Volume 2 number 6 June 1975
Nucleic Acids Research
Metal ton-mediated specific interactions between nucleic acid bases of polynucleotides and amino acid side chains of polypeptides. Claude Helene Centre de Btophystque MolEculaire, 45045 Orlfans C&dex, France
Received 29 April 1975 Summary Interactions between copolypeptides containing Glu and Tyr residues and polynucleotides can be mediated through divalent metal ions such as Zn2+ and CuZ+. Circular dichrotsm studies show that the binding of metal ion - polypeptide complexes to poly(A) induces an unstacking of adenine bases. Fluorescence investigations demonstrate that Tyrosine - AdeniAe interactions result from the formation of ternary complexes polypeptide-Zn -polynucleotide. Introduction The selective recognition of nucleic acid base sequences by proteins rests upon structural complementarity and specific interactions between amino acid side-chains and nucleic acid bases. Structural requirements are certainly of major importance in the recognition of, e. g., tRNAs by amino
acyl-tRNA synthetases. The interactions between the amino acid residues of the recognition site and the bases of the interacting nucleic acid region may be direct or indirect (i. e. promoted by a third species acting as an intermediate).
Stacking interactions involving aromatic amino acids were first demonstrated in frozen aqueous solutions , then in concentrated aqueous solutions at room temperature ' ) and more recently in oligopeptide nucleic acid complexes (4-11) . Hydrogen bonding between a few amino acid side chains and nucleic acid bases has also been shown to take place in organic solvents ). As to electrostatic interactions between basic amino acids and phosphate groups they may contribute to the specificity of protein-nucleic acid interactions inasmuch as the relative orientation of phosphate groups depends on the base sequence.
Stacking, hydrogen bonding and electrostatic binding result from direct interactions. One might also consider the possibility that the interaction between the amino acid side chain and the base is mediated through
961
Nucleic Acids Resarch a
third species which might be
a
metal ion. Numerous studies have been devo-
ted to the study of metal ion binding to polynucleotides, nucleic acids, peptides and proteins. It has been shown that transition metal ions bind
not only to
the phosphates but also to the bases of nucleic acids. The mode of binding depends
the nature of both the metal ion and the base (see references 13-15
on
and references cited therein). For example Cu
ions
and Zn
are
able to
form chelates between phosphates and bases (especially N7 of guanine in DNA) and possibly sandwich complexes between consecutive G-C base pairs( Certain
of amino acids
sequences
complexes with Zn
2+
and Cu
2+.
may
ions in
also form aqueous
very
16)
strong and specific
solutions (see references 17
and 18 for recent reviews). It is thus possible to contemplate that specific
interactions between amino acid and base
sequences
such metal ions. Moreover the formation of metal ion
a
could be promoted by
ternary complex amino acid
-
base (or phosphate) might induce selective interactions between
-
amino acid residues and bases
even
far removed from the site of metal
attachment. The purpose of this preliminary report is to present experimental evidence for interactions promoted by Zn
or
Cu
ions
between copolypep-
tides containing Glu and Tyr residues and polynucleotides. These copolypeptides
were
chosen because they do not interact with polynucleotides
in
the
absence of metal ions. Experimental Polynucleotides poly(A) and poly(C)
were
purchased from Miles.
Copolypeptides of glutamic acid and tyrosine have been synthesized in laboratory
(1
9) .
The following sequences were available
(Glu-Tyr-Glu)n,
(Glu-Tyr-Tyr-Glu)
.
our
Ty- Glu),
(Tyr
These polypeptides are soluble in
water at pH 7 (after neutralizing the carboxylic groups) and adopt a random
coil conformation at this pH value. The synthesis and characterization of
these polypeptides have been reported elsewhere(19 20). Circular dichroism measurements were carried out with a Roussel-Jouan II dichrograph. Fluorescence spectra were recorded with a
Jobin-Yvon spectrofluorometer modified tion wavelength
=
275 nm).
All solutions
962
as described in reference 9 (excita-
were
made in a pH 7 buffer containing 1 mM
Nucleic Acids Research sodium cacodylate and either 1 mM out at
a
or
0. 2 M NaCl. Measurements
were
carried
temperature of 230C.
Results and Discussion The binding of Cu
ions to poly(A) and poly(C) in their
and Zn
single-stranded helical conformation (pH 7, 20°C) induces the
bases(l5).
an
unstacking of
The resulting changes in absorbance and optical rotatory dis-
persion (or circular dichrotsm,
see
below)
by raising the temperature of the solution and poly(C) than Cu
ions
strength. These ions have
are
(15)
similar to those brought about Zn
2+
ions
and their binding is much a
bind less to poly(A)
more
sensitive to ionic
fairly strong affinity for both the bases and the
phosphates and the formation of chelates between phosphates and bases has been
suggested(
).
In contrast Mg
and Ca
ions
which bind chiefly to phos-
phates (and not to the bases) stabilize the ordered structure of the polynucleotides. The binding of Cu
investigated here The binding of Cu
can
and Zn
ions to the Glu-Tyr copolypeptides
be conveniently followed by fluorescence measurements.
ions
to (Glu-Tyr-Glu)
fluorescence whereas Zn
ions
leads to
a
quenching of the tyrosine
~~~n enhance the fluorescence intensity of tyrosine
residues by about 35 %. The binding sites of Cu and Zn ions on the copolypeptides are not known. The carboxyl groups of Glu residues are certainno
evidence from
group
of t*yrosine. It
ly involved (and perhaps also the peptide bonds) but there is absorbance measurements for ionization of the hydroxyl is. known that the binding of Cu
ions to
the amino and carboxyl groups of free
tyrosine leads to fluorescence quenching at pH 7(l). The fluorescence enhancement due to Zn
2+
binding might be ascribed to
fluorescence quenching by ionized carboxylic groups In the absence of divalent metal ions,
a
decrease of tyrosine
(22) we
have no evidence for
the binding of Glu-Tyr copolypeptides to poly(A) neither at high
strength
as
expected from the electrostatic repulsion between negatively char-
ged carboxylic and phosphate groups. Circular dichrolsm that a
a
nor at low ionic
ternary complex
was
formed when Zn
or Cu
was used to show
ions were present in
mixture of polynucleotide and polypeptide. As shown in figure 1, addition of
Cu
ions to a poly(A) solution at high ionic strength (0. 2 M NaCl) leads to a
963
Nucleic Acids Research
+
ic
Figure 1 Circular dichrotsm spectra of 2 x 10- M poly(A) in the absence (a) and the presence of CuCl2 at. Cu2+) / LAJ ratio of 4 (b). Spectra (c) -(d) (e) were obtained upon addition of (Glu-Tyr-Glu)n to solution (b). Concentrations of (Glu-Tyr-Glu)n expressed as moles tyrosine per liter are 8 x 10-6 M (c), 1.65 x 10-5 M (d) and 4.15 x 10-5 M (e). All solutions were made in a pH 7 buffer containing 1 mM sodium cacodylate and 0.2 M NaCl. decrease of the circular dichroism amplitude of both the positive and negative
bands. At a ratio [Cu
] / [A] equal to 4, this decrease amounts to about 2+ 25 %. Addition of (Glu-Tyr-Glu) n to this Cu - poly(A) mixture leads to a
large decrease of the CD amplitude of poly(A) (figure 1). On the other hand, in 2+ the absence of Cu ions, the addition of (Glu-Tyr-Glu) has no effect on the poly(A) CD spectrum. Therefore these CD experiments show that a ternary complex is formed. The changes induced in the CD spectrum of poly(A) above 2+ 230 nm in the presence of both (Glu-Tyr-Glu) and Cu ions are equivalent to those effected by much higher Cu concentrations (or by the same Cu concentration at lower ionic strength). Below 230 nm, the negative contribution to the CD spectrum of the (Glu-Tyr-Glu) n - Cu complex is clearly indicated (In a separate experiment it has been shown that the positive CD band of 2+ (Glu-Tyr-Glu)n at 225 nm is replaced by a negative band upon Cu binding). The CD spectra of the ternary mixtures do not depend on the order of addition of the reactants. Spectra similar to those presented in figure 1 were obtained upon addition of Cu ions to a poly(A) + (Glu-Tyr-Glu) n mixture. Furthermore the quenching of Tyr fluorescence is not removed by addition of 2+ poly(A) to a (Glu-Tyr-Glu) - CuI2+ complex, indicating that Cu ions remain
bound to the polypeptide.
964
Nucleic Acids Research Results similar to those reported above other copolypeptides (Glu-Tyr) and (Glu-Tyr-Tyr-Glu)
were
obtained with the
although there
differences in the relative concentrations required to observe the In all
cases
same
are
effects.
addition of 1 mM EDTA restores both the original
CD spectrum of poly(A) and the original tyrosine fluorescence of the poly-
peptide. Similar investigations ionic strengths
were
were
carried out with Zn
used since the binding of Zn
2+
ions.
Lower
ions to polynucleotides
strongly decreases when the ionic strength increases(l5), As shown in figure 2, addition of (Glu-Tyr-Glu) to
a
Zn
-
poly(A) mixture induces
a
large decrease
n
2
in the CD amplitude of poly(A) similar to that effected by much higher Zn
concentrations in the absence of (Glu-Tyr-Glu)
Similar results were obtained
. n
with poly(C). An important decrease of the CD amplitude and +
a
slight red-
(a)
20
+ 10
0'
roW~~~~~C 240
260
280
X(nm)
-10
Figure 2 Circular dichroism spectra of 5. 7 x 10-5 M poly(A) in the absence (a) and in the presence of 2 x 10-4 M ZnCl2 (b). Spectra (c) and (d) were obtained after addition to solution (b) of 1. 2 x 10-5 M and 3. 5 x 10-5 M (Glu-Tyr-Glu)n, respectively. Spectrum (e) (broken line) was obtained after further addition of 1 mM EDTA to solution (d). All solutions were made in a pH 7 buffer containing 1 mM sodium cacodylate and 1 mM NaCl. shift of the CD spectrum of poly(C)
added to
a
poly(C)
-
Zn
were
observed when (Glu-Tyr-Glu)
was
mixture. More interesting is the result obtained by
fluorescence investigations of the ternary mixtures. As said above, the bin'2+. ding of Zn ions to (Glu-Tyr-Glu) enhances the fluorescence intensity of Tyr residues. Addition of poly(A) to this (Glu-Tyr-Glu) - Zn complex leads to a n
965
Nucleic Acids Research decrease in the intensity of tyrosine fluorescence which
is much
than that due to the screening effect of the polynucleotide
at
thd
more
excitation
shown in figure 3, poly(A)
wavelength (275 nm). In the experiments
added to (Glu-Tyr-Glu) and the screening effect decrease of the fluorescence intensity. When
Zn
important
was
first
of the polynucleotide led ions
were
added
to
to
a
this
poly(A) + (Glu-Tyr-Glu) mixture, a strong quenching of tyrosine fluorescence was
observed. This quenching
was
reversed by adding 1 mM EDTA.
the formation of the ternary complex (Glu-Tyr-Glu)-
-
Zn
-
Therefore
poly(A) leads
to
n
a
quenching of Tyr fluorescence which is
(Glu-Tyr-Glu)
-
Zn
c
not observed in the binary
omplex.
n~~~~~~~~~~~
60-
'4X4 C~~~~~ 4,0
3
bi
340 320 '(nm) 300
Fluorescence spectra of (1) 8. 2 x 10- M (Glu-Tyr-Glu)n, (2) solu(1 + . 2 x10-4 M poly(A), (3) solution (2) + 3. 7 x 10- 4 M Z nC 1? and (4) tio solution (3) + 1 mM EDTA. Same buffer as in figure Z. The decrease in fluorescence intensity observed upon addition of poly(A) (spectrum 2) is due to the screening effect of the polynucleotide at the excitation wavelength (275 nm). The increase in scattered light observed with solution 3 was also observed when ZnCl2was added to poly(A) alone. It should be noted that upon addition of low concentrations of
Figure 3
(Glu- Tyr -Glu) (8. 2 x 1 0 -5M) and poly(A) (1.2 x 10 eM),the fluorescence of tyrosine residues first increases indicating that Zn ions bind to the polypeptide. There is no change in the absorp-
Zn2Z+ ions -s ift red
to a mixture of
f
a
s
ho
uldi
noe tedu
tion spectrum of the mixture. When the concentration of Zn ions reaches a value corresponding to one Zn ion per tyrosine, one observes a quenching of Tyr fluorescence together with an increase in absorbance at 260 mnm and a ZnGsif
966
added
tbsorpoty(
alone.t
f
poly(A) (as observed when Zn
ions
Nucleic Acids Research bind to poly(A) )(
5).
These three effects
EDTA. These results indicate that the
peptide has bound amounts of Zn static repulsion of phosphate
required, this might
sequence is
reversed by addition of 1 mM
ternary complex forms only when the poly-
ions
groups.
are
large enough
Since
one
Zn2+
suggest that Zn
to overcome the electro-
ion
per
Glu-Tyr-Glu
is coordinated by
two Glu
residues. The fluorescence of Tyr residues is quenched when basic oligo-
peptides containing tyrosine (e. g., Lys-Tyr-Lys) bind to poly(A) or DNA(4' 10) In the case of Lys-Tyr-Lys complexes with poly(A), this quenching is due to a
stacking interaction of tyrosine with adenine bases
Lys-Tyr-Lys complexes
with DNA, such
a
stacking is
Tyr fluorescence quenching might be ascribed bases (quenching by phosphate
eliminated by residues
;
a
or
to
).
not
In the
case
of
observed (8b) and
an energy
transfer to the
through hydrogen bond formation has been
comparative study of peptides containing Tyr and Tyr(OMe)
unpublished results). In the
case
of the ternary complexes investiga-
ted here, the quenching of Tyr fluorescence might be due to
any
of these possi-
bilities. Further experiments using Nuclear Magnetic Resonance spectroscopy should help clarify this point. C onclusiQn
The changes in the poly(A) structure responsible for the CD
modifications shown in figures land 2 bases
).
are
due to Cu
or
Zn
binding to the
These results show that transition metal ions such as Zn
might be able to act
as
or Cu
intermediate species in the interaction between amino
acid side chains and nucleic acid bases. Moreover the formation of such a ternary complex may also induce other types of interactions between neighbo-
ring residues of proteins and nucleic acids (e. g., tyrosine-adenine interac-
poly(A) complexces). The role of metal ions nucleic acid complexes remains to be elucidated in most cases.
tions in (Glu-Tyr-Glu)n
in protein
-
-
ZnZ+
-
participate in phosphate binding but they could also contribute to base sequence recognition by more specific interactions involving the bases themselves. It is known that Zn ions are required for the activity of many tin(23 -26)aswl as well enzymes involved in nucleic acid replication or transcription They
as
can
of reverse transcriptase(
).
Whether the ions play a structural role in
maintaining the correct enzyme conformation or whether they are involved in
967
Nucleic Acids Resarch the interaction with the nucleic acid is yet unknown. The results presented above show that they could promote specific interactions between amino acid side chains and nucleic acid bases.
Acknowledgment I wish to thank Dr. Y. Trudelle for a gift of copolypeptides and helpful discussions and Prof. J. Baraduc for careful reading of the manuscript.
References
(1) T. Montenay-Garestier and C. H6l1ne, Nature 217, 844-845 (1968) Biochemistry 10, 300-306 (1971) J. Chim. Phys. 70, 1385-1390 (1973) (2) J. L. Dimicoli and C. Hel'ne, Biochimie 53, 331-345 (1971) J. Am. Chem. Soc. 95, 1036-1044 (1973) (3) R. Lawaczeck and K. G. Wagner, J. Magn. Res. 8, 164-174 (1972) (4) C. H6lne and J. L. Dimicoli, FEBS Letters 26, 6-10 (1972)
(5) E.J. Gabbay, K. Sanford and C.S. Baxter, Biochemistry 11, 3429-3435 (1972) (6) E. J. Gabbay, K. Sanford, C. S. Baxter and L. Kapicak, Biochemistry 12, 4021-4029 (1973) (7) R. L. Novak and J. Dohnal, Nature 243, 155-157 (1973) (8) J. L. Dimicoli and C. Hgl'ne, a. Biochemistry 13, 714-723 (1974) b. Biochemistry 13, 723-730 (1974) (9) J. J. Toulm4, M. Charlier and C. He1hne, Proc. Nat. Acad. Sci. U. S. 71, 3185-3188 (1974) (10) F. Brun, J. J. Toulme and C. Hlv1ne, Biochemistry 14, 558-563 (1975) (11) M. Durand, J. C. Maurizot, H. N. Borazan and C. H6lene, Biochemistry 14, 563-570 (1975) (12) H. Sellini, J. C. Maurizot, J. L. Dimicoli and C. H61bne, FEBS Letters 30, 219-224 (1973) (13) M. Daune in "Metal Ions in Biological Systems", H. Sigel Ed., M. Dekker , 1-43 (1974) (14) C. Zimmer, G. Luck and H. Triebel, Biopolyrners 13, 425-453 (1974) (15) a. Y.A. Shin, J.M. Heim and G. L. Eichhorn, Bioorg. Chem. 1, 149-163 (1972) b. Y. A. Shin, Biopolymers 12, 2459-2475 (1973) (16) J. P. Schreiber and M. Daune, Biopolymers 8, 139-152 (1969) (17) R. P. Martin, M. M. Petit-Ramel and J. P. Scharff in "Metal Ions in Biological Systems", H. Sigel Ed., M. Dekker, 2, 1-61 (1973) (18) H. Sigel, in ref. 17 pp. 63-125 968
Nucleic Acids Research (19) Y. Trudelle, J. Chem. Soc. Perkin Trans. I, 1001-1005 (1973) (20) a. Y. Trudelle, Polymer 16, 9-15 (1975) b. Y. Trudelle and G. Spach, Polymer 16, 16-20 (1975)
(21) C.K. Luk, Biopolymers 10, 1229-1241 (1971) (22) A. Pesce, E. Bodenheimer, K. Norland and G. D. Fasman, J. Am. Chem. Soc. 86, 5669-5675 (1964) (23) J. P. Slater, A.S. Mildvan and L.A. Loeb, Biochem. Biophys. Res. Comm. 44, 37-43 (1971) (24) M. C. Scrutton, C. W. Wu and D. A. Goldthwait, Proc. Nat. Acad. Sci. U. S. 68, 2497-2501 (1971) (25) H. Rubin, Proc. Nat. Acad. Sci. U.S. 69, 712-716 (1972)
(26) C. F. Springgate, A.S. Mildvan, R. Abramson, J. L. Engle and L. A. Loeb, J. Biol . Chem. 248, 5987-5993 (1973) (27) B. J. Poiesz, G. Seal and L. A. Loeb, Proc. Nat. Acad. Sci. U.S.A. 71, 4892-4896 (1974) (28) D. S. Auld, H. Kawaguchi, D. M. Livingston and B. L. Vallee, Proc. Nat. Acad. Sci. U.S.A. 71, 2091-2995 (1974) Biochem. Biophys. Res. Comm. 62, 296-302 (1975.
969
Nucleic Acids Research
Metal ton-mediated specific interactions between nucleic acid bases of polynucleotides and amino acid side chains of polypeptides. Claude Helene Centre de Btophystque MolEculaire, 45045 Orlfans C&dex, France
Received 29 April 1975 Summary Interactions between copolypeptides containing Glu and Tyr residues and polynucleotides can be mediated through divalent metal ions such as Zn2+ and CuZ+. Circular dichrotsm studies show that the binding of metal ion - polypeptide complexes to poly(A) induces an unstacking of adenine bases. Fluorescence investigations demonstrate that Tyrosine - AdeniAe interactions result from the formation of ternary complexes polypeptide-Zn -polynucleotide. Introduction The selective recognition of nucleic acid base sequences by proteins rests upon structural complementarity and specific interactions between amino acid side-chains and nucleic acid bases. Structural requirements are certainly of major importance in the recognition of, e. g., tRNAs by amino
acyl-tRNA synthetases. The interactions between the amino acid residues of the recognition site and the bases of the interacting nucleic acid region may be direct or indirect (i. e. promoted by a third species acting as an intermediate).
Stacking interactions involving aromatic amino acids were first demonstrated in frozen aqueous solutions , then in concentrated aqueous solutions at room temperature ' ) and more recently in oligopeptide nucleic acid complexes (4-11) . Hydrogen bonding between a few amino acid side chains and nucleic acid bases has also been shown to take place in organic solvents ). As to electrostatic interactions between basic amino acids and phosphate groups they may contribute to the specificity of protein-nucleic acid interactions inasmuch as the relative orientation of phosphate groups depends on the base sequence.
Stacking, hydrogen bonding and electrostatic binding result from direct interactions. One might also consider the possibility that the interaction between the amino acid side chain and the base is mediated through
961
Nucleic Acids Resarch a
third species which might be
a
metal ion. Numerous studies have been devo-
ted to the study of metal ion binding to polynucleotides, nucleic acids, peptides and proteins. It has been shown that transition metal ions bind
not only to
the phosphates but also to the bases of nucleic acids. The mode of binding depends
the nature of both the metal ion and the base (see references 13-15
on
and references cited therein). For example Cu
ions
and Zn
are
able to
form chelates between phosphates and bases (especially N7 of guanine in DNA) and possibly sandwich complexes between consecutive G-C base pairs( Certain
of amino acids
sequences
complexes with Zn
2+
and Cu
2+.
may
ions in
also form aqueous
very
16)
strong and specific
solutions (see references 17
and 18 for recent reviews). It is thus possible to contemplate that specific
interactions between amino acid and base
sequences
such metal ions. Moreover the formation of metal ion
a
could be promoted by
ternary complex amino acid
-
base (or phosphate) might induce selective interactions between
-
amino acid residues and bases
even
far removed from the site of metal
attachment. The purpose of this preliminary report is to present experimental evidence for interactions promoted by Zn
or
Cu
ions
between copolypep-
tides containing Glu and Tyr residues and polynucleotides. These copolypeptides
were
chosen because they do not interact with polynucleotides
in
the
absence of metal ions. Experimental Polynucleotides poly(A) and poly(C)
were
purchased from Miles.
Copolypeptides of glutamic acid and tyrosine have been synthesized in laboratory
(1
9) .
The following sequences were available
(Glu-Tyr-Glu)n,
(Glu-Tyr-Tyr-Glu)
.
our
Ty- Glu),
(Tyr
These polypeptides are soluble in
water at pH 7 (after neutralizing the carboxylic groups) and adopt a random
coil conformation at this pH value. The synthesis and characterization of
these polypeptides have been reported elsewhere(19 20). Circular dichroism measurements were carried out with a Roussel-Jouan II dichrograph. Fluorescence spectra were recorded with a
Jobin-Yvon spectrofluorometer modified tion wavelength
=
275 nm).
All solutions
962
as described in reference 9 (excita-
were
made in a pH 7 buffer containing 1 mM
Nucleic Acids Research sodium cacodylate and either 1 mM out at
a
or
0. 2 M NaCl. Measurements
were
carried
temperature of 230C.
Results and Discussion The binding of Cu
ions to poly(A) and poly(C) in their
and Zn
single-stranded helical conformation (pH 7, 20°C) induces the
bases(l5).
an
unstacking of
The resulting changes in absorbance and optical rotatory dis-
persion (or circular dichrotsm,
see
below)
by raising the temperature of the solution and poly(C) than Cu
ions
strength. These ions have
are
(15)
similar to those brought about Zn
2+
ions
and their binding is much a
bind less to poly(A)
more
sensitive to ionic
fairly strong affinity for both the bases and the
phosphates and the formation of chelates between phosphates and bases has been
suggested(
).
In contrast Mg
and Ca
ions
which bind chiefly to phos-
phates (and not to the bases) stabilize the ordered structure of the polynucleotides. The binding of Cu
investigated here The binding of Cu
can
and Zn
ions to the Glu-Tyr copolypeptides
be conveniently followed by fluorescence measurements.
ions
to (Glu-Tyr-Glu)
fluorescence whereas Zn
ions
leads to
a
quenching of the tyrosine
~~~n enhance the fluorescence intensity of tyrosine
residues by about 35 %. The binding sites of Cu and Zn ions on the copolypeptides are not known. The carboxyl groups of Glu residues are certainno
evidence from
group
of t*yrosine. It
ly involved (and perhaps also the peptide bonds) but there is absorbance measurements for ionization of the hydroxyl is. known that the binding of Cu
ions to
the amino and carboxyl groups of free
tyrosine leads to fluorescence quenching at pH 7(l). The fluorescence enhancement due to Zn
2+
binding might be ascribed to
fluorescence quenching by ionized carboxylic groups In the absence of divalent metal ions,
a
decrease of tyrosine
(22) we
have no evidence for
the binding of Glu-Tyr copolypeptides to poly(A) neither at high
strength
as
expected from the electrostatic repulsion between negatively char-
ged carboxylic and phosphate groups. Circular dichrolsm that a
a
nor at low ionic
ternary complex
was
formed when Zn
or Cu
was used to show
ions were present in
mixture of polynucleotide and polypeptide. As shown in figure 1, addition of
Cu
ions to a poly(A) solution at high ionic strength (0. 2 M NaCl) leads to a
963
Nucleic Acids Research
+
ic
Figure 1 Circular dichrotsm spectra of 2 x 10- M poly(A) in the absence (a) and the presence of CuCl2 at. Cu2+) / LAJ ratio of 4 (b). Spectra (c) -(d) (e) were obtained upon addition of (Glu-Tyr-Glu)n to solution (b). Concentrations of (Glu-Tyr-Glu)n expressed as moles tyrosine per liter are 8 x 10-6 M (c), 1.65 x 10-5 M (d) and 4.15 x 10-5 M (e). All solutions were made in a pH 7 buffer containing 1 mM sodium cacodylate and 0.2 M NaCl. decrease of the circular dichroism amplitude of both the positive and negative
bands. At a ratio [Cu
] / [A] equal to 4, this decrease amounts to about 2+ 25 %. Addition of (Glu-Tyr-Glu) n to this Cu - poly(A) mixture leads to a
large decrease of the CD amplitude of poly(A) (figure 1). On the other hand, in 2+ the absence of Cu ions, the addition of (Glu-Tyr-Glu) has no effect on the poly(A) CD spectrum. Therefore these CD experiments show that a ternary complex is formed. The changes induced in the CD spectrum of poly(A) above 2+ 230 nm in the presence of both (Glu-Tyr-Glu) and Cu ions are equivalent to those effected by much higher Cu concentrations (or by the same Cu concentration at lower ionic strength). Below 230 nm, the negative contribution to the CD spectrum of the (Glu-Tyr-Glu) n - Cu complex is clearly indicated (In a separate experiment it has been shown that the positive CD band of 2+ (Glu-Tyr-Glu)n at 225 nm is replaced by a negative band upon Cu binding). The CD spectra of the ternary mixtures do not depend on the order of addition of the reactants. Spectra similar to those presented in figure 1 were obtained upon addition of Cu ions to a poly(A) + (Glu-Tyr-Glu) n mixture. Furthermore the quenching of Tyr fluorescence is not removed by addition of 2+ poly(A) to a (Glu-Tyr-Glu) - CuI2+ complex, indicating that Cu ions remain
bound to the polypeptide.
964
Nucleic Acids Research Results similar to those reported above other copolypeptides (Glu-Tyr) and (Glu-Tyr-Tyr-Glu)
were
obtained with the
although there
differences in the relative concentrations required to observe the In all
cases
same
are
effects.
addition of 1 mM EDTA restores both the original
CD spectrum of poly(A) and the original tyrosine fluorescence of the poly-
peptide. Similar investigations ionic strengths
were
were
carried out with Zn
used since the binding of Zn
2+
ions.
Lower
ions to polynucleotides
strongly decreases when the ionic strength increases(l5), As shown in figure 2, addition of (Glu-Tyr-Glu) to
a
Zn
-
poly(A) mixture induces
a
large decrease
n
2
in the CD amplitude of poly(A) similar to that effected by much higher Zn
concentrations in the absence of (Glu-Tyr-Glu)
Similar results were obtained
. n
with poly(C). An important decrease of the CD amplitude and +
a
slight red-
(a)
20
+ 10
0'
roW~~~~~C 240
260
280
X(nm)
-10
Figure 2 Circular dichroism spectra of 5. 7 x 10-5 M poly(A) in the absence (a) and in the presence of 2 x 10-4 M ZnCl2 (b). Spectra (c) and (d) were obtained after addition to solution (b) of 1. 2 x 10-5 M and 3. 5 x 10-5 M (Glu-Tyr-Glu)n, respectively. Spectrum (e) (broken line) was obtained after further addition of 1 mM EDTA to solution (d). All solutions were made in a pH 7 buffer containing 1 mM sodium cacodylate and 1 mM NaCl. shift of the CD spectrum of poly(C)
added to
a
poly(C)
-
Zn
were
observed when (Glu-Tyr-Glu)
was
mixture. More interesting is the result obtained by
fluorescence investigations of the ternary mixtures. As said above, the bin'2+. ding of Zn ions to (Glu-Tyr-Glu) enhances the fluorescence intensity of Tyr residues. Addition of poly(A) to this (Glu-Tyr-Glu) - Zn complex leads to a n
965
Nucleic Acids Research decrease in the intensity of tyrosine fluorescence which
is much
than that due to the screening effect of the polynucleotide
at
thd
more
excitation
shown in figure 3, poly(A)
wavelength (275 nm). In the experiments
added to (Glu-Tyr-Glu) and the screening effect decrease of the fluorescence intensity. When
Zn
important
was
first
of the polynucleotide led ions
were
added
to
to
a
this
poly(A) + (Glu-Tyr-Glu) mixture, a strong quenching of tyrosine fluorescence was
observed. This quenching
was
reversed by adding 1 mM EDTA.
the formation of the ternary complex (Glu-Tyr-Glu)-
-
Zn
-
Therefore
poly(A) leads
to
n
a
quenching of Tyr fluorescence which is
(Glu-Tyr-Glu)
-
Zn
c
not observed in the binary
omplex.
n~~~~~~~~~~~
60-
'4X4 C~~~~~ 4,0
3
bi
340 320 '(nm) 300
Fluorescence spectra of (1) 8. 2 x 10- M (Glu-Tyr-Glu)n, (2) solu(1 + . 2 x10-4 M poly(A), (3) solution (2) + 3. 7 x 10- 4 M Z nC 1? and (4) tio solution (3) + 1 mM EDTA. Same buffer as in figure Z. The decrease in fluorescence intensity observed upon addition of poly(A) (spectrum 2) is due to the screening effect of the polynucleotide at the excitation wavelength (275 nm). The increase in scattered light observed with solution 3 was also observed when ZnCl2was added to poly(A) alone. It should be noted that upon addition of low concentrations of
Figure 3
(Glu- Tyr -Glu) (8. 2 x 1 0 -5M) and poly(A) (1.2 x 10 eM),the fluorescence of tyrosine residues first increases indicating that Zn ions bind to the polypeptide. There is no change in the absorp-
Zn2Z+ ions -s ift red
to a mixture of
f
a
s
ho
uldi
noe tedu
tion spectrum of the mixture. When the concentration of Zn ions reaches a value corresponding to one Zn ion per tyrosine, one observes a quenching of Tyr fluorescence together with an increase in absorbance at 260 mnm and a ZnGsif
966
added
tbsorpoty(
alone.t
f
poly(A) (as observed when Zn
ions
Nucleic Acids Research bind to poly(A) )(
5).
These three effects
EDTA. These results indicate that the
peptide has bound amounts of Zn static repulsion of phosphate
required, this might
sequence is
reversed by addition of 1 mM
ternary complex forms only when the poly-
ions
groups.
are
large enough
Since
one
Zn2+
suggest that Zn
to overcome the electro-
ion
per
Glu-Tyr-Glu
is coordinated by
two Glu
residues. The fluorescence of Tyr residues is quenched when basic oligo-
peptides containing tyrosine (e. g., Lys-Tyr-Lys) bind to poly(A) or DNA(4' 10) In the case of Lys-Tyr-Lys complexes with poly(A), this quenching is due to a
stacking interaction of tyrosine with adenine bases
Lys-Tyr-Lys complexes
with DNA, such
a
stacking is
Tyr fluorescence quenching might be ascribed bases (quenching by phosphate
eliminated by residues
;
a
or
to
).
not
In the
case
of
observed (8b) and
an energy
transfer to the
through hydrogen bond formation has been
comparative study of peptides containing Tyr and Tyr(OMe)
unpublished results). In the
case
of the ternary complexes investiga-
ted here, the quenching of Tyr fluorescence might be due to
any
of these possi-
bilities. Further experiments using Nuclear Magnetic Resonance spectroscopy should help clarify this point. C onclusiQn
The changes in the poly(A) structure responsible for the CD
modifications shown in figures land 2 bases
).
are
due to Cu
or
Zn
binding to the
These results show that transition metal ions such as Zn
might be able to act
as
or Cu
intermediate species in the interaction between amino
acid side chains and nucleic acid bases. Moreover the formation of such a ternary complex may also induce other types of interactions between neighbo-
ring residues of proteins and nucleic acids (e. g., tyrosine-adenine interac-
poly(A) complexces). The role of metal ions nucleic acid complexes remains to be elucidated in most cases.
tions in (Glu-Tyr-Glu)n
in protein
-
-
ZnZ+
-
participate in phosphate binding but they could also contribute to base sequence recognition by more specific interactions involving the bases themselves. It is known that Zn ions are required for the activity of many tin(23 -26)aswl as well enzymes involved in nucleic acid replication or transcription They
as
can
of reverse transcriptase(
).
Whether the ions play a structural role in
maintaining the correct enzyme conformation or whether they are involved in
967
Nucleic Acids Resarch the interaction with the nucleic acid is yet unknown. The results presented above show that they could promote specific interactions between amino acid side chains and nucleic acid bases.
Acknowledgment I wish to thank Dr. Y. Trudelle for a gift of copolypeptides and helpful discussions and Prof. J. Baraduc for careful reading of the manuscript.
References
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