DOI 10.1515/bmc-2013-0037 BioMol Concepts 2014; 5(1): 21–43
Review Subrata Batabyal, Susobhan Choudhury, Dilip Sao, Tanumoy Mondol and Samir Kumar Pal*
Dynamical perspective of protein-DNA interaction Abstract: The interactions between protein-DNA are essential for various biological activities. In this review, we provide an overview of protein-DNA interactions that emphasizes the importance of dynamical aspects. We divide protein-DNA interactions into two categories: nonspecific and specific and both the categories would be discussed highlighting some of our relevant work. In the case of nonspecific protein-DNA interaction, solvation studies (picosecond and femtosecond-resolved) explore the role environmental dynamics and change in the micropolarity around DNA molecules upon complexation with histone protein (H1). While exploring the specific protein-DNA interaction at λ-repressor-operator sites interaction, particularly OR1 and OR2, it was observed that the interfacial water dynamics is minimally perturbed upon interaction with DNA, suggesting the labile interface in the proteinDNA complex. Förster resonance energy transfer (FRET) study revealed that the structure of the protein is more compact in repressor-OR2 complex than in the repressorOR1 complex. Fluorescence anisotropy studies indicated enhanced flexibility of the C-terminal domain of the repressor at fast timescales after complex formation with OR1. The enhanced flexibility and different conformation of the C-terminal domain of the repressor upon complexation with OR1 DNA compared to OR2 DNA were found to have pronounced effect on the rate of photoinduced electron transfer. Keywords: different dynamical conformations; molecular recognition; operator DNA; photoinduced electron transfer; protein-DNA interactions; protein-DNA interface; resonance energy transfer; solvation dynamics. *Corresponding author: Samir Kumar Pal, Department of Chemical, Biological and Macromolecular Sciences, S. N. Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Kolkata 700 098, India, e-mail: [email protected] Subrata Batabyal, Susobhan Choudhury, Dilip Sao and Tanumoy Mondol: Department of Chemical, Biological and Macromolecular Sciences, S. N. Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Kolkata 700 098, India
Introduction Protein and DNA are two of the most important biomolecules in any living organism (1–5). DNA stores the genetic information, while protein takes care of executing and regulating the life processes. Protein-DNA interactions therefore play a crucial role in central biological processes, ranging from the mechanism of replication, transcription and recombination to enzymatic events utilizing nucleic acids as substrates (6–8). Protein-DNA interaction can be broadly classified into two types – nonspecific and specific interaction (9). Structural proteins that bind DNA are well-understood examples of nonspecific DNA-protein interactions. From a mechanistic standpoint, the non-sequence-specific interaction between nucleic acids and proteins can be qualitatively understood on the basis of relatively simple physicochemical principle. Histone (protein)-DNA interactions are an example of such interactions, and they occur between functional groups on the protein and the sugar-phosphate backbone of DNA. In the case of nonspecific interactions, the sequence of nucleotides does not matter, as far as the binding interactions are concerned. Through nonspecific interaction, structural proteins help to organize DNA into compact ‘chromatin’ form (Figure 1). Specific DNA-protein interactions, however, depend upon the sequence of bases in the DNA. Specific protein-DNA interaction (9) is governed by sequence recognition (base readout) through direct contacts between protein’s amino acid side chains and DNA bases (10), as well as the spatial geometry of the two biomolecules (shape readout) (10). Recognition of specific DNA sequences by specific DNA-binding proteins often leads to the formation of large protein complexes responsible for regulation of gene expression. Biological function of the protein-DNA conjugates often depend on the conformational dynamics, local solvation, and micropolarity after complexation. In a variety of biological processes, the conformational flexibility of the protein upon DNA binding is the key signal for the further propagation of the process. The molecular bases of the remarkably accurate recognition of particular nucleotide sequences by site-specific proteins comprise one of the major
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22 S. Batabyal et al.: Dynamics of protein-DNA interactions
Figure 1 Schematic representation of non-specific (DNA-histone I) and specific protein-DNA (operator DNA with lambda/crorepressor) interaction and their implications.
unsolved puzzles of present day molecular biology. A long-standing but yet unattained objective in structural biology is to elucidate the physicochemical basis of the specific and nonspecific interaction between various protein-protein and protein-DNA (biomolecular recognition), leading to the regulation of gene expression. In this regard, dynamics of biomolecular recognition is of crucial importance. Many factors may contribute in this process of complex recognition process. Recent works on specific and nonspecific protein-DNA interactions have demonstrated effects of binding rigidity, local solvation and micropolarity on the biological function of the complexes (11–17). In the recent past, there has been some advancement in the exploration of the dynamical perspective of protein-DNA interaction. An interesting series of works have been published by Kalodimos et al. on the binding of DNA to several variants of the catabolite activator protein (CAP), which differentially populate the inactive and active DNA-binding domain states (18, 19). The allosteric control of the CAP protein has been comprehensively elaborated in several of their recent publications (20) and interested readers are highly encouraged to go through the original publications. DNA-protein interactions are studied using a variety of methods such as gel-shift assays, foot-printing, NMR structural studies (21), X-ray crystallographic studies (21) and fluorescence spectroscopy. Among all the possible methodologies, fluorescence polarization/anisotropy provides a rapid, non-radioactive method for accurately measuring DNA-protein interaction directly in solution. In this review, we provide a revised view of proteinDNA interactions that emphasizes the importance of the dynamical aspects and conformational changes of both macromolecules. The experimental techniques are mainly based on fluorescence spectroscopy. In order to reassess protein-DNA interaction, we have divided this review into two main sections. For the nonspecific protein-DNA
interaction, the interaction of histone protein with DNA is elaborated in great detail (17, 22). The studies of the nature of binding interactions and dynamics of protein histone I (H1) with ligands in solution and as a complex with DNA, an important biological process for the higher-order structure in chromatin are discussed. While elaborating the important aspects of specific protein-DNA interaction, the complexation of lambda repressor protein with different operators is explored (Figure 1). Structurally λ-repressor is a two domain protein. The N-terminal domain interacts with the DNA (16), while the C-terminal domain is responsible for most protein-protein interactions (23); these interactions are essential for the co-operative binding (24, 25) and for the functioning of the genetic switch (11, 26).
Methodology In this review, the experimentally obtained results are the outcome of the various florescence spectroscopic tool and analytical methods. Steady-state absorption and emission spectrophotometers were used to measure the sample concentration and steady-state fluorescence emission. More sophisticated time-resolved fluorescence techniques were used to monitor dynamical aspects in the femtosecond to picosecond time domain (17, 22). Details of the time-resolved techniques are available in the published literature (27, 28). In order to explore biomolecular recognition, microfluidics approach coupled to fluorescence microscopy was employed (27, 29). The most useful technique, often termed as the molecular ruler, FRET (30, 31), was used to monitor the distance between donor and acceptor molecules. Furthermore solvation analysis of the excited fluoroprobe was used to envisage the immediate environments and hence to detect the effect of the interaction of protein-DNA (31).
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S. Batabyal et al.: Dynamics of protein-DNA interactions 23
Results and discussion Nonspecific protein-DNA interaction In this section we have highlighted the structural and dynamical aspects of DNAs from various sources including synthetic oligonucleotides and as a complex with histone1 (H1) in the nonspecific protein-DNA interaction.
Picosecond-resolved dynamics at the protein-DNA interface: histone-DNA complexes in aquesous solution (22) Genomic DNAs extracted from calf functional thymus and salmon testes are known to form complexes with the linker histone H1 through a nonspecific interaction. The schematic representation of the complexes is shown in Figure 1. Though both calf thymus and salmon sperm DNA interact with H1, the ultimate structure of the complexes differs substantially. TEM study reveals the compact nature of the calf thymus DNA-H1 complex (DNA and protein are from the same species; intra-species) compared to that of the salmon sperm DNA-H1 (interspecies) and were found to be caused by the greater compactness of intra-species DNA condensates than that of the interspecies DNA condensates. In order to provide detailed dynamical aspects of such protein-DNA condensates, minor groove binder dye Hoechst 33258 (H33258) was used. Figure 2(A) shows the emission spectra of the probe H33258 in the calf thymus DNA and DNA-H1 complex. In hydrophobic environments the dye shows blue shift (32). The emission maxima of the ligand in buffer and in DNA are 515 and 470 nm, respectively. The ligand bound to the DNA-H1 complex shows an emission maximum at 460 nm, indicating more non-polar environments of the probe in the DNA upon complexation with H1. The fluorescence transients of H33258-calf thymus DNA in buffer at three characteristic wavelengths are shown in Figure 2(B). The numerical fittings (solid lines) of the data are made up to 35 ns. The change in the transient in the blue side of the emission spectra from that in the red side, are shown in the 6 ns time window. The transients with a systematic series of wavelength detection from 410 to 600 nm with a 10 nm regular wavelength interval show three distinct time scales of ~ 150 ps, ~ 2.2 ns, and ~ 4 ns. The blue wavelength (410 nm) decays with 150 ps time constant and rises with a similar time constant at the red side (600 nm), which is the signature of solvation dynamics (33). Other two time constants remain similar
in all of the wavelengths of detection. The time-resolved emission spectra (TRES) of the H33258-calf thymus DNA complex up to 15 ns is shown in Figure 2(C). The net spectral shift is 1715/cm (from 21 514 to 19 799/cm in 35 ns). It should be noted that the emission peak of H33258 in nonpolar dioxane is 22 935/cm (34). The difference of the peak value of H33258 in DNA environments at t = 0 from that of the peak in the non-polar medium reflects the missing dynamics of the corresponding environments within the instrumental resolution (35). Figure 2(D) demonstrates that the time evolution of the spectral peak frequency of H33258 attached to calf thymus DNA and in the DNA-H1 complex. The data points are fitted with a biexponential decay function yielding different solvent relaxation time constants for the DNA in different environments. The dynamics of the H33258-calf thymus DNA complex shows two distinct time constants of 250 ps and 13.50 ns. The time scales obtained from our experiment are similar to the relaxation times seen in the coumarin-modified DNA (36). The time constants are much slower than the low-frequency vibrational motion of DNA or quasiharmonic oscillation of a fluorophore within a static DNA structure and are close to the diffusive reorganization of a liquid (36). The faster time constant of 250 ps may be caused by the relaxation of the DNA structure. The longer time constant (13.50 ns) arises from the diffusive motion. As evidenced in Figure 2(E), H33258 in salmon sperm DNA shows two dynamical time constants of 70 ps and 13.60 ns. The dynamical time constants of H33258 in DNA-H1 are 60 ps and 10.44 ns, which are similar to that of the DNA without H1. The solvation features in the different environments are similar to that in the respective environments of the calf thymus DNA. Structural X-ray crystallographic and NMR studies (37) on the ligand with duplex1 show that the specific sequence of (CGCAAATTTGCG) of the DNA is extremely important for the ligand H33258 binding. Solvation dynamics study (Figure 2F) of the ligand bound oligonucleotide reveals two time constants of 60 ps and 8.50 ns. In the duplex1-H1 complex, the time constants change to 120 ps and 25 ns. The observation of slower relaxation dynamics of the probe in the duplex1-H1 complex is not consistent with those in the genomic DNAs. The poly GC sequence of duplex2 (GCGCGCGCGCGC)2 is ideal for the intercalative mode of EtBr-binding (38). The significant change in the fluorescence intensity and lifetime of the probe EtBr upon binding to DNA and DNA-H1 complex is used to explore the perturbation of intercalative interaction of the ligand in the DNA condensate. The significant decrease in emission intensity and shift toward the red wavelength (622 nm) of EtBr in the DNA-H1 complex compared to EtBr-DNA in the bulk buffer
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24 S. Batabyal et al.: Dynamics of protein-DNA interactions
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Figure 2 Solvation relaxation of H33258 in DNA and in DNA-Histone complex. (A) Steady-state emission spectra of H33258 in calf thymus DNA and the DNA-H1 complex. (B) Fluorescence transients of H33258-calf thymus DNA at three different detection wavelengths. (C) Time-resolved emission spectra of H33258 in calf thymus DNA. Time-resolved fluorescence spectral peak frequency of H33258 in (D) calf thymus DNA and DNA-H1 complex, (E) salmon sperm DNA and its condensate, and (F) duplex1 and duplex1-H1 complex are shown. Solid lines indicate biexponential numerical fitting of the experimental data points. [Reprinted with permission from ref. (22) Copyright 2007, American Chemical Society.]
as evidenced from Figure 3(A) is indicative of the reduction of binding strength of EtBr-binding to the DNA upon complexation with H1. The observations find similarity with other reports in the literature (39). Picosecondresolved transients of EtBr-calf thymus DNA complexes are shown in Figure 3(B). As shown in Figure 3(B) the
maximum population [22 ns (73%)] of the dye is intercalated to the DNA. The transient of EtBr in the DNA-H1 complex shows that the contribution of a longer ~ 22 ns (2%) decay component decreases considerably, reflecting significant decrease in the binding affinity of EtBr in the DNA condensate. The binding constants of EtBr in
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S. Batabyal et al.: Dynamics of protein-DNA interactions 25
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Figure 3 FRET study in DNA-Histone I complex. (A) Steady-state emission spectra of EtBr in calf thymus DNA and DNA-H1 complex are shown. Picosecond-resolved fluorescence transients of EtBr in (B) calf thymus DNA, (C) salmon sperm DNA, and (D) duplex2 and their condensates are shown. Solid lines indicate biexponential numerical fitting of the experimental data points. [Reprinted with permission from ref. (22) Copyright 2007, American Chemical Society.]
the DNA and DNA-H1 complex are shown in Table 1. In order to investigate the ligand binding of the interspecies DNA-H1 complex, the complexation of EtBr-salmon sperm DNA with calf thymus H1 was investigated. The decrease in fluorescence intensity (data not shown) and change of the contribution of a longer [22 ns (34%)] time constant of the EtBr-DNA-H1 complex [Figure 3(C) and Table 1] are less than that of the calf thymus DNA condensate. The observation is consistent with the fact that the intercalative interaction of EtBr with the salmon sperm DNA condensate is much stronger than that with the calf thymus DNA condensate. The stronger interaction in the salmon sperm DNA condensate is correlated with the less-compact structure of the DNA-H1 complex as evidenced from TEM experiments. In order to study the effect of a base pair sequence in the molecular recognition of the DNA-H1 complex, oligonucleotide sequence of duplex2 was deliberately chosen. EtBr strongly intercalates to GC enriched homo oligonucleotides. From Figure 3(D) and Table 1, it is
evident that the perturbation of EtBr-binding (change in 22 ns component) to the duplex2 upon complexation with H1 is less compared to both calf thymus and salmon sperm DNA condensates. From Table 1, it is evident that the faster time constant (
Review Subrata Batabyal, Susobhan Choudhury, Dilip Sao, Tanumoy Mondol and Samir Kumar Pal*
Dynamical perspective of protein-DNA interaction Abstract: The interactions between protein-DNA are essential for various biological activities. In this review, we provide an overview of protein-DNA interactions that emphasizes the importance of dynamical aspects. We divide protein-DNA interactions into two categories: nonspecific and specific and both the categories would be discussed highlighting some of our relevant work. In the case of nonspecific protein-DNA interaction, solvation studies (picosecond and femtosecond-resolved) explore the role environmental dynamics and change in the micropolarity around DNA molecules upon complexation with histone protein (H1). While exploring the specific protein-DNA interaction at λ-repressor-operator sites interaction, particularly OR1 and OR2, it was observed that the interfacial water dynamics is minimally perturbed upon interaction with DNA, suggesting the labile interface in the proteinDNA complex. Förster resonance energy transfer (FRET) study revealed that the structure of the protein is more compact in repressor-OR2 complex than in the repressorOR1 complex. Fluorescence anisotropy studies indicated enhanced flexibility of the C-terminal domain of the repressor at fast timescales after complex formation with OR1. The enhanced flexibility and different conformation of the C-terminal domain of the repressor upon complexation with OR1 DNA compared to OR2 DNA were found to have pronounced effect on the rate of photoinduced electron transfer. Keywords: different dynamical conformations; molecular recognition; operator DNA; photoinduced electron transfer; protein-DNA interactions; protein-DNA interface; resonance energy transfer; solvation dynamics. *Corresponding author: Samir Kumar Pal, Department of Chemical, Biological and Macromolecular Sciences, S. N. Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Kolkata 700 098, India, e-mail: [email protected] Subrata Batabyal, Susobhan Choudhury, Dilip Sao and Tanumoy Mondol: Department of Chemical, Biological and Macromolecular Sciences, S. N. Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Kolkata 700 098, India
Introduction Protein and DNA are two of the most important biomolecules in any living organism (1–5). DNA stores the genetic information, while protein takes care of executing and regulating the life processes. Protein-DNA interactions therefore play a crucial role in central biological processes, ranging from the mechanism of replication, transcription and recombination to enzymatic events utilizing nucleic acids as substrates (6–8). Protein-DNA interaction can be broadly classified into two types – nonspecific and specific interaction (9). Structural proteins that bind DNA are well-understood examples of nonspecific DNA-protein interactions. From a mechanistic standpoint, the non-sequence-specific interaction between nucleic acids and proteins can be qualitatively understood on the basis of relatively simple physicochemical principle. Histone (protein)-DNA interactions are an example of such interactions, and they occur between functional groups on the protein and the sugar-phosphate backbone of DNA. In the case of nonspecific interactions, the sequence of nucleotides does not matter, as far as the binding interactions are concerned. Through nonspecific interaction, structural proteins help to organize DNA into compact ‘chromatin’ form (Figure 1). Specific DNA-protein interactions, however, depend upon the sequence of bases in the DNA. Specific protein-DNA interaction (9) is governed by sequence recognition (base readout) through direct contacts between protein’s amino acid side chains and DNA bases (10), as well as the spatial geometry of the two biomolecules (shape readout) (10). Recognition of specific DNA sequences by specific DNA-binding proteins often leads to the formation of large protein complexes responsible for regulation of gene expression. Biological function of the protein-DNA conjugates often depend on the conformational dynamics, local solvation, and micropolarity after complexation. In a variety of biological processes, the conformational flexibility of the protein upon DNA binding is the key signal for the further propagation of the process. The molecular bases of the remarkably accurate recognition of particular nucleotide sequences by site-specific proteins comprise one of the major
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22 S. Batabyal et al.: Dynamics of protein-DNA interactions
Figure 1 Schematic representation of non-specific (DNA-histone I) and specific protein-DNA (operator DNA with lambda/crorepressor) interaction and their implications.
unsolved puzzles of present day molecular biology. A long-standing but yet unattained objective in structural biology is to elucidate the physicochemical basis of the specific and nonspecific interaction between various protein-protein and protein-DNA (biomolecular recognition), leading to the regulation of gene expression. In this regard, dynamics of biomolecular recognition is of crucial importance. Many factors may contribute in this process of complex recognition process. Recent works on specific and nonspecific protein-DNA interactions have demonstrated effects of binding rigidity, local solvation and micropolarity on the biological function of the complexes (11–17). In the recent past, there has been some advancement in the exploration of the dynamical perspective of protein-DNA interaction. An interesting series of works have been published by Kalodimos et al. on the binding of DNA to several variants of the catabolite activator protein (CAP), which differentially populate the inactive and active DNA-binding domain states (18, 19). The allosteric control of the CAP protein has been comprehensively elaborated in several of their recent publications (20) and interested readers are highly encouraged to go through the original publications. DNA-protein interactions are studied using a variety of methods such as gel-shift assays, foot-printing, NMR structural studies (21), X-ray crystallographic studies (21) and fluorescence spectroscopy. Among all the possible methodologies, fluorescence polarization/anisotropy provides a rapid, non-radioactive method for accurately measuring DNA-protein interaction directly in solution. In this review, we provide a revised view of proteinDNA interactions that emphasizes the importance of the dynamical aspects and conformational changes of both macromolecules. The experimental techniques are mainly based on fluorescence spectroscopy. In order to reassess protein-DNA interaction, we have divided this review into two main sections. For the nonspecific protein-DNA
interaction, the interaction of histone protein with DNA is elaborated in great detail (17, 22). The studies of the nature of binding interactions and dynamics of protein histone I (H1) with ligands in solution and as a complex with DNA, an important biological process for the higher-order structure in chromatin are discussed. While elaborating the important aspects of specific protein-DNA interaction, the complexation of lambda repressor protein with different operators is explored (Figure 1). Structurally λ-repressor is a two domain protein. The N-terminal domain interacts with the DNA (16), while the C-terminal domain is responsible for most protein-protein interactions (23); these interactions are essential for the co-operative binding (24, 25) and for the functioning of the genetic switch (11, 26).
Methodology In this review, the experimentally obtained results are the outcome of the various florescence spectroscopic tool and analytical methods. Steady-state absorption and emission spectrophotometers were used to measure the sample concentration and steady-state fluorescence emission. More sophisticated time-resolved fluorescence techniques were used to monitor dynamical aspects in the femtosecond to picosecond time domain (17, 22). Details of the time-resolved techniques are available in the published literature (27, 28). In order to explore biomolecular recognition, microfluidics approach coupled to fluorescence microscopy was employed (27, 29). The most useful technique, often termed as the molecular ruler, FRET (30, 31), was used to monitor the distance between donor and acceptor molecules. Furthermore solvation analysis of the excited fluoroprobe was used to envisage the immediate environments and hence to detect the effect of the interaction of protein-DNA (31).
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S. Batabyal et al.: Dynamics of protein-DNA interactions 23
Results and discussion Nonspecific protein-DNA interaction In this section we have highlighted the structural and dynamical aspects of DNAs from various sources including synthetic oligonucleotides and as a complex with histone1 (H1) in the nonspecific protein-DNA interaction.
Picosecond-resolved dynamics at the protein-DNA interface: histone-DNA complexes in aquesous solution (22) Genomic DNAs extracted from calf functional thymus and salmon testes are known to form complexes with the linker histone H1 through a nonspecific interaction. The schematic representation of the complexes is shown in Figure 1. Though both calf thymus and salmon sperm DNA interact with H1, the ultimate structure of the complexes differs substantially. TEM study reveals the compact nature of the calf thymus DNA-H1 complex (DNA and protein are from the same species; intra-species) compared to that of the salmon sperm DNA-H1 (interspecies) and were found to be caused by the greater compactness of intra-species DNA condensates than that of the interspecies DNA condensates. In order to provide detailed dynamical aspects of such protein-DNA condensates, minor groove binder dye Hoechst 33258 (H33258) was used. Figure 2(A) shows the emission spectra of the probe H33258 in the calf thymus DNA and DNA-H1 complex. In hydrophobic environments the dye shows blue shift (32). The emission maxima of the ligand in buffer and in DNA are 515 and 470 nm, respectively. The ligand bound to the DNA-H1 complex shows an emission maximum at 460 nm, indicating more non-polar environments of the probe in the DNA upon complexation with H1. The fluorescence transients of H33258-calf thymus DNA in buffer at three characteristic wavelengths are shown in Figure 2(B). The numerical fittings (solid lines) of the data are made up to 35 ns. The change in the transient in the blue side of the emission spectra from that in the red side, are shown in the 6 ns time window. The transients with a systematic series of wavelength detection from 410 to 600 nm with a 10 nm regular wavelength interval show three distinct time scales of ~ 150 ps, ~ 2.2 ns, and ~ 4 ns. The blue wavelength (410 nm) decays with 150 ps time constant and rises with a similar time constant at the red side (600 nm), which is the signature of solvation dynamics (33). Other two time constants remain similar
in all of the wavelengths of detection. The time-resolved emission spectra (TRES) of the H33258-calf thymus DNA complex up to 15 ns is shown in Figure 2(C). The net spectral shift is 1715/cm (from 21 514 to 19 799/cm in 35 ns). It should be noted that the emission peak of H33258 in nonpolar dioxane is 22 935/cm (34). The difference of the peak value of H33258 in DNA environments at t = 0 from that of the peak in the non-polar medium reflects the missing dynamics of the corresponding environments within the instrumental resolution (35). Figure 2(D) demonstrates that the time evolution of the spectral peak frequency of H33258 attached to calf thymus DNA and in the DNA-H1 complex. The data points are fitted with a biexponential decay function yielding different solvent relaxation time constants for the DNA in different environments. The dynamics of the H33258-calf thymus DNA complex shows two distinct time constants of 250 ps and 13.50 ns. The time scales obtained from our experiment are similar to the relaxation times seen in the coumarin-modified DNA (36). The time constants are much slower than the low-frequency vibrational motion of DNA or quasiharmonic oscillation of a fluorophore within a static DNA structure and are close to the diffusive reorganization of a liquid (36). The faster time constant of 250 ps may be caused by the relaxation of the DNA structure. The longer time constant (13.50 ns) arises from the diffusive motion. As evidenced in Figure 2(E), H33258 in salmon sperm DNA shows two dynamical time constants of 70 ps and 13.60 ns. The dynamical time constants of H33258 in DNA-H1 are 60 ps and 10.44 ns, which are similar to that of the DNA without H1. The solvation features in the different environments are similar to that in the respective environments of the calf thymus DNA. Structural X-ray crystallographic and NMR studies (37) on the ligand with duplex1 show that the specific sequence of (CGCAAATTTGCG) of the DNA is extremely important for the ligand H33258 binding. Solvation dynamics study (Figure 2F) of the ligand bound oligonucleotide reveals two time constants of 60 ps and 8.50 ns. In the duplex1-H1 complex, the time constants change to 120 ps and 25 ns. The observation of slower relaxation dynamics of the probe in the duplex1-H1 complex is not consistent with those in the genomic DNAs. The poly GC sequence of duplex2 (GCGCGCGCGCGC)2 is ideal for the intercalative mode of EtBr-binding (38). The significant change in the fluorescence intensity and lifetime of the probe EtBr upon binding to DNA and DNA-H1 complex is used to explore the perturbation of intercalative interaction of the ligand in the DNA condensate. The significant decrease in emission intensity and shift toward the red wavelength (622 nm) of EtBr in the DNA-H1 complex compared to EtBr-DNA in the bulk buffer
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24 S. Batabyal et al.: Dynamics of protein-DNA interactions
5000 H33258 in calf thymus DNA
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Figure 2 Solvation relaxation of H33258 in DNA and in DNA-Histone complex. (A) Steady-state emission spectra of H33258 in calf thymus DNA and the DNA-H1 complex. (B) Fluorescence transients of H33258-calf thymus DNA at three different detection wavelengths. (C) Time-resolved emission spectra of H33258 in calf thymus DNA. Time-resolved fluorescence spectral peak frequency of H33258 in (D) calf thymus DNA and DNA-H1 complex, (E) salmon sperm DNA and its condensate, and (F) duplex1 and duplex1-H1 complex are shown. Solid lines indicate biexponential numerical fitting of the experimental data points. [Reprinted with permission from ref. (22) Copyright 2007, American Chemical Society.]
as evidenced from Figure 3(A) is indicative of the reduction of binding strength of EtBr-binding to the DNA upon complexation with H1. The observations find similarity with other reports in the literature (39). Picosecondresolved transients of EtBr-calf thymus DNA complexes are shown in Figure 3(B). As shown in Figure 3(B) the
maximum population [22 ns (73%)] of the dye is intercalated to the DNA. The transient of EtBr in the DNA-H1 complex shows that the contribution of a longer ~ 22 ns (2%) decay component decreases considerably, reflecting significant decrease in the binding affinity of EtBr in the DNA condensate. The binding constants of EtBr in
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S. Batabyal et al.: Dynamics of protein-DNA interactions 25
A
B
10 000
Emission spectra of ethidium in calf thymus DNA
DNA (Calf thymus) 1000
DNA DNA-H1
6e+3
Counts
Fluorescence intensity
8e+3
4e+3
100
DNA-H1
10
2e+3 0 550
600
650
700
1
750
0
10
20
Wavelength (nm)
C
D
10 000
10 000 DNA (Salmon sperm)
DNA (duplex2)
1000
1000 DNA-H1
Counts
Counts
40
30
Time (ns)
100
10
1
IRF
0
DNA-H1 100
10
10
20 Time (ns)
30
40
1
0
10
20 Time (ns)
30
40
Figure 3 FRET study in DNA-Histone I complex. (A) Steady-state emission spectra of EtBr in calf thymus DNA and DNA-H1 complex are shown. Picosecond-resolved fluorescence transients of EtBr in (B) calf thymus DNA, (C) salmon sperm DNA, and (D) duplex2 and their condensates are shown. Solid lines indicate biexponential numerical fitting of the experimental data points. [Reprinted with permission from ref. (22) Copyright 2007, American Chemical Society.]
the DNA and DNA-H1 complex are shown in Table 1. In order to investigate the ligand binding of the interspecies DNA-H1 complex, the complexation of EtBr-salmon sperm DNA with calf thymus H1 was investigated. The decrease in fluorescence intensity (data not shown) and change of the contribution of a longer [22 ns (34%)] time constant of the EtBr-DNA-H1 complex [Figure 3(C) and Table 1] are less than that of the calf thymus DNA condensate. The observation is consistent with the fact that the intercalative interaction of EtBr with the salmon sperm DNA condensate is much stronger than that with the calf thymus DNA condensate. The stronger interaction in the salmon sperm DNA condensate is correlated with the less-compact structure of the DNA-H1 complex as evidenced from TEM experiments. In order to study the effect of a base pair sequence in the molecular recognition of the DNA-H1 complex, oligonucleotide sequence of duplex2 was deliberately chosen. EtBr strongly intercalates to GC enriched homo oligonucleotides. From Figure 3(D) and Table 1, it is
evident that the perturbation of EtBr-binding (change in 22 ns component) to the duplex2 upon complexation with H1 is less compared to both calf thymus and salmon sperm DNA condensates. From Table 1, it is evident that the faster time constant (
