Volume 4 Number 3 March 1977 Vlum
4
Nucleic Acids Research
Nubr3Mrh17ucecAisRsac
Specificity of DNA-basic polypeptide interactions. I1+. Influence of aromatic amino acid residues investigated with agarose bound lysine copolypeptides Klaus Wehling, Hans-Adolf Arfmann, Gerhard Seipke and Karl G.Wagner
Gesellschaft fur Biotechnologische Forschung, Abteilung Molekularbiologie, 3300 BraunschweigSt6ckheim, GFR Received 8 November 1976
ABSTRACT Binding affinities towards DNA and base pair specificities of lysine copolymers, containing different amounts of Phe, Tyr or Trp residues, were estimated using a previously described chromatographic method. Incorporation of few aromatic residues into polylysine causes a decrease in the binding affinity, however, further raising the aromatic residue lysine ratio results in a continous increase of affinity, which is most pronounced with the Tyr copolymers and not observed with polymers containing neutral aliphatic amino acid residues. AT-specificity increases concomitant with binding affinity in the case of the Tyr copolymers but not with the Phe copolymers. The interaction of DNA with the alternating Phe-Lys polymer is significantly stronger than with the random copolymer of equal residue composition. The molecular and conformational reasons determining specificity are discussed. INTRODUCT ION The recognition of base pair sequences on double stranded DNA by proteins is one of the most challenging problems in the field of protein-nucleic acid interaction. Only few conceptions have emerged up to now, trying to explain specificity in terms of contributions of amino acid residues. While some authors emphazised the formation of specific hydrogen bonds2-4 others stressed the chemical properties of aromatic amino acid side chains in the interaction with the DNA surface or even in intercalation5'6For the elucidation of specific contributions caused by aromatic side chains, model investigations were mostly performed with oligopeptides and DNA6 20 or with nucleotides and basic copolypeptides21'22 Extraction of
+Part
I is Ref. 1
C Information Retrieval Limited I Falconberg Court London Wl V 5FG England
513
Nucleic Acids Research equilibrium data from DNA and basic polypeptides, i. e. from polymerpolymer interactions, is experimentally more difficult. As an approach we developed in a previous work (part I) a method for the estimation of binding affinities by coupling basic polypeptides to agarose and measuring the ionic strength required to dissociate DNA-polypeptide complexes. Using DNAs of different base pair composition, base pair directed specificities could be evaluated. It was shown in this work how neutral aliphatic amino acid residues reduce the affinity and base pair specificity of their host polylysine or polyarginine towards DNA. In the present work lysine copolymers, containing different amounts of aromatic amino acid residues,were probed and specific contributions exerted by the aromatic side chains were elucidated. MATERIALS AND METHODS Synthesis of polylysine, the random copolypeptides and the alternating (Lys-Phe)n was described previously22-24. Apparent average molecular weights estimated by gel filtration on Biogel P100, ranged from 40 000 to 90 000 dalton22-24. DNA from Micrococcus lysodeicticus (ML) and Clostridium perfringens (CP) were a gift from Dr. K. -H. C. Standke (Forschungsanstalt fUr Landwirtschaft, Braunschweig). Their fragmentation and fractionation on Sepharose 4B, already described in the previous paper1, yielded fractions with the following specifications: ML-DNA (25 mole % AT) 5.9 S and 23.5 % hyperchromicity; CP-DNA (69 mole % AT) 6.1 S and 30.4 % hyperchromicity.
Coupling of the polypeptides to CNBr-activated Sepharose 4B and elution of the applied DNA by linear NaCl gradients, containing 0.01 M sodium acetate of pH 6.7, was accomplished as previously described1. For standardization coupling was performed with constant ratios of CNBr-activated Sepharose to the basic polypeptides. Because of their limited solubility copolypeptides with a higher content of aromatic amino acids had to be coupled in suspension (these are marked with a star in Table 1). The coupling yield, however, was not significantly affected by this fact. NaCl gradients from 0.5 to 2.0 and 1.0 to 3.0 were used depending on the relative affinity of DNA towards the respective basic polypeptides. Fractions containing the eluted DNA were analyzed for absorbance at 260 nm and for conductivity, to determine the exact NaCl 514
Nucleic Acids Research concentration in the peak maximum.
RESULTS The method used in the present work and described in more details in part I of this series allows the determination of affinities in terms of a NaCl concentration which dissociates the DNA-polypeptide complex. Applying DNAs of extremly different base pair composition on the same polypeptide-agarose column, the resulting differences in the NaCl concentration (A AT-values) found for the elution profiles of these DNAs can serve as a measure for base pair specificities. As an example Fig. 1 shows the elution profiles obtained for a polylysine and a (Lys88, Phe12)n-Sepharose column.-From both columns the AT-rich CP-DNA (69 mole%) was eluted at higher ionic strength than ML-DNA (25 mole% AT), exhibiting AT-specificity for both polypeptides. The incorporation of 12 mole% Phe residues into polylysine reduces both the affinity and AT-specificity towards DNA. These results are in agreement with those obtained from the lysine copolypeptides containing aliphatic amino acid residues, and are apparently caused by the dilution of the positive -
OD260
(Lys88, Phel2)n
0.10
Polylysine 0.06
0.02
1.0
2.0
3.0 NaCi(M)
Figure 1: Comparison of the affinities and base pair specificities of polylysine and poly(Lys8, Phe') towards DNA. The elution profiles of ML-DNA (o) and CP-DNA (s) obtained with Sepharose 4B coated with the respective polypeptides are shown. 515
Nucleic Acids Research charges along the basic polymer. Gaps in the lysine matrix obviously reduce both affinity towards DNA and the AT-specificity of the lysine
side chains. Enhanced incorporation of neutral residues into polylysine results in further and continuous decrease in the DNA affinity, when aliphatic amino acid residues are concerned (i. e. alanine, Fig. 2); however in the case of the aromatic amino acid residues affinity increases starting at about 30 mole%. This is illustrated with the Phe and Tyr copolymers in Fig. 2, and the extracted data from the different copolypeptides are listed in Table 1. a
The order of increasing affinities paralells the order of AT-specificity: Ala
4
Nucleic Acids Research
Nubr3Mrh17ucecAisRsac
Specificity of DNA-basic polypeptide interactions. I1+. Influence of aromatic amino acid residues investigated with agarose bound lysine copolypeptides Klaus Wehling, Hans-Adolf Arfmann, Gerhard Seipke and Karl G.Wagner
Gesellschaft fur Biotechnologische Forschung, Abteilung Molekularbiologie, 3300 BraunschweigSt6ckheim, GFR Received 8 November 1976
ABSTRACT Binding affinities towards DNA and base pair specificities of lysine copolymers, containing different amounts of Phe, Tyr or Trp residues, were estimated using a previously described chromatographic method. Incorporation of few aromatic residues into polylysine causes a decrease in the binding affinity, however, further raising the aromatic residue lysine ratio results in a continous increase of affinity, which is most pronounced with the Tyr copolymers and not observed with polymers containing neutral aliphatic amino acid residues. AT-specificity increases concomitant with binding affinity in the case of the Tyr copolymers but not with the Phe copolymers. The interaction of DNA with the alternating Phe-Lys polymer is significantly stronger than with the random copolymer of equal residue composition. The molecular and conformational reasons determining specificity are discussed. INTRODUCT ION The recognition of base pair sequences on double stranded DNA by proteins is one of the most challenging problems in the field of protein-nucleic acid interaction. Only few conceptions have emerged up to now, trying to explain specificity in terms of contributions of amino acid residues. While some authors emphazised the formation of specific hydrogen bonds2-4 others stressed the chemical properties of aromatic amino acid side chains in the interaction with the DNA surface or even in intercalation5'6For the elucidation of specific contributions caused by aromatic side chains, model investigations were mostly performed with oligopeptides and DNA6 20 or with nucleotides and basic copolypeptides21'22 Extraction of
+Part
I is Ref. 1
C Information Retrieval Limited I Falconberg Court London Wl V 5FG England
513
Nucleic Acids Research equilibrium data from DNA and basic polypeptides, i. e. from polymerpolymer interactions, is experimentally more difficult. As an approach we developed in a previous work (part I) a method for the estimation of binding affinities by coupling basic polypeptides to agarose and measuring the ionic strength required to dissociate DNA-polypeptide complexes. Using DNAs of different base pair composition, base pair directed specificities could be evaluated. It was shown in this work how neutral aliphatic amino acid residues reduce the affinity and base pair specificity of their host polylysine or polyarginine towards DNA. In the present work lysine copolymers, containing different amounts of aromatic amino acid residues,were probed and specific contributions exerted by the aromatic side chains were elucidated. MATERIALS AND METHODS Synthesis of polylysine, the random copolypeptides and the alternating (Lys-Phe)n was described previously22-24. Apparent average molecular weights estimated by gel filtration on Biogel P100, ranged from 40 000 to 90 000 dalton22-24. DNA from Micrococcus lysodeicticus (ML) and Clostridium perfringens (CP) were a gift from Dr. K. -H. C. Standke (Forschungsanstalt fUr Landwirtschaft, Braunschweig). Their fragmentation and fractionation on Sepharose 4B, already described in the previous paper1, yielded fractions with the following specifications: ML-DNA (25 mole % AT) 5.9 S and 23.5 % hyperchromicity; CP-DNA (69 mole % AT) 6.1 S and 30.4 % hyperchromicity.
Coupling of the polypeptides to CNBr-activated Sepharose 4B and elution of the applied DNA by linear NaCl gradients, containing 0.01 M sodium acetate of pH 6.7, was accomplished as previously described1. For standardization coupling was performed with constant ratios of CNBr-activated Sepharose to the basic polypeptides. Because of their limited solubility copolypeptides with a higher content of aromatic amino acids had to be coupled in suspension (these are marked with a star in Table 1). The coupling yield, however, was not significantly affected by this fact. NaCl gradients from 0.5 to 2.0 and 1.0 to 3.0 were used depending on the relative affinity of DNA towards the respective basic polypeptides. Fractions containing the eluted DNA were analyzed for absorbance at 260 nm and for conductivity, to determine the exact NaCl 514
Nucleic Acids Research concentration in the peak maximum.
RESULTS The method used in the present work and described in more details in part I of this series allows the determination of affinities in terms of a NaCl concentration which dissociates the DNA-polypeptide complex. Applying DNAs of extremly different base pair composition on the same polypeptide-agarose column, the resulting differences in the NaCl concentration (A AT-values) found for the elution profiles of these DNAs can serve as a measure for base pair specificities. As an example Fig. 1 shows the elution profiles obtained for a polylysine and a (Lys88, Phe12)n-Sepharose column.-From both columns the AT-rich CP-DNA (69 mole%) was eluted at higher ionic strength than ML-DNA (25 mole% AT), exhibiting AT-specificity for both polypeptides. The incorporation of 12 mole% Phe residues into polylysine reduces both the affinity and AT-specificity towards DNA. These results are in agreement with those obtained from the lysine copolypeptides containing aliphatic amino acid residues, and are apparently caused by the dilution of the positive -
OD260
(Lys88, Phel2)n
0.10
Polylysine 0.06
0.02
1.0
2.0
3.0 NaCi(M)
Figure 1: Comparison of the affinities and base pair specificities of polylysine and poly(Lys8, Phe') towards DNA. The elution profiles of ML-DNA (o) and CP-DNA (s) obtained with Sepharose 4B coated with the respective polypeptides are shown. 515
Nucleic Acids Research charges along the basic polymer. Gaps in the lysine matrix obviously reduce both affinity towards DNA and the AT-specificity of the lysine
side chains. Enhanced incorporation of neutral residues into polylysine results in further and continuous decrease in the DNA affinity, when aliphatic amino acid residues are concerned (i. e. alanine, Fig. 2); however in the case of the aromatic amino acid residues affinity increases starting at about 30 mole%. This is illustrated with the Phe and Tyr copolymers in Fig. 2, and the extracted data from the different copolypeptides are listed in Table 1. a
The order of increasing affinities paralells the order of AT-specificity: Ala
