the absence
of DNAbands in slots d,e,h and i. In conclusion, calf thymus HMGproteins which consist mainly of four proteins can be classified into two groups. In one group we have IMGl and HMG2proteins which are highly structured and globular and are very similiar to each other. In another class are HMG14and HMG17which have random structure and resemble each other in physicochemical characteristics. HMGproteins of trout testis consist of two proteins (14). One is similiar to HMGl and the other to HMG17. It remains to be seen whether the diversity of these proteins through evolution
has biological
significance.
ACKNOWLFDGEMENTS This investigation was supported by grants from the University and Ministry of Science of IRAN. 1390
of Tehran
Vol. 85, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. Goodwin, G.H., Sanders, C. and Johns, E.W. (1973) Eur. J. Biochem. 38, 14-19. 2. Goodwin, G.H., Walker, J.M. and Johns, E.W. (1978) Biochem. Biophys. Acta, 519, 233-242. 3. Marushige, K. and Dixon, G.H. (1971) J. Biol. Chem. 246, 5799-5805. G. (1978) J. Biol. Chem. 253, 3830-3836. 4. Sterner, R., Boffa, L.C. and Vidali, 5. Rabbani, A., Goodwin, G.H. and Johns, E.W. (1978) Biochem. Biophys. Res. Commun. 81, 351-358. 6. Spiker, S., Mardian, J.K.W. and Isenberg, I. (1978) Biochem. Biophys. Res. Commun. 82, 129-135. 7. Javaherian, K. in " Organization and Expression of Eukaryotic Genome". ed. E.M. Bradbury and K. Javaherian (1977). Academic Press, London. 8. Javaherian, K., Liu, L.F. and Wang, J.C. (1978) Science, 199, 1345-1346. 9. Javaherian, K. and Amini, S. (1977) Biochem. Biophys. Acta. 478,295-304. lO.Abercombie, B.D., Kneale, G.G., Crane-Robinson, C., Bradbury, E.M., Coodwin, G.H. and Johns, E.W. (1978) Eur. J. Biochem. 84, 173-177. ll.Sanders, C. (1977). Biochem. Biophys. Res. Commun. 78,1034-1042. lZ.Laemmli, U.K. (1970) Nature 227, 680-685. Chem. 253,1694-1699. 13.Bustin, M., Hopkins, R.B. and Isenberg, I. (1978) J.Biol. 14.Watson, D.C., Peters, E.H. and Dixon, G.H. (1977) Eur. J. Biochem. 74, 53-60.
1391
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages
December 29,1978
Conformational
Study of Calf Thymus lMGl4 Nonhistone
1385-1391
Protein
Kashayar Javaherian and Shohreh Amini Institute of Biochemistry and Biophysics University of Tehran, P.O. Box 314-1700, Tehran, IRAN Received
October
18, 1978
Physicochemical study of nonhistone protein HMG14 from calf thymus has been undertaken. The protein has a random structure with a molecular weight of approximately 10,000. On interaction with DNA, it behaves like histones and nonhistone protein HMG17. Both circular dichroism and melting absorption technics show that the protein has an ionic interaction with DNA without causing significant changes in DNA structure. In conrast to HMGl and HMG2 which reduce linking number of circular DNA, nonhistone protein HMG14 and HMG17 do not introduce any changes in topological winding number of DNA. Introduction The present model of chromatin structure provides us with a framework to investigate the properties of chromosomal macromolecules. In particular, we are now in a better position to learn about the structural roles of some of the nonhistone proteins. A group of such proteins were isolated from calf thymus chromatin at 0.35M Nacl by Johns and his collaborators(l). The proteins were termed HMG (High Mobility Group). Calf thymus HMG consists mainly of four proteins designated HMGl, HMG2, HMG14 and HMG17 (2). HMG proteins have been isolated from a number of higher organisms and they are believed to be present in all tissues (3,4,5,6). HMGl and HMG2 proteins are shown to be very similar to each other in structure and sequence (for a review on this subject see ref. 7).Recently it was demonstrated that these two proteins reduce the linking number of circular DNA pointing to unwinding or supercoiling of DNA molecule by HMGl and HMG2 proteins (8). Some physicochemical study of HMG17 has been undertaken before and was found out that it behaved very much like histones (9,lO). We now report our observations on conformational aspects of HMG14 and its interactions with DNA. From our results we conclude that HMG14 demonstrates a great deal of similarity to HMG17 nonhistone protein. Methods HMG14 was prepared according to the method of Sanders (11). 0l-Sephadex C 25 was used for final separation of different HMG proteins. Slab gel electrophoresis was performed according to Laemmli (12) using lo-15% acrylamide gradient. Molecular 0006-291X/78/0854-1385$01.00/0 1385
Copyright 0 I978 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 85, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
weight of the protein was determined by using Beckman Model E analytical ultracentrifuge equipped with an optical scanner. To reduce the time of equilibrium, short columns were employed. Measurements were made at 23Onm. Calf thymus DNA was purchased from Sigma. T7 DNA was a gift from Dr. T. Kovacic, Harvard University Protein concentration was determined by weighing the dry powder. For DNA concentration E260mg/ml = 20 was used. Circular dichroism measurements were carried out at room temperature using Jobin Yvon Mark III dichrograph. Ellipticity calculations were done in terms of mean residue weight (DNA=340, HMG14 =llO). Thermal denaturation results were obtained on a Gilford spectrophotometer 2400-2 equipped with a thermoprogrammer. The unit was connected to a computer for calculating and plotting the derivative curves. Temperature scanning rate was 1 C/min. To determine whether HMG14 and HMG17 reduce the linking number of DNA, we employed a procedure described elsewhere (8). Briefly, 0.5pg of nicked PM2 DNA was used in each reaction mixture. After adding appropriate amounts of HMG14 and HMG17 to DNA, the circular DNA was closed enzymatically by E. coli ligase. The products of the reaction were examined on 0.7% agarose at 4'C, 40 volts using 5mM magnesium acetate, 40mM Tris-Hcl, pH=8.0, lmM EDNA as electrophoresis buffer. RESULTS and DISCUSSION Electrophoresis of HMG14 is shown in Fig. 1. Slots (a) and (b) contain HMGl and HMG2 respectively. Slot (c) is HMG complex which includes all four HMG proteins. As one can see from slot (d), HMG14 is contaminated with upper bands, very likely HMG2. However, the impurity does not affect the results presented here. Amino acid analysis of purified HMG14 was in complete agreement with earlier results (ref.(ll), data not shown). Both HMG14 and HMG17 contain no aromatic residues. We would like to point out that SDS gel electrophoresis of HMG14 and HMG17 show pink color in the presence of Coomassie Blue when the gel is allowed to remain at room temperature. The reason for this color change is not clear. Both HMG14 and HMG17 show anomalous positions on SDS gels (13). Sedimentation equilibrium investigation resulted in the molecular weight calculation of 8500-10500 for HMG14. The buffer was 0.1 M Tris, pH=8 and the time of run was 22 hours at 20°C. The protein does not show aggregation. With regard to the calculated molecular weight, it should be pointed out that sedimentation equilibrium result for HMG17 was 7500-8500 (9) which is below the value determined from amino acid sequence (9200). In view of the fact that HMG14 has a higher molecular weight than HMG17, we believe the upper limit 10500 determined for HMG14 is closer to the true molecular weight of the protein. DNA-protein precipitation curve is shown in Fig. 2. Increasing ionic strength from 0.1 to 0.3M Tris reduces the DNA-protein interaction. The data is consis-
1386
Vol. 85, No. 4, 1978
BIOCHEMICAL
8
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
b
C
d
Fig. 1. Sodium dodecyl sulfate slab gel electrophoresis the intense band is HMGl4.
of HMG14. In slot
(d)
Fig. 2. Precipitation curve of DNA-HMG14 complexes. Approximately lml of calf thymus DNAwas taken ( A26O=2) and HMG14was added. After centrifugation at 32OOpMfor 1 hour the absorption of supernatant was measured. 0.1 M Tris, pH=8. (-------) 0.3 M Tris, pH=8. ( ------)
1387
Vol. 85, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0.01 hi Hcl
-
pH=8.3
---
0.2 MNacl,pH=8.3.-.-
Fig.
3. Circular complex.
dichroism of HMG14. (a) far ultraviolet spectrum (b) DNA-W14 The buffer was 0.1 M Tris, pH=8.0. All ratios are by weights.
tent with the hypothesis that HMG14 interaction with DNA is mainly ionic. The general shape of the curve is similar to W17 with DNA(g). However it seems that more HI%14 is required, in comparison with HMG17, to neutralize DNA. Circular dichroism results are presented in Fig. 3. Far ultraviolet data reveal that HMG14 has a random structure under different ionic conditions. Circular dichroism measurements of DNA-protein complex in near ultraviolet show that the HMG14 protein introduces some minor changes in DNA spectrum. Clearly, the stru cture of DNA bases is not changed significantly upon complex formation with HMG14. T7 DNA melting derivative curves are similar to HMG17 and histone proteins(Fig.4). Addition of HN.24 gives rise to a biphasic profile. The first transition is due to DNA and the second one is DNA-HMG14 complex. The presence of protein stabilizes DNA against heat denaturation. Finally in Fig. 5, the agarose gel electrophoresis of FM2 DNA in the presence of HMG14 and HMG17 is shown. Each band differs by&=1 from its neighboring band(&s linking number). The results show that HMG14 and HMG17,in contrast to HMGl and HMGZ, do not change the topological winding number of DNA. The observed slight change ofO( for HMG14 (slots b and c) is due to For both HMG14 and HMG17 when the ratio contamination from other proteins.
1388
Vol. 85, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DNA Protein/DNA=+ Protein/DNA 210
220
230
240 Circular
250 dichroism
Figure
50
55
60
65
260 h(nml
270
200
290
----
= 1 -300
310
320
of DNA.HMG,&
3 (continued)
7o T@‘)
75
60
65
90
I
95
Fig. 4. Absorption melting curves of T7 DNA-HMG14.Buffer was 2.5 x 10e7 M FDTA, pH=8.0. (
) DNA, ( - - - - - ) +&4
1389
= $, ( ------
) HMG17=1. --Dir
Vol. 85, No. 4, 1978
8lOCHEMlCAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
abcdefghi
Fig. 5. Agarose gel electrophoresis
of covalently
HMG14and HMG17. (a) control
closed PM? in the presence of
DNA. b-e are DNA-HMG14 at different
weight ratios,(b)l,(c)Z,(d)3(e)4.f-i
are for y$
HMG14 DNA weight ratios, (f)l,
k)2,01)3,(il4.
of protein
to DNAexceeds 2, precipitation
occurs which explains the absence
of DNAbands in slots d,e,h and i. In conclusion, calf thymus HMGproteins which consist mainly of four proteins can be classified into two groups. In one group we have IMGl and HMG2proteins which are highly structured and globular and are very similiar to each other. In another class are HMG14and HMG17which have random structure and resemble each other in physicochemical characteristics. HMGproteins of trout testis consist of two proteins (14). One is similiar to HMGl and the other to HMG17. It remains to be seen whether the diversity of these proteins through evolution
has biological
significance.
ACKNOWLFDGEMENTS This investigation was supported by grants from the University and Ministry of Science of IRAN. 1390
of Tehran
Vol. 85, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. Goodwin, G.H., Sanders, C. and Johns, E.W. (1973) Eur. J. Biochem. 38, 14-19. 2. Goodwin, G.H., Walker, J.M. and Johns, E.W. (1978) Biochem. Biophys. Acta, 519, 233-242. 3. Marushige, K. and Dixon, G.H. (1971) J. Biol. Chem. 246, 5799-5805. G. (1978) J. Biol. Chem. 253, 3830-3836. 4. Sterner, R., Boffa, L.C. and Vidali, 5. Rabbani, A., Goodwin, G.H. and Johns, E.W. (1978) Biochem. Biophys. Res. Commun. 81, 351-358. 6. Spiker, S., Mardian, J.K.W. and Isenberg, I. (1978) Biochem. Biophys. Res. Commun. 82, 129-135. 7. Javaherian, K. in " Organization and Expression of Eukaryotic Genome". ed. E.M. Bradbury and K. Javaherian (1977). Academic Press, London. 8. Javaherian, K., Liu, L.F. and Wang, J.C. (1978) Science, 199, 1345-1346. 9. Javaherian, K. and Amini, S. (1977) Biochem. Biophys. Acta. 478,295-304. lO.Abercombie, B.D., Kneale, G.G., Crane-Robinson, C., Bradbury, E.M., Coodwin, G.H. and Johns, E.W. (1978) Eur. J. Biochem. 84, 173-177. ll.Sanders, C. (1977). Biochem. Biophys. Res. Commun. 78,1034-1042. lZ.Laemmli, U.K. (1970) Nature 227, 680-685. Chem. 253,1694-1699. 13.Bustin, M., Hopkins, R.B. and Isenberg, I. (1978) J.Biol. 14.Watson, D.C., Peters, E.H. and Dixon, G.H. (1977) Eur. J. Biochem. 74, 53-60.
1391
