Helix Formation in Methylated Copolymers of Lysine and Alanine

JAKE BELL0

Department of Chemistry, Roswell Park Division of the Graduate School, Sate University of New York, Buffalo, New York 14263

SYNOPSIS

Random copolymers of lysine and alanine, 2 : 1 and 1 : 1, were trimethylated on the lysine amino groups to quaternary ammonium groups. Methylated and unmethylated polymers were prepared with C1- or C10; as the counterion. CD spectra were measured for increasing concentration of peptide without added salt, and at constant peptide concentration in increasing NaCl or NaC104. Unmethylated peptides, as the chloride, form a-helix more readily than do the methylated peptides. The opposite occurs with C10; as counterion. The helix-promoting effect of methylated lysine residues (C10; counterion) is diminished by the presence of alanine, as compared with effects when lysine is the only type of residue. The effect of methylation of proteins on helix formation may depend on the types of anionic groups with which the protein may be involved.

INTRODUCTION

Some lysine residues of some proteins are methyl- ated after translation.' The effects of such methyl- ation on the properties of proteins are not well un- derstood. We have presented data on the properties of methylated poly (L-lysine) [ ( K ),I, chiefly poly- (N',N',N'-trimethyl-L-lysine) [ ( MeaK),], and to a lesser extent on poly (dimethyl-L-lysine) and a partially monomethylated poly ( L-lysine ) .2-5 Ya- mamoto and Yang' reported the synthesis and some properties of poly (N'-methyl-L-lysine) . Our earlier results on ( Me3K ), HClO, may be summarized briefly as follows: in the absence of salt the meth- ylated polymer forms a-helix at much lower peptide concentration, by a factor of about 50, than does (K), - HClO,; ( Me3K), * HClO, at constant peptide concentration in different concentrations of NaC10, adopts the a-helix at about one-thirtieth of the NaC104 concentration required for (K), * HClO,. There are also differences in binding to polynucle- otides and in the CD spectra of complexes of the peptides with polynucleotides.7~s Now we report on the a-helix-forming properties at 0°C of two random

~~ ~ ~

Biopolymers, Vol. 32, 491-496 (1992) 0 1992 John Wiley & Sons, Inc. CCC oooS-3525/92/050491-06$04.00

copolymers of lysine and alanine, a 2 : 1 and a 1 : 1 copolymer in the unmethylated and methylated forms, as the chloride and as the perchlorate.

EXPERIMENTAL

Copolymers of lysine and alanine ( hydrobromides ) were from Sigma. Molecular weights according to the vendor were 52 kDa for the 1 : 1 polymer and 49 kDa for the 2 : 1 polymer. The absorption spectra showed no residual carbobenzyloxy blocking groups. Methylation with dimethyl sulfate was done as de- scribed earlier.3 Methylation was complete, as shown by the absence of lysine spots on thin layer chro- matography plates, after hydrolysis, by the method of Pillay and Mehdi? A trimethyl-L-lysine spot was present. Reaction mixtures were dialyzed against NaC104 or NaC1, then against water. Unmethylated controls were put through mock reactions, and were dialyzed in the same way. Concentrations of peptide were estimated from absorbance at 191 nm, using

reported by Rosenheck and Doty1° for the random coil (details in Ref. 5) .

CD spectra were recorded with a Jasco J-500A. Temperature was measured with a Pt resistance element and a digital temperature indicator (Omega

49 1

the molecular absorbance of 7100 cm-'M-' for(K)n

492 BELL0

Model 199P2). Solutions of peptides were about pH 5.5-6; no buffer was used. Light scattering was mea- sured at 90" in an Aminco spectrofluorometer, at 550 nm, with the emission monochromator supple- mented with a 550-nm interference filter. The orig- inal detector was replaced with a photon counter (SSR photomultiplier and preamplifier, and a Beckman Universal counter UClO) .

RESULTS

All the CD results were obtained at 0-0.2"C. The CD spectra showed the classical random coil or he- lical characteristics; the former showed a positive extremum at 218 nm, and the latter the two negative extrema at 222 and 208 nm. ( K), HBr at high con- centration, above about 30%, in water, goes over to the a-helix, "J' in hexagonal arrays. In earlier work we found that when the counterion is ClO,, 50% conversion to a-helix occurs at about 0.15Mpeptide5 and ( Me3K), - HC104 attains 50% helix at 2-4 mM, depending on molecular weight, at 0°C. When one- third of the lysine residues is replaced by alanine and the counterion is C1- (but with no added NaCl) , no a-helix was detected to the highest peptide con- centration examined, 0.1 M, at 0°C. Stokrova et al.13 found about 10% helix at room temperature for ( K2,A1),. HC1 in 0.01M NaC1, 0.01M Tris at pH 7. Kubota et al.I4 found no significant helix forma- tion for ( K ,A l ), - HC1.

-301

0 0.5 10 4.5 2 0 2 5 3.0 35 NaCI, M

Figure 1. EIlipticity of (K*,A'), and (Me3K2,A')" as the chlorides in NaCl solutions. 0: (K2,A'),; 0: ( MesK2

- 'a - E U

0

Q) U

% c5 c

M

I0 - X n CD U

200 220 240 260

X, nm Figure 2. Zero NaC1; 2: 0.8M NaC1; and 3: 3.2M NaC1.

CD spectra of ( MesKZ ,A')" * HC1 in NaC1.1:

We measured CD spectra of ( K-2 ,A' ), HC1 and (Me3K2,A'),.HC1 in the presence of NaC1. For ( Me3K2,A1), - C1 there is a change from a positive extremum at 217 nm, with [8] = 6 X lo3 , to a neg- ative extremum at 222 nm with 8 = -6 X l o 3 deg cm2 dmole-', constant at the latter value from 1.5 to 3.2 M NaCl (Figure 1). The spectra of ( Me3K2, A'),.HCl in NaCl for 0, 0.8 and 3.2M NaCl are shown in Figure 2; little, if any, a-helix is formed. The positive extremum seen for the (K), random coil a t 218 nm is seen at zero NaCl for (Me3K2, A'),-HCl. (K2,A1), in NaCl forms a-helix more readily, with [8]222 reaching a value of -26 X lo3 deg cm2 dmole-' a t 3.2 M NaCl (Figure 1 ) , and ap- parently still increasing in magnitude.

were also prepared as the perchlorates. In the absence of added NaC104, a-helix formation in (K2,A1),-HC104 is small at up to 16 mMpeptide, the ellipticity increasing grad- ually with polypeptide concentration (Figure 3) . For ( Me3K2 ,A1 ), HCIOl there is 50% helix at about 4 m M and nearly complete helix at 14 m M (Figure 3) . For comparison, data from Ref. 5 for ( Me3K), HC104 are shown for two molecular weights.

With added NaC104 and a constant concentration of 0.6 m M peptide, both (K2,A1),.HC104 and

( K ' ,A1 ), and ( Me3K ,A1

HELIX FORMATION IN METHYLATED COPOLYMERS 493

I. Peptidel, mM

Figure 3. Ellipticity of (K)n , (Me,K),, (K2,A1),, and ( Me3KZ as the perchlorates, without added salt, at 0°C. 1: (Me,K),, 400 kDa; 2: (Me3K),, 40 kDa; 3: (K),, 400kDa;4: (K2,A1),;5: (Me3K2,A1),.Datafor (K).and ( Me3K), from Ref. 5 . For 1 and 2, the molecular weights are for the parent (K), - HBr polymers.

( Me3K ,A1 ), become 50% helical at 15 m M (Figure 4). Before complete helix can be reached in ( Me3K2,A1), there is a decrease in ellipticity a t about 0.15M NaC104, followed by an increase a t about 0.6-1M (Figure 4 ) . Light scattering mea- surements, at 550 nm, showed that scattering by ( Me3K2,A1), increased 100-fold a t 0.25M over the scattering a t 0.01M NaC104 (Figure 4 ) . Scattering decreases from about 0.6M NaClO,, presumably as a result of dissociation of aggregates by the chao- tropic effect of ClO;, This effect in ( MesK2 ,A'), is like the stronger effect we reported with (Me,K), in NaC104? There was no increase in light scattering in solutions of (K2,A1),. (K2,A'), in NaC104 is converted to a-helix, about as easily as is ( Me3K2 ,A1),. This is quite different from the be- havior of the pair (K), and (Me3K),. The NaC104 concentrations needed for 50% conversion to a-helix

-401

NaCIOs, M

300

260 .c, h

cn

5 220 2 e c .- 480 $ 440 =

LT -I

U

.- 100 '0

s E

60 5 20 0

Figure 4. Ellipticity and light scattering of ( K2,A'), and ( Me3K2,A1), as perchlorates in NaC104 solutions. 1: Ellipticity of (K2,A'),; 2: ellipticity of (Me3K2,A1),; 3: light scat- tering of (Me3K2,A1),; 4: light scattering of (K2,A1),.

494 BELL0

is about 30 times as large for (K), as for ( Me3 ,K),? Next, a 1 : 1 copolymer was studied with C1- or

C10; as counterion. With no added NaC1, (K ' ,A1), * HC1 shows linearly increasing helix con- tent as the peptide concentration is increased, with [ 0]222 going from -11 X lo3 deg cm2 dmole-' at 0.2 m M t o -19 X l o3 at 12.8 m M (Figure 5) . Kubota et al.14 found a slight helical extent for 0.3 m M (K1,A'),.HC1 at 25"C, with about -2 X lo3 deg cm2 dmole-'. For ( Me3K1,A'),-HCl the helix content is smaller at all peptide concentrations, with [ 0 ] 2 2 2 going from -4 X l o3 deg cm2 dmole-' at 0.3 m M t o -9 X lo3 at 10 m M (Figure 5) .

In the presence of added NaC1, helix formation is promoted for both the parent and methylated peptides. Helix is more readily formed in (K', A') * HC1 than in ( Me3K1,A') * HCI (Figure 6), with 50% a-helix being formed at 0.3 m M NaCl for the former and 1 m M for the latter. For (Me3K1, A') * HCl, an unusual feature is the initial steep rise in - [0 ]222 , although not as steep as for ( K ' ,A' ), * HC1, followed by a gradual, continuous rise to at least 0.128M NaC1.

Turning now to the C10, forms of the 1 : 1 co- polymers, in the absence of NaC104, we see in Figure

5 that with the higher content of alanine, helix for- mation is promoted, compared with the 2 : 1 copoly- mer (Figure 3 ) and with (K),, and that the meth- ylated ( Me3K ' ,A1 ), - C104 has only a moderately greater helix-forming tendency than does (K' , A l) , - c104. Helix formation in ( Me3K1 ,A') is 50% at about 1.7 mM, compared with 4 m M for (Me3K),,' while for (K',A1), and (K), corre- sponding values are about 2.5 m M and 0.5 M, a ratio of 200. Thus, incorporation of alanine promotes he- lix formation so much that methylation has only a small further effect. In the presence of increasing concentrations of NaC104 ( Me3 ,K' ,A1 ) also attains the helical state somewhat more easily than does ( K' ,A1) (Figure 7) , but by a much smaller margin than in the case of ( Me3K), vs ( K),.' The require- ments for helix formation are tabulated in Tables I and 11, for more convenient comparison.

DISCUSSION

Several conclusions appear from the data. First, in- creasing the proportion of alanine residues (or de-

-40

C Peptide I , mM

Figure 5. Ellipticity of ( K1,A') and ( Me3K',A1)n as the chloride andperchlorate, without added salt. 1: (Me3K',A'),-HC1O4; 2: (K',A'),.HClO,; 3: (K',A'),-HC1; 4: (Me3K1, A'), - HCI.

HELIX FORMATION IN METHYLATED COPOLYMERS 496

-40t

[NaCll mM

Figure 6. Ellipticity of (K' 0 (K1,A1)=; 0: (Me3K',A1),.

HC104 and ( Me3K1 ,A')n HC10, in NaC104 solutions.

creasing the proportion of lysine residues ) lessens the difference between the methylated and unmeth- ylated polypeptides. Second, the greater the pro- portion of alanine, the lower the salt concentration required for helix formation. Third, the ease of helix formation by methylated peptide vs unmethylated is dependent on the nature of the counterion. In the presence of NaC1, unmethylated peptide forms helix more readily than does methylated. In the presence of NaClO,, the reverse is found. The third result may be relevant to the effect of methylation of pro- teins. It cannot be stated that methylated lysine residues will, as a rule, promote or hinder helix for-

mation. The effect may depend on the types and concentrations of ions present, and also on the na- ture of the anionic groups on the other biopoly- mer(s) with which the methylated amino groups (and perhaps also methylated arginine and histi- dine) interact. Some anionic groups may have dif- ferent conformational effects than do others. Protein conformation may be significantly influenced by concentrations of different ions inside and outside cell compartments.

Table I Salt Concentration €or 50% Helix Formation

NoC1O4, m M

Figure 7. Ellipticity of (K1,A1)n and (Me3K1,A1),, as the chlorides, in added NaC1.0 (K',A1),; 0: ( Me3KL,A'),.

Salt

NaCl Peptidea

5OOb 30 3 4 b

3 2

a All peptide concentrations about 0.6 mh4 (residues).

' High, if at all. Ref. 4.

496 BELL0

Table I1 Peptide Concentration for 50% Helix Formation

Counterion," (mM)

Peptide c1- c10;

(K)n lOOOb 150 (K2, A), High = 40' (K2, 10 2 (Me,K), High 4 (Me&', A1)n High 4 (Me&', A1), 30d 1.5

a No added salt. Ref. 12.

'By extrapolation (from Figure 2). By extrapolation (from Figure 4).

REFERENCES

1. Paik, W. K. & Kim, S. (1990) Protein Methylation,

2. Granados, E. N. & Bello, J. (1979) Biopolymers 18, CRC Press, Boca Raton, FL, pp. 1-22.

1479-1486.

3. Bello, J., Granados, E. N., Lewinski, S., Bello, H. R. & Trueheart, T. (1985) J. Biomol. Struct. Dynum. 2 , 899-9 13.

4. Bello, J. (1988) Biopolymers 27, 1627-1640. 5. Bello, J. ( 1 9 9 ~ ) Biopolymers, in press. 6. Yamamoto, H. & Yang, J. T. (1974) Biopolymers 13,

7. Granados, E. N. & Bello, J. (1981) Biochemistry 20,

8. Granados, E. N. & Bello, J. (1980) Biochemistry 19,

9. Pillay, D. T. & Mehdi, R. (1970) J. Chromatogr. 47,

10. Rosenheck, K. & Doty, P. (1961) Proc. Nutl. Acud.

11. Kondo, Y., Ukai, Y. & Iizuka, E. ( 1978) Polym. J. 10,

12. Darke, A. &Finer, E. G. (1975) Biopolymers 14,441- 456.

13. Stokrova, S., Sponar, J., Havranek, M., Sedlacek, B. & Blaka, K. (1975) Biopolymers 14,1231-1244.

14. Kubota, S., Ikeda, K. & Yang, J. T. (1983) Bwpolymers

1093-1107.

4761-4765.

3227-3233.

119-123.

Sci. USA 47,1775-1785.

631-636.

22, 2219-2236.

Received September 24, 1991 Accepted November 21, 1991