Accepted Manuscript Title: THE INTERACTION OF AMINO ACIDS, PEPTIDES, AND PROTEINS WITH DNA Author: Andrey Y. Solovyev Svetlana I. Tarnovskaya Irina A. Chernova Larisa K. Shataeva Yury A. Skorik PII: DOI: Reference:
S0141-8130(15)00206-8 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.03.054 BIOMAC 4988
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
9-1-2015 19-3-2015 20-3-2015
Please cite this article as: A.Y. Solovyev, S.I. Tarnovskaya, I.A. Chernova, L.K. Shataeva, Y.A. Skorik, THE INTERACTION OF AMINO ACIDS, PEPTIDES, AND PROTEINS WITH DNA, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.03.054 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Abstract Amino acids that carry charges on their side groups can bind to double stranded DNA (dsDNA) and change the strength of the double helix. Measurement of the DNA melting temperature (Tm) confirmed that acidic amino acids (Glu, Asp) weaken the H-bonds between DNA strands, whereas basic amino acids (Arg, Lys) strengthen the interaction
ip t
between the strands. A rank correlation exists between the amino acid isoelectric points and the observed changes in Tm. A similar dependence of the hyperchromic effect on the isoelectric point of a protein (pepsin, insulin, cortexin, and protamine) was observed for
cr
DNA-protein complexes at room temperature. Short peptides (KE, AEDG, and KEDP) containing a mixture of acidic and basic amino acid residues also affect Tm and the
us
stability of the double helix. A model for binding Glu and Lys to dsDNA was explored by a docking simulation. The model shows that Glu, in an untwisted shape, binds to dsDNA in
an
its major groove and disrupts three H-bonds between the strands, thereby destabilizing the double helix. Lys, in an untwisted shape, binds to the external side of the dsDNA and forms two bonds with O atoms of neighboring phosphodiester groups, thereby
M
strengthening the DNA helix.
Ac ce p
te
d
Keywords: amino acid, regulatory peptide, protein, DNA binding, melting temperature
1 Page 1 of 26
THE INTERACTION OF AMINO ACIDS, PEPTIDES, AND PROTEINS WITH DNA Andrey Y. Solovyev1, Svetlana I. Tarnovskaya2, Irina A. Chernova1, Larisa K. Shataeva1, Yury A. Skorik1,3,* 1Institute
of Macromolecular Compounds of the Russian Academy of Sciences, Bolshoy pr. VO
2St.
Petersburg State Polytechnical University, Polytekhnicheskaya ul. 29, St. Petersburg, 195251 Russian Federation
Petersburg State Chemical Pharmaceutical Academy, ul. Prof. Popova 14, St. Petersburg,
us
197022 Russian Federation
cr
3St.
ip t
31, St. Petersburg, 199004 Russian Federation
an
1. Introduction
In the 1970s, much interest was generated in minor components of tissue extracts,
M
and especially low molecular weight peptides, as these displayed regulatory activities for the control of tissue-specific proliferation [1-3]. Today, the selection and purification of peptides has expanded to include searches for regulatory peptides in new native sources
d
such as wheat sprouts [4] and sea urchin eggs [5].
te
Short regulatory peptides consist of amino acid residues with charged side groups in various alternations. These peptides are advantageous in medical use for several reasons:
Ac ce p
they show precise tissue-specific selectivity, have no allergic or other side effects, and display therapeutic activity at ultrasmall doses as low as 20 ng/mL [6]. Small peptides readily penetrate cell and nuclear membranes, bind to DNA, and take part in DNA initiation, transcription, and replication processes. Extensive in vivo research and in vitro study on organotypic cultures has not yet explained the molecular mechanisms of peptide-DNA binding [7-10].
Native regulatory peptides are present in a large variety in physiological liquids and
tissue extracts and form part of the functional continuum of the eukaryotic organism [11]. The origin of the regulator peptide pool in healthy organisms became obvious with the discovery of ubiquitin-mediated protein degradation by Ciechanover, Hershko, and Rose *Corresponding author.
Tel.: + 7 812 3283224; Fax: +7 812 3286869 E-mail address: [email protected]
2 Page 2 of 26
(Nobel Prize in Chemistry 2004), who showed that a single macromolecular protein can be hydrolyzed in proteasomes in several different ways to form a variety of short peptides. This mechanism may produce peptides with biological functions that differ greatly from those of the initial macromolecule [12]. For example, short peptides in the form of autocrine hormones and neuropeptides are important for disseminating biological
ip t
information.
DNA is a macromolecular polyampholyte: the amino groups of purines and pyrimidines are the donor sites for hydrogen bonds and carry a net positive charge. At the
cr
same time, the O atoms of keto groups and the N atoms of the nucleobases serve as acceptors for hydrogen bonds. By virtue of its special structure, DNA may participate in significance in the formation of the double helix.
us
various intermolecular interactions, and particularly hydrogen bonding, which is of great
an
Research into the thermodynamics of DNA has shown that gradual increases in temperature result in a phase transition to the “helix-coil,” referred to as DNA melting [13, 14]. As a result of this phase transition, the double helix separates into two single strands
M
in the form of random coils. A similar reversible helix-coil transition also occurs at room temperature, without heating, when DNA is exposed to acidic and alkaline solutions [15].
d
This random coil transition is accompanied by an increase in absorbance at 260 nm—the
te
so-called hyperchromic effect [16].
This hyperchromic effect was also observed at neutral pH 7.1, in 1979, when natural
Ac ce p
DNA was added to a nuclear enzyme, DNA-dependent RNA polymerase [17]. The absorbance at 260 nm increased linearly with increases in enzyme concentration. This result was explained by affinity binding of the nuclear DNA and RNA polymerase through polar, hydrophobic, and hydrogen bonds, which disrupted the bonds between the DNA strands, leading to DNA melting. Research into the biochemical functions of DNA has shown that local melting of DNA
strands is necessary to initiate the processes of transcription and replication. In recent years, DNA melting studies have drawn attention to organic ligands that are capable of competing for hydrogen bonds, thereby influencing DNA melting regardless of their hydrophobicity. These components, which include amino acids and peptides, are called “osmolytes,” and may influence important cellular functions and intracellular regulation [18-20]. Several recent papers have studied the DNA binding of synthetic oligopeptides and nucleobase–containing peptides [21-23].
3 Page 3 of 26
Amino acids are the main structural components of peptides, while peptides form protein
macromolecules.
This
homology
hierarchy
preserves
the
diversity
of
intermolecular binding modes. A related question is whether amino acids, peptides, and proteins might also interact with and regulate the properties of dsDNA. A simple method for the detection of ligand binding to nucleic acids is based on the
ip t
commonly used thermal denaturation method, in which ligand binding is registered as changes in the nucleic acid melting temperature (Tm). The purpose of the present work was to study the specificity and typical molecular peculiarities of amino acids, short peptides,
cr
and proteins when interacting with DNA, by measuring hyperchromicity and DNA melting
us
temperature. 2. Experimental
an
2.1. Materials
Synthetic dsDNA poly[dA-dT]poly[dA-dT] and single strands poly[dA] and poly[dT]
M
were purchased from Sigma-Aldrich; Svedberg sedimentation coefficient S=12S20 that corresponds to MW 520000 or 825 base pairs. The dsDNA poly[dA]poly[dT] was obtained by mixing equimolar amounts of poly[dA] and poly[dT] chains. The mixture was heated to
d
50oC and then slowly cooled for 2 hours to 20oC. The dsDNA concentration was determined
te
by UV spectroscopy and was expressed as mole bp/L. The native DNA sample was calf thymus DNA from Sigma-Aldrich. The native
Ac ce p
structure of the DNA sample was controlled with a hyperchromic shift (35-40%) after heating the DNA solution for 10 minutes in boiling water, followed by cooling in ice. Amino acids were purchased from Sigma-Aldrich; synthetic oligopeptides KE, AEDG,
and AEDP were from the St. Petersburg Institute of Bioregulation and Gerontology (Russia). Porcine pepsin (MW 36400, pI 2.0), crystalline insulin (MW 12700, pI 5.2), and cortexin from calf cortex (MW ˂10000, pI 9.5) were from Samson Ltd. (St. Petersburg, Russia). Protamine sulfate (MW 700, pI 12.0) was obtained from Merck Schuchardt OHG. All other reagents were of analytical grade and were used without further purification. Peptide and protein solutions were prepared in double distilled water. Amino acid and peptide concentrations were determined with ninhydrin. Protein concentrations were calculated using UV absorbance at the characteristic wavelength of each protein. 2.2. UV melting experiments UV experiments were performed on a Shimadzu UV-1700 spectrophotometer with programmable temperature block, in 10 mm quartz cells. DNA stock solutions at a 4 Page 4 of 26
concentration of 0.05 mg/mL were prepared in distilled water and were stored at -18°C. The DNA concentrations were determined by absorbance at 260 nm, while peptide concentrations were determined with ninhydrin. DNA solutions for melting curves and hyperchromicity measurements were prepared from stock solutions in 2 mM Tris buffer + as the molar concentration of AT base pairs.
ip t
15 mM NaCl (ionic strength 0.017 M) at pH 7.0-7.1. The DNA concentration was expressed UV melting curves were recorded in the temperature range of 20–70 °C for the the maximum of the first derivative of the melting curve. 2.3. Molecular modeling
cr
heating mode, with a temperature rate increase of 0.6–1.6oC·min-1. Tm was calculated using
us
The MOE 2012.10 software (Molecular Operating Environment 2012, Chemical Computing Group, Inc.) was used for molecular modeling. A sequence AAAA was built as a
an
fragment of B-DNA. Amino acids Glu and Lys also were built. All molecules were protonated at pH 7, T 300 K, NaCl concentration 0.017 M, and minimized in force field MMFF94x. The generalized Born model was used for implicit water simulation. Docking was used to
M
predict the preferred orientation of the amino acids within the DNA. The DNA was tethered, while amino acids were flexible. The full molecule of DNA was chosen as an active
Ac ce p
3. Results
te
energy.
d
site. London dG was used as the first and second scoring functions for prediction of bond
3.1. DNA thermal degradation in the presence of L-amino acids A number of L-amino acids with various isoelectric points (pI), namely Arg, Lys, Pro,
Glu, and Asp, were used at 300 µM concentration to study their interactions with dsDNA. Fig. 1 shows melting curves of poly[dA]poly[dT] at 260 nm in the presence of different amino acids; Tm values are listed in Table 1. Fig. 1. Table 1. The hyperchromic effect (Hyp) was evaluated from the difference in absorbance at 260 nm before and after phase transition (Table 1). A reversible transition is usually assumed to occur between double stranded and single stranded DNA (ssDNA), and .the molar fractions of ssDNA and dsDNA at temperature T are α and (1–α), respectively. The 5 Page 5 of 26
ratio of molar fractions (α/1-α) represents the equilibrium coefficient Ke at a given temperature. At α=0.5, T equals to Tm and standard state free energy changes ΔGm0 for DNA melting were calculated using the following equation [24]: , where [DNA] is the molar concentration of AT base pairs, Tm is the melting temperature in
ip t
K, and R is the gas constant 8.314 Jmol-1.
cr
The van’t Hoff melting enthalpy was calculated as:
The van’t Hoff entropy is given by:
us
[25]. All numerical data are listed in Table 1.
The comparison of DNA melting temperatures in the presence of amino acids reveals
an
a small increase in Tm in the case of basic amino acids (Arg, Lys), whereas the acidic amino acids (Glu, Asp) decrease the Tm. A neutral amino acid (Pro) caused a small decrease in the
M
Tm value. Thus, DNA melting depends on the net charge of the amino acid at a given pH, which is related to the isoelectric point of the amino acid. We observed that the Tm of DNA did not show a linear dependence on the pI value of the amino acid; therefore, we used a
d
nonparametric rank correlation method, which indicates how well the relationship
te
between two variables can be described using a monotonic function. The calculated Spearman’s coefficient (r = 0.90; p
S0141-8130(15)00206-8 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.03.054 BIOMAC 4988
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
9-1-2015 19-3-2015 20-3-2015
Please cite this article as: A.Y. Solovyev, S.I. Tarnovskaya, I.A. Chernova, L.K. Shataeva, Y.A. Skorik, THE INTERACTION OF AMINO ACIDS, PEPTIDES, AND PROTEINS WITH DNA, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.03.054 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Abstract Amino acids that carry charges on their side groups can bind to double stranded DNA (dsDNA) and change the strength of the double helix. Measurement of the DNA melting temperature (Tm) confirmed that acidic amino acids (Glu, Asp) weaken the H-bonds between DNA strands, whereas basic amino acids (Arg, Lys) strengthen the interaction
ip t
between the strands. A rank correlation exists between the amino acid isoelectric points and the observed changes in Tm. A similar dependence of the hyperchromic effect on the isoelectric point of a protein (pepsin, insulin, cortexin, and protamine) was observed for
cr
DNA-protein complexes at room temperature. Short peptides (KE, AEDG, and KEDP) containing a mixture of acidic and basic amino acid residues also affect Tm and the
us
stability of the double helix. A model for binding Glu and Lys to dsDNA was explored by a docking simulation. The model shows that Glu, in an untwisted shape, binds to dsDNA in
an
its major groove and disrupts three H-bonds between the strands, thereby destabilizing the double helix. Lys, in an untwisted shape, binds to the external side of the dsDNA and forms two bonds with O atoms of neighboring phosphodiester groups, thereby
M
strengthening the DNA helix.
Ac ce p
te
d
Keywords: amino acid, regulatory peptide, protein, DNA binding, melting temperature
1 Page 1 of 26
THE INTERACTION OF AMINO ACIDS, PEPTIDES, AND PROTEINS WITH DNA Andrey Y. Solovyev1, Svetlana I. Tarnovskaya2, Irina A. Chernova1, Larisa K. Shataeva1, Yury A. Skorik1,3,* 1Institute
of Macromolecular Compounds of the Russian Academy of Sciences, Bolshoy pr. VO
2St.
Petersburg State Polytechnical University, Polytekhnicheskaya ul. 29, St. Petersburg, 195251 Russian Federation
Petersburg State Chemical Pharmaceutical Academy, ul. Prof. Popova 14, St. Petersburg,
us
197022 Russian Federation
cr
3St.
ip t
31, St. Petersburg, 199004 Russian Federation
an
1. Introduction
In the 1970s, much interest was generated in minor components of tissue extracts,
M
and especially low molecular weight peptides, as these displayed regulatory activities for the control of tissue-specific proliferation [1-3]. Today, the selection and purification of peptides has expanded to include searches for regulatory peptides in new native sources
d
such as wheat sprouts [4] and sea urchin eggs [5].
te
Short regulatory peptides consist of amino acid residues with charged side groups in various alternations. These peptides are advantageous in medical use for several reasons:
Ac ce p
they show precise tissue-specific selectivity, have no allergic or other side effects, and display therapeutic activity at ultrasmall doses as low as 20 ng/mL [6]. Small peptides readily penetrate cell and nuclear membranes, bind to DNA, and take part in DNA initiation, transcription, and replication processes. Extensive in vivo research and in vitro study on organotypic cultures has not yet explained the molecular mechanisms of peptide-DNA binding [7-10].
Native regulatory peptides are present in a large variety in physiological liquids and
tissue extracts and form part of the functional continuum of the eukaryotic organism [11]. The origin of the regulator peptide pool in healthy organisms became obvious with the discovery of ubiquitin-mediated protein degradation by Ciechanover, Hershko, and Rose *Corresponding author.
Tel.: + 7 812 3283224; Fax: +7 812 3286869 E-mail address: [email protected]
2 Page 2 of 26
(Nobel Prize in Chemistry 2004), who showed that a single macromolecular protein can be hydrolyzed in proteasomes in several different ways to form a variety of short peptides. This mechanism may produce peptides with biological functions that differ greatly from those of the initial macromolecule [12]. For example, short peptides in the form of autocrine hormones and neuropeptides are important for disseminating biological
ip t
information.
DNA is a macromolecular polyampholyte: the amino groups of purines and pyrimidines are the donor sites for hydrogen bonds and carry a net positive charge. At the
cr
same time, the O atoms of keto groups and the N atoms of the nucleobases serve as acceptors for hydrogen bonds. By virtue of its special structure, DNA may participate in significance in the formation of the double helix.
us
various intermolecular interactions, and particularly hydrogen bonding, which is of great
an
Research into the thermodynamics of DNA has shown that gradual increases in temperature result in a phase transition to the “helix-coil,” referred to as DNA melting [13, 14]. As a result of this phase transition, the double helix separates into two single strands
M
in the form of random coils. A similar reversible helix-coil transition also occurs at room temperature, without heating, when DNA is exposed to acidic and alkaline solutions [15].
d
This random coil transition is accompanied by an increase in absorbance at 260 nm—the
te
so-called hyperchromic effect [16].
This hyperchromic effect was also observed at neutral pH 7.1, in 1979, when natural
Ac ce p
DNA was added to a nuclear enzyme, DNA-dependent RNA polymerase [17]. The absorbance at 260 nm increased linearly with increases in enzyme concentration. This result was explained by affinity binding of the nuclear DNA and RNA polymerase through polar, hydrophobic, and hydrogen bonds, which disrupted the bonds between the DNA strands, leading to DNA melting. Research into the biochemical functions of DNA has shown that local melting of DNA
strands is necessary to initiate the processes of transcription and replication. In recent years, DNA melting studies have drawn attention to organic ligands that are capable of competing for hydrogen bonds, thereby influencing DNA melting regardless of their hydrophobicity. These components, which include amino acids and peptides, are called “osmolytes,” and may influence important cellular functions and intracellular regulation [18-20]. Several recent papers have studied the DNA binding of synthetic oligopeptides and nucleobase–containing peptides [21-23].
3 Page 3 of 26
Amino acids are the main structural components of peptides, while peptides form protein
macromolecules.
This
homology
hierarchy
preserves
the
diversity
of
intermolecular binding modes. A related question is whether amino acids, peptides, and proteins might also interact with and regulate the properties of dsDNA. A simple method for the detection of ligand binding to nucleic acids is based on the
ip t
commonly used thermal denaturation method, in which ligand binding is registered as changes in the nucleic acid melting temperature (Tm). The purpose of the present work was to study the specificity and typical molecular peculiarities of amino acids, short peptides,
cr
and proteins when interacting with DNA, by measuring hyperchromicity and DNA melting
us
temperature. 2. Experimental
an
2.1. Materials
Synthetic dsDNA poly[dA-dT]poly[dA-dT] and single strands poly[dA] and poly[dT]
M
were purchased from Sigma-Aldrich; Svedberg sedimentation coefficient S=12S20 that corresponds to MW 520000 or 825 base pairs. The dsDNA poly[dA]poly[dT] was obtained by mixing equimolar amounts of poly[dA] and poly[dT] chains. The mixture was heated to
d
50oC and then slowly cooled for 2 hours to 20oC. The dsDNA concentration was determined
te
by UV spectroscopy and was expressed as mole bp/L. The native DNA sample was calf thymus DNA from Sigma-Aldrich. The native
Ac ce p
structure of the DNA sample was controlled with a hyperchromic shift (35-40%) after heating the DNA solution for 10 minutes in boiling water, followed by cooling in ice. Amino acids were purchased from Sigma-Aldrich; synthetic oligopeptides KE, AEDG,
and AEDP were from the St. Petersburg Institute of Bioregulation and Gerontology (Russia). Porcine pepsin (MW 36400, pI 2.0), crystalline insulin (MW 12700, pI 5.2), and cortexin from calf cortex (MW ˂10000, pI 9.5) were from Samson Ltd. (St. Petersburg, Russia). Protamine sulfate (MW 700, pI 12.0) was obtained from Merck Schuchardt OHG. All other reagents were of analytical grade and were used without further purification. Peptide and protein solutions were prepared in double distilled water. Amino acid and peptide concentrations were determined with ninhydrin. Protein concentrations were calculated using UV absorbance at the characteristic wavelength of each protein. 2.2. UV melting experiments UV experiments were performed on a Shimadzu UV-1700 spectrophotometer with programmable temperature block, in 10 mm quartz cells. DNA stock solutions at a 4 Page 4 of 26
concentration of 0.05 mg/mL were prepared in distilled water and were stored at -18°C. The DNA concentrations were determined by absorbance at 260 nm, while peptide concentrations were determined with ninhydrin. DNA solutions for melting curves and hyperchromicity measurements were prepared from stock solutions in 2 mM Tris buffer + as the molar concentration of AT base pairs.
ip t
15 mM NaCl (ionic strength 0.017 M) at pH 7.0-7.1. The DNA concentration was expressed UV melting curves were recorded in the temperature range of 20–70 °C for the the maximum of the first derivative of the melting curve. 2.3. Molecular modeling
cr
heating mode, with a temperature rate increase of 0.6–1.6oC·min-1. Tm was calculated using
us
The MOE 2012.10 software (Molecular Operating Environment 2012, Chemical Computing Group, Inc.) was used for molecular modeling. A sequence AAAA was built as a
an
fragment of B-DNA. Amino acids Glu and Lys also were built. All molecules were protonated at pH 7, T 300 K, NaCl concentration 0.017 M, and minimized in force field MMFF94x. The generalized Born model was used for implicit water simulation. Docking was used to
M
predict the preferred orientation of the amino acids within the DNA. The DNA was tethered, while amino acids were flexible. The full molecule of DNA was chosen as an active
Ac ce p
3. Results
te
energy.
d
site. London dG was used as the first and second scoring functions for prediction of bond
3.1. DNA thermal degradation in the presence of L-amino acids A number of L-amino acids with various isoelectric points (pI), namely Arg, Lys, Pro,
Glu, and Asp, were used at 300 µM concentration to study their interactions with dsDNA. Fig. 1 shows melting curves of poly[dA]poly[dT] at 260 nm in the presence of different amino acids; Tm values are listed in Table 1. Fig. 1. Table 1. The hyperchromic effect (Hyp) was evaluated from the difference in absorbance at 260 nm before and after phase transition (Table 1). A reversible transition is usually assumed to occur between double stranded and single stranded DNA (ssDNA), and .the molar fractions of ssDNA and dsDNA at temperature T are α and (1–α), respectively. The 5 Page 5 of 26
ratio of molar fractions (α/1-α) represents the equilibrium coefficient Ke at a given temperature. At α=0.5, T equals to Tm and standard state free energy changes ΔGm0 for DNA melting were calculated using the following equation [24]: , where [DNA] is the molar concentration of AT base pairs, Tm is the melting temperature in
ip t
K, and R is the gas constant 8.314 Jmol-1.
cr
The van’t Hoff melting enthalpy was calculated as:
The van’t Hoff entropy is given by:
us
[25]. All numerical data are listed in Table 1.
The comparison of DNA melting temperatures in the presence of amino acids reveals
an
a small increase in Tm in the case of basic amino acids (Arg, Lys), whereas the acidic amino acids (Glu, Asp) decrease the Tm. A neutral amino acid (Pro) caused a small decrease in the
M
Tm value. Thus, DNA melting depends on the net charge of the amino acid at a given pH, which is related to the isoelectric point of the amino acid. We observed that the Tm of DNA did not show a linear dependence on the pI value of the amino acid; therefore, we used a
d
nonparametric rank correlation method, which indicates how well the relationship
te
between two variables can be described using a monotonic function. The calculated Spearman’s coefficient (r = 0.90; p
