Biomacromolecules 2008, 9, 21–28
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Collagen–DNA Complex David V. Svintradze,*,†,‡ George M. Mrevlishvili,‡ Nunu Metreveli,†,‡ Ketevan Jariashvili,‡ Luisa Namicheishvili,‡ Joana Skopinska,§ and Alina Sionkowska§ Faculty of Physics and Mathematics, Ilia Chavchavadze State University, Chavchavadze Av. 32, Tbilisi 0157, Georgia, Faculty of Exact and Natural Sciences, Institute of Physics, Tbilisi State University, Chavchavadze Av. 3, Tbilisi 0128, Georgia, and Faculty of General Chemistry, N. Copernicus University, Gagarin 7, Torun 87-100, Poland Received April 5, 2007; Revised Manuscript Received September 23, 2007
Previously presented models of collagen–DNA7 and collagen–siRNA8 complexes point to a general description of delivery systems and indicate to what specific topology that system should be equipped with to effectively deliver the gene into the living body via in vivo and in vitro injection. We focused our interest on the nature of collagen–DNA complex structure and the molecular and environmental determinants of the self-association processes of collagen with the presence of DNA. In this aspect, the self-association of collagen–DNA complex offers an opportunity to characterize a unique system, which may be related to the general mechanisms of selfassociation of fiber macromolecules by water bridges. For characterizing the collagen–DNA interaction, we used FTIR-ATR, NMR, and AFM experiments done on a separate collagen film, DNA film, and on the peptide–DNA aqueous solution. We demonstrate that collagen–DNA spontaneously forms self-assembling complex systems in aqueous solution. Such self-association of the complex could be induced by electrostatic interactions between neutral collagen cylinders, having strong dipole moment, and negatively charged DNA cylinders. A final complex could be formed by hydrogen bonds between specified donor groups of collagen and phosphate acceptor groups of DNA. According to FTIR measurements, a collagen triple helix should not change global conformation during collagen–DNA complex formation.
Introduction During the last decades, analysis of the mechanism of delivering properties of type I collagen triple helices in the gene delivery systems attracted huge interest.1,2 Accordingly, it has become also very important to study the principles of organization of the molecular complex between collagen triple helices and DNA double helices. Furthermore, detailed analysis of the mechanisms of interactions between collagen triple helix and double helix of nucleic acids will lead to better understanding of the mechanisms of drug and gene delivery systems, which is crucial for gene therapy. Nowadays, there are known several types of gene delivering systems: viral-vector mediated delivery,3,4 lipid-based delivery,5,6 and atelocollagen (atelocollagen is a highly purified type I collagen of calf dermis with pepsin treatment) mediated delivery systems.1,2 All of the systems have one principal common characteristic: virus-viral delivery system has original common fittings for virus structures, where the gene is packed into some protein; lipid-based delivery, where the gene is packed in a cationic lipid bilayer, and finally, atelocollagen-mediated gene delivery, where the gene also has to be packed in collagen fibers as is demonstrated in molecular models.7,8 However, it was reported earlier that DNA causes more twisted fiber formation of collagen fibrils,9 but the mechanism of such influence of DNA on collagen aggregation is still unclear. Previously presented results suggested that the * Address for Correspondence. E-mail:
[email protected]. Telephone: +995 99 644500. Fax: +995 32 588780. David V. Svintradze, Dr. of Sci., Faculty of Exact and Natural Sciences, Institute of Physics, Tbilisi State University, Chavchavadze Av. 3, Tbilisi 0128, Georgia. † Faculty of Physics and Mathematics, Ilia Chavchavadze State University. ‡ Faculty of Exact and Natural Sciences, Institute of Physics, Tbilisi State University. § Faculty of General Chemistry, N. Copernicus University.
complex aggregation and the structure of the collagen–DNA complex depend on the DNA types (DNA types: ssDNA singlestranded DNA, dsDNA double-stranded DNA, lin-DNA linear DNA, and cycle-DNA), and the structure and mechanical properties of the complex can be effectively controlled by using various types of DNA.9 Thus the interest toward the ability of proteins and peptides to self-associate into aggregates, both in normal and pathological processes, is a field of increasing interest. Normal selfassociation processes include fibril formation of collagen, while pathological aggregation of collagen could be implicated in some collagen diseases. However, during some pathological processes, DNA could be released from the nucleus and placed in the matrix between the cells in the connective tissue.10,11 It has been recently shown that old and archeological bovine leather may represent a useful source of genetic information.10 Futhermore, other work demonstrates the presence of a privileged niche within fossil bone, which contains DNA in a better state of preservation than the DNA presents in the total bone.11 This counterintuitive approach to extracting relatively well-preserved DNA from bones, significantly improves the chances of obtaining authentic ancient DNA sequences, especially from human bones.11 These results indicate that collagen can relatively well preserve DNA, which indirectly means that DNA can be released from the nucleus and be placed in the matrix between the cells in the connective tissue. According to molecular biology data, the assembly of the complex between collagen and DNA determines the stability of DNA against the nucleases.1,2 We focused on the nature of collagen aggregation and the molecular and environmental determinants of the self-association processes with the presence of DNA. In this aspect, selfassociation of the collagen–DNA functional complex offers an opportunity to characterize a unique system, which may relate
10.1021/bm7008813 CCC: $40.75 2008 American Chemical Society Published on Web 12/04/2007
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to the physiological self-association of collagen molecules by water bridges, as shown in ref 12. Such self-association of the complex could be induced by electrostatic interactions between neutral collagen molecules, having strong dipole moment, and negatively charged DNA cylinders. Detailed description of the electrostatic interactions between helical macromolecules is given below in the section of mechanism of interactions between collagen and DNA. The problem in gene therapy and drug and gene delivery systems is that a gene vector, containing DNA or RNA, immediately after administration into the living body becomes deactivated due to immunological reaction or enzymatic attack.1 It is very difficult to control the expression of the gene introduced into the cell. These problems resemble those encountered in control and delivery of bioactive proteins that are unstable in the living body. If a preparation in which a gene vector is embedded in inactive biomaterial (like collagen) is administrated into the living body, the gene vector, like bioactive protein, is protected from immunological reaction and enzymatic attack. In this case, release of the gene vector from the biomaterial can be controlled and then the gene expression period can be controlled. The problem solving was found in atelocollagen (highly purified type I collagen of calf dermis with pepsin treatment), due to its bioinactivity and ability of drug and gene delivery.1,2 It has been pointed that DNA double helix and collagen triple helix could create the unusual supramolecular self-assembly system in aqueous solution.13 This nanoscale selfassembling structure can determine the rise of stability of DNA against the nucleases, growth of collagen unusual fibrils in the presence of DNA,9 and development of autoimmune reaction.13 A molecular model of the DNA-collagen complex7 was also presented, and it was proposed that during the complex formation, the hydration shell of a collagen triple helix rearranges, creating water bridges between phosphate groups of DNA and hydration shells of the macromolecules.14 However, neither experimental evidence supporting collagen–DNA molecular model were presented before nor were FTIR-ATR, AFM, and NMR experiments performed to clarify the mechanisms of unusual collagen aggregation in the presence of DNA. The experimental data of collagen triple helix and DNA double helix self-association in the solutions are presented and some specific inhomogeneous surfaces of collagen–DNA films are clarified. General Description of Collagen Triple-Helix Hydration. Collagen, the major structural protein in the extracellular matrix, presented as not merely a stiff rodlike structure, about lcollagen ) 300 nm in length and dcollagen ) 1.5 nm in diameter,15 has a characteristic triple helical conformation consisting of three polyproline II-like chains that are supercoiled around a common axis.16–18 According to structural biology data, each triple helix is surrounded by a hydration cylinder, with an extensive hydrogen bonding network between water molecules and peptide acceptor groups.19,20 Thus the H-bond network in the triplehelix hydration shell determines not only the stability of this structure but the mechanism of interaction between molecules in the hierarchical assembly of helices at different levels of organization. A very specific arrangement of amino-imino acids20–23 in the triple helix of collagen determines the summary dipole moment of the macromolecule. Furthermore, specific arrangement of acceptor CdO groups in the triple-helical structure24–27 and orientation of amino-imino acids’ dipole moments induces a specific arrangement of water molecules’ dipole moments orientation, situated in the triple-helical structure.14 The close packing of the three chains near the central axis generates a requirement for Gly as every third residue, (Gly
Svintradze et al.
X-Y)n, while the high content of imino acids Pro and Hyp stabilize the individual polyproline II-like helices. While imino acids are highly favorable for the triple helix, the posttranslational modification of Pro to Hyp in the Y position confers an additional stabilizing contribution. This further stabilization of Hyp is likely to result from steroelectronic promotion of more favorable exo-ring pucker for the Y position and Hyp involvement in solvent-mediated hydrogen bonding.19,28,29 The favorable enthalpy of collagen indicates that the hydrogen bonding is a major contributor to stability, and this consists of one direct interchain peptide bond for each Gly X-Y unit, together with an extensive water-mediated hydrogen bonding network.19,30 The ordered water network seen in crystal structures links the available backbone carbonyls of the triple-helix and Hyp groups. General Description of DNA Hydration. With respect to the role of hydration of proteins, water molecules are also critical to the conformation and function of nucleic acids:31 DNA double helix in aqueous solution stabilized by H-bounds between complementary bases (AdT, GtC), stacking interactions along the helix axis between adjacent base pairs, and interactions with surrounding water layers. Hydration plays a major role not only in the stability of the 3D structure of double helix32–39 but in the assembly of different forms (A-, B-, Z-) of DNA and their conformational dynamics.40,41 The bound water in the multilayer hydration shell of double-stranded DNA,40–42 which has two possible orientations42 depending on the content of base pairs plays an important role in the duplex stability and is proposed to have a crucial role in collagen–DNA functional complex formation.7,13,14 Water constrains the conformation of a DNA molecule, as reflected by the transition from B-DNA to A-DNA upon dehydration. DNA undergoes conformational transitions in some polar solvents. The hydration of DNA depends not only on the DNA conformation but also on its sequence.43,44 The DNA interior is mainly hydrophobic and is stabilized by the stacking interactions between the consecutive base pairs, and its surface is rich with hydrophilic groups from the phosphates and sugars. The fundamental forces that cause proteins and nucleic acids to fold to unique structures are the same; however, the energetic contributions from free energies of salvation for DNA are stronger then in proteins.31 Without water to screen the electrostatic repulsions between phosphate groups, the classic double-helical structure of DNA is no longer stable. In addition to hydrating the backbone phosphates, the water molecules in the grooves are ordered and vital to stability. Because of the regular repeating structure of DNA, hydrating water is held in a cooperative manner along the double helix in both the major and minor grooves. The water density in the first hydration shell is much larger than that in bulk water and is the outcome of the many strongly solvated sites. Changes in the hydrogen-bonding network between the hydration shell and DNA can assist ligand binding or release of ions.31 A model for double-stranded DNA in diluted aqueous solution is the wormlike chain. This chain represents intermediate behavior between the rigid rod and random coil, thus taking into account the local stiffness but long-range flexibility of the double helix.40
Theory Discussion of Mechanisms of Interactions between Collagen Triple Helix and DNA Double Helix. Electrostatic interaction between DNA and collagen molecules exhibits strong dependence on the patterns of molecular surfaces’ groups, adsorbed counterions, and collagen triple-helix amino-imino
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bτcollagen ) bµDNA ×
b b ∑ (Eφ i(collagen) + Ewi(collagen)) i
τDNAis the torque that tries Figure 1. Summary torque b τ)b τcollagen + b to rearrange water molecules situated in the hydration shell of collagen triple helix and DNA double helix. The range of intercation energy is equal to collagen hydration energy. The hydration energy of DNA is more then proteinhydration energy from the point of view of DNA hydration and protein hydration data. It means that during collagen–DNA complex formation, collagen hydration has to destroy and to rearrange, creating water bridges between the double helix of DNA and the triple helix of collagen (detailed description in the text).
acid dipole moments. As a result, it is affected by such structural parameters as the helical pitch (both DNA double helix and collagen triple helix), groove width, etc. DNA double helix is negatively charged, while collagen triple helix is variously charged (sometimes negative, sometimes positive, sometimes apolar). Variation of charge along the collagen molecule depends on the content of collagen. Here we focused on the case when collagen has a neutral nature with a strong dipole moment. So DNA-collagen interaction in that case can be described as it is done in refs 45–48 although with small correlation, besides, Poisson electrostatic potential of collagen should be taken as electrostatic potential induced by amino-imino acid dipole moments. Electrostatic interactions between collagen and DNA stimulate collagen–DNA complex formation, but a complex could arise due to H-bonds between some donor groups of collagen and phosphate groups of DNA.7,8 According to the description, collagen and DNA have similar hydration. Taking into account the electric field induced by phosphate groups, adsorbed counterions and water molecule dipole moments situated in the minor groove of DNA, we can consider that torque b τDNA, exerted on dipole moment b µcollagen (b µcollagen is the dipole moment of water molecules situated in the hydration shells of collagen triple helix, in collagen–DNA interactions) is
bτDNA ) bµcollagen ×
b) Epi + Ew ∑ (b i i
where ∑i b Epi is the summary electric field induced by adsorbed counterions, phosphate groups, and dipole moment of base pairs, b is an electric field induced by dipole moments of ∑i Ew i water molecules situated in the minor groove of DNA. On bDNA the other hand, torque τbcollagen exerted on dipole moment µ (b µDNA is the dipole moment of water molecules, situated on the hydration shells of DNA double helix, in collagen–DNA interactions) is
b where Eφ i(collagen) electric field is created by dipole moments of b collagen cylinder and Ew i(collagen) is an electric field induced by dipole moments of water molecules situated in the hydration of collagen triple helix (Figure 1). Summary torque b τ)b τcollagen +b τDNA is the torque that tries to rearrange water molecules situated in the hydration shell of collagen triple helix and DNA double helix. As we mentioned, the range of interaction energy is equal to the collagen hydration energy (hydrogen bonding energy) at r ∼ dµ(water) ) 1–3 nm distances (dµ(water) is the size of the water molecule dipole moment). From the point of view of DNA and protein hydration data, it is well-known that the energetic of DNA hydration is stronger then energetic of protein hydration. This means that during collagen–DNA complex formation, the collagen hydration shell has to destroy and the reduced hydration shell in the complex has to rearrange creating water bridges between the DNA double helix and the collagen triple helix as it was predicted in previous works.7,14 The range of dipole–dipole interactions energy between water molecules is equal Einteraction ∼ Ecollagen to each other (where Ecollagen is water molecule hydrogen bonding energy situated in the triple helix hydration shell), which means that during complex formation, collagen hydration destroys and rearranges, creating water bridges between the DNA double helix and the collagen triple helix (Figure 2).49 Molecular Model of Collagen–DNA Complex. Electrostatic interactions between collagen and DNA stimulate collagen–DNA complex formation, but a complex could arise due to H-bonds between some donor groups of collagen and DNA phosphate groups.7,8 The similarity of collagen triple-helix and DNA double-helix topology dictates that formation of hydrogen bonds between collagen donor groups and DNA acceptor groups is possible. Furthermore, if we calculate distances between donor groups of collagen and acceptor phosphate groups of DNA, we will find that these distances are similar dcollagen(CH2,CHOH,CH) ≈ 0.64 nm ≈ dDNA(PO4-2).7,8 Topology and some geometrical parameters of B helical structure of DNA and triple-helical structure of collagen points to the possibilities of formation fiber molecular structures similar to previously represented molecular models.7,8 To avoid problems connected to contour length of DNA and length of collagen, we discussed interaction between the structural unit (Pro,Pro,Gly)10 of collagen triple helix and dodecamer of DNA (a turn of double-helix DNA). The models are done for (Pro,Pro,Gly)10 and DNA dodecamer. From the point of view of biochemistry, most principle conformations for double-stranded DNA in aqueous solution is the B-form, with an average of 10 base pairs per turn and diameter of d(DNA) ) 2.0 nm, height of a turn 3.6 nm. Consequently, specific geometry is a 10-face prism per turn for DNA (B-form). Base side length of the prism is a projection length of distances between phosphate groups.7 The projection of cross-section of the transverse to the common axis of distance between phosphate groups is l(P) ≈ 0.62 nm for DNA.7 The projections contain information about 3D arrangement of phosphate groups on the surface of nucleic acids (in this case, we are concentrated on B-DNA). After simple geometric calculations,7 it is clear that the B-form is suitable to form fiber complexes with collagen-like triple helices (Figure 2). Our model is based on the topology of collagen and DNA surfaces, including their acceptor and donor groups of hydrogen bonding. Only donor CH,CH2 groups of hydrogen bonding (white spheres on the Figure 2) are situated on the surface of the collagen cylinder (content (Pro,Pro,Gly)10), while only
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Figure 2. Molecular model of collagen–DNA complex (in the model is discussed structural unit (Pro,Pro,Gly)10 of collagen-like triple helix and structural unit, dodecamer crystal structures, of DNA). Hydrogen bonds can arise between CH2 group of collagen and four phosphate group per turn of double-stranded DNA. Structural well arrangement of collagen CH2 groups and phosphate groups of DNA can induce formation of possible hydrogen bonds between CH and phosphate groups (PO4). Water-mediated intermolecular hydrogen bonding contacts between the unit of collagen triple helix and the unit of B-DNA (dodecamer) can be restricted to the backbone phosphates and water spines situated in the minor groove of the B-DNA. 1,3-Phosphate groups are situated on the first chain of the DNA and other 2,4-phosphate groups are situated on another chain. Crystal structures of (Pro,Pro,Gly)10 of a collagen-like triple helix and dodecamer of DNA are taken from the Protein Data Bank www.PDB.org.
acceptor phosphate groups of hydrogen bonding are placed on the surface of the DNA’s cylinder. The structural well arrangement of donor–acceptor groups induces formation of possible hydrogen bonds between collagen (CH,CH2) and DNA(PO4) groups. If we take into account that a water spine with defined dipole moment orientation is situated in the groove of the DNA and torque τ tries to rearrange hydration–water of collagen and DNA, then we could consider that water-mediated intermolecular hydrogen bonding contacts between collagen and DNA are restricted to the backbone phosphate groups and water spine (Figure 2). From the point of view of quantum chemistry, the energy of CH,CH2---PO4 will be about 1 kcal/mol (weak hydrogen bonds) but (Pro,Pro,Gly)10 could make hydrogen bonds with four phosphate groups of DNA dodecamer, which means that minimum bonding energy of the complex could be 4 kcal/mol. As it is known, one phosphate group can form four hydrogen bonds; accordingly we will have maximum 16 hydrogen bonds per turn of DNA. The maximum energy of collagen–DNA interaction (per turn of the DNA) could be about 16 kcal/mol. Although according to the molecular model, each segment of DNA having similar length to collagen length could interact to five collagen molecules. Taking into account the molecular weight of DNA, the number of the interacted segments on DNA can be calculated according to the equation N ) L/lcollagen, where L is contour length of DNA and lcollagen is the length of collagen. Our experiments were done on type I collagen extracted from rat tail tendon (lcollagen ) 300 nm) and salmon sperm DNA (L ≈ 1000 nm). A maximum number of whole collagen molecules involved in interactions with single DNA molecule could be about Ntotal ) 5 · N ) 5 · L/lcollagen (for example, for salmon sperm, DNA with molecular weight 106 Dalton, having a contour length of about 1000 nm can interact with 15 collagen molecules). Involvement of collagen–DNA complex in collagen aggregation depends on Ntotal ) 5 · N for a small N collagen–DNA complex could more easily form unusual twisted complex aggregates that have different geometry than native collagen fibers.9 Consequently, the geometry of collagen–DNA
complex aggregates depends on the molecular weight of DNA. Furthermore, taking into account that collagen in the tissue may not be a merely stiff-rod,50 and the collagen helix probably undergoes shifts in symmetry along its length depending on the local amino acid content (not just immnio acid concentration) and bends at least as much if not more then DNA due to thermally induced superhelix dissociation at physiological temperatures,51 then it can describe the more twisted nature of collagen–DNA complex aggregates. In another words, collagen is more flexible at physiological temperature then a stiff, rodlike structure. According to the above-mentioned above and that DNA is a wormlike chain with high flexibility (persistence length 50 nm) in aqueous solution, we can consider that the high twisted nature of collagen–DNA complex aggregates could be induced by coiled-coil structure of collagen–DNA side-byside complex. Thus donor-acceptor groups involved in collagen–DNA complex were predicted by molecular models,7,8 but there were not any experimental evidence to support theoretical predictions. The donor-acceptor groups are experimentally identified in this paper, and it is demonstrated that secondary structures of collagen and DNA remain.
Experimental Section Materials. Type I collagen was obtained in the laboratory of the Department of General Chemistry, M. Copernicus University, from rat tail tendon of young albino rats. Low-molecular-weight salmon sperm DNA (with molecular weight of about 106 Dalton) was obtained from Sigma. Peptide (Pro,Pro,Gly)10 (collagen-like triple helix) was bought from Peptide International and used for only NMR experiments. FTIRATR and AFM measurements were done on type I collagen from rat tail tendon and salmon sperm DNA. Acetic acid solution of the collagen was dialyzed in aqueous solution for three days, so final pH for the collagen solution was pH ) 4.88. The most important thing about it was the solution without any aggregation of collagen. Aggregation started after mixing with the DNA aqueous solution (practically at the same time), so there were two possibilities: (1) the aggregation was
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Table 1. Positions and Areas of Packs in FTIR-ATR Spectra of Collagen, DNA, and Collagen–DNA Filmsa collagen -1
wavenumber (cm
a
DNA
collagen–DNA 5:1 -1
) area wavenumber (cm
3310 3082 2939 1632
2.99 0.08 0.17 2.29
1549
1.21
1441
0.24
1396 1337 1237 1205 1164 1080 1032 969 934
0.13 0.09 0.64 0.64 0.02 0.48 0.48 0.01 0.02
-1
) area wavenumber (cm
collagen–DNA 10:1 -1
) area wavenumber (cm
collagen–DNA 20:1
) area wavenumber (cm-1) area
3314 3086 2940 1635
3.02 0.08 0.12 2.77
3314 3070 2936 1634
1.56 0.07 0.11 1.78
3318 3088 2939 1634
3.10 0.11 0.14 3.63
1550
0.75
1549
0.55
1549
1.14
1452
0.32
1452
0.16
1376 1338 1277 1229
0.02 0.05 1.67 1.67
1378 1337 1276 1229
0.01 0.03 1.15 1.15
1452 1404 1378 1337 1276 1231
0.27 0.01 0.02 0.09 2.12 2.12
1681 1609 1539 1506 1472 1417 1380 1323 1277 1207
4.3 4.3 0.03 0.01 0.56 0.56 0.56 0.02 0.12 1.57
1053 1007 962
0.96 0.24 0.28
1058
1.76
1058
1.22
1059
2.16
963
0.22
964
0.16
963
0.25
883 824 777 722
0.11 0.26 0.20 0.07
813
0.02
813
0.02
778
0.03 605
0.01
605
0.03
Error of area calculations is 0.001. Error of wavenumber 4 cm-1.
induced by formation of collagen–DNA complex or (2) the aggregation was induced by further increasing pH as a result of adding aqueous solution. Considering these points, we did further small experiments. We added the same volume of just aqueous solution to collagen solution (dialyzed acetic acid solution pH ) 4.88), but formation of any collagen aggregates was not obtained. However, collagen aggregation is temperature-dependent but the aggregation was not obtained before mixing collagen and DNA solutions (experiments were done at room temperature). So, there is no doubt that collagen aggregation was induced by the presence of the DNA. collagen–DNA blends were prepared by mixing of dialyzed acetic acid solution of collagen in aqueous solution (final pH for collagen solution pH ) 4.88, concentration Ccollagen ) 2.4 mg/ml) and an aqueous solution of DNA (concentration CDNA ) 0.24 mg/ml). DNA is in B-conformation in aqueous solution, which was shown in the recent work.52 The final weight ratio of collagen: DNA in films was 20:1, 10:1, and 5:1, respectively. There were observed formation of fiber-like structures in the mixture after mixing of type I collagen and salmon sperm DNA aqueous solutions at a room temperature. The mixture was centrifuged (by Eppendorf centrifuge 5804 R) at 10000g at 4 °C to separate formed aggregates from solution. After centrifugation, the aggregate was gently taken and put on the CaF2 specrophotometric window for drying and incubated for 96 h at room temperature. A glass plate was also used for AFM films preparation. Formed aggregates in the collagen–DNA mixture were gently taken (immediately after mixing) and put on the glass plate and incubated 96 h at a room temperature. FTIR-ATR Measurements. The IR spectra were obtained using a Mattson Genesis II spectrophotometer with an ATR prism crystal. The collagen–DNA films’ FTIR-ATR spectra were compared with FTIRATR spectra of type I collagen and salmon sperm DNA films. All spectra were recorded at the resolution of 4 cm-1 and 100 times scanning. NMR Measurements. The NMR spectra were obtained using a Bruker Avance 300 MHz spectrophotometer. The spectra of salmon sperm DNA in D2O solution and the peptide (Gly,Pro,Pro)10 in D2O solutions were compared with the peptide–DNA mixture in D2O. All spectra were recorded at 300 Mhz frequency. AFM Observation. The atomic force microscope Veeco (Digital Instruments) was used for taking images of type I collagen, salmon sperm DNA and collagen–DNA films. The images were taken at various resolutions.
Results and Discussion FTIR-ATR Spectroscopy Results. FTIR-ATR spectra of the collagen, the DNA, and the collagen–DNA blends show that the positions of amid A, B, I, and II are at the same wavenumbers, but changes in some peak positions are observed at the region of 800–1600 cm-1. It points at interactions between collagen and DNA molecules and collagen–DNA complex formation. Some hydrogen bond formation could determine collagen–DNA complex structure in films. The positions of A, B, I, and II amide bands in collagen–DNA films are nearly unchanged in comparison with pure collagen and pure DNA films bands. However, the unchanged peak positions in the bands and small changes in integral absorbance of the peaks’ (A, B, I, II amide bands) region indicates that collagen triple helix conformation in collagen–DNA film is almost the same but with differences in energy of formation of collagen triple helix (which indicates changes of numbers of both direct and indirect intramolecular hydrogen bonds in collagen triple helix). The amide I band at around 1632 cm-1 (CdO stretching) in FTIR-ATR spectra of the collagen is sensitive to the secondary structure of the collagen.53,54 Unchanged position of the amide I band of the collagen in the collagen–DNA films clarifies unchanged secondary structure of the collagen in the complex (Figure 3a-c). The most interesting changes were observed in the interval of 800–1600 cm-1. Amide I and II regions of collagen, which are 1632 and 1549 cm-1, respectively, were not changed. The peak of DNA at 1682 cm-1 was not observed at all. The position of the peak at 1441 cm-1 is changed in the collagen–DNA film and appears at 1452 cm-1, while the peak at 1396 cm-1 of the collagen disappears. However, there are no peaks at 1472 and 1417 cm-1 of the DNA in collagen–DNA films (Table 1). The peak at 1378 cm-1 could be a peak of the DNA and changed peak of the collagen (1396 cm-1). However, appearance of the peak at 1276 cm-1, which is the characterized peak of the sugar–phosphate backbone of the DNA, indicates involvement of phosphate groups in collagen–DNA complex formation. Some chemical shifts in the region from 800 to 1200 cm-1 could indicate the rearrangement of quasistructural water of the
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Figure 3. FTIR-ATR spectra of collagen, DNA, and collagen–DNA films with various mass distributions. Red line corresponds to pure collagen, blue, pure DNA, and black, collagen-DNA complex.
collagen hydration shell and formation–water bridges between the collagen and the DNA. The chemical shifts, detailed in Table 1, show possible hydrogen bonding interactions between type I collagen CH2, CH(OH), CH groups and phosphate groups of salmon sperm DNA. Furthermore, by taking into account that strong absorbance in the region of 1400–1465 cm-1 and 1425–1475 cm-1 indicates sensitivity to backbone conformation of Pro(CH2) groups,55–57 we can suggest that formation of collagen–DNA complex is induced by hydrogen bonds between Pro(CH2), Gly(CH), Hyp(CHOH) donor groups and acceptor PO4-2 groups of DNA. On the other hand, such a small signal of the DNA in the collagen–DNA films is caused by very low weight distribution of the DNA in the collagen aggregates. Although, according to the molecular model,7 each segment of the DNA, having similar length to the collagen length, could interact with five collagen molecules. DNA could be packed in collagen aggregates. According to theoretical predictions, salmon sperm DNA (with molecular weight 106 Dalton) could interact with 15 collagen molecules. The DNA segments (the segments’ length is similar to collagen length 300 nm) are packed in the collagen molecules and form a fiberlike complex (Figure 2) and the complex is wrapped in collagen aggregates. One-Dimensional 1D NMR Experimental Results. The spectra of salmon sperm DNA in D2O solution and the peptide (Pro,Pro,Gly)10 in D2O solutions were compared to the peptide– DNA mixture in D2O. However, it is impossible to analyze the
Figure 4. 1D NMR spectra of DNA, peptide (Pro,Pro,Gly)10, and peptide–DNA mixture (the signal was stronger in the peptide–DNA mixture).
whole spectra of salmon sperm DNA in 1D NMR experiments, which is why we did not analyze below-described spectra of NMR experiments of salmon sperm DNA. Concentration of the DNA in D2O is 7 mg/ml, concentration of the peptide is 20
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Figure 5. AFM images (tapping mode) of pure collagen, DNA, and collagen–DNA films. On the graphics are indicated dependences of height on width through blue, red, and green points.
mg/ml, and concentration of the DNA and the peptide in the DNA-peptide mixture is 4 and 20 mg/ml, respectively. At 4 °C, the spectra of the peptide in the peptide–DNA mixture have features similar to pure (Pro-Pro-Gly)10 spectra in D2O but with change of intensity of each peak in the region of 0–4 ppm. However, these changes could indicate some possible interactions between the peptide (Pro,Pro,Gly)10 and the DNA in aqueous solution and possible complexity of the peptide (collagen like triple helix) and the DNA. When recorded at the same concentration, all of the peptides in the mixture have an increase in the intensity of the peaks at 0–4.2 ppm, which is assigned to the proline CδH, CγH, and CβH groups in the triple-helical conformation and CRH of Gly, which is not involved in intermolecular hydrogen bonds in the collagen-like triple helix of the peptide (Figure 4). On the other hand, 1D spectra of (Pro,Pro,Gly)10 is similar to the spectra of (Pro, Hyp,Gly)10, which indicates that the peptide in D2O solution has triple-helical conformation similar to (Pro,Hyp,Gly)1058 at 4 °C temperature. However, a chemical shift does not change at 7.62 ppm, which belongs to the NH group of the peptide, showing that triple-helical conformation of the collagen-like triple helix should not be changed in the peptide–DNA interactions. Thus theoretically predicted possibilities of formation hydrogen bonds between peptide(CH,CH2) and DNA(PO4) groups are validated by NMR experiments. AFM Images of Collagen–DNA Films. AFM images of the pure collagen (type I collagen), the DNA (salmon sperm), and the collagen–DNA complex films at various mass distributions are shown in Figure 5. The surface morphologies of the collagen,
the DNA, and the collagen–DNA complex films are different. Pure collagen and DNA films show relatively well distributions of the collagen and the DNA at the films surface. The collagen/ DNA films are more irregular films with specific height distributions (Figure 5). However, the surface of the collagen/ DNA films confirms spontaneously formed collagen–DNA complex aggregate. The collagen–DNA aggregates are inserted in unusual collagen aggregates that are relatively well shown in the Figure 5E.
Conclusions FTIR-ATR spectra showed unchanged position of the amide I band of the collagen in the collagen–DNA films that clarified unchanged secondary structure of the collagen in the complex. It confirms that the triple helix of the collagen is protected from denaturation during the formation of collagen–DNA complex. On the other hand, NMR measurements indicate that there is some interaction between the peptides (Pro,Pro,Gly)10 (collagenlike triple helix) and DNA in D2O solution. Changes in peak intensity and some chemical shifts of proline CH2 groups indicate that they are involved in interaction with the DNA surface that nontrivially show the possibilities of formation H-bonds between donors CH2, COH, CH, and acceptor phosphate groups of DNA, which was predicted in a molecular model.7 Thus theoretically predicted possibilities of formation of hydrogen bonds between the peptide (CH,CH2) groups and DNA(PO4) groups are validated by NMR. On the other hand, FTIR experiments indicate that in the collagen–DNA com-
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Biomacromolecules, Vol. 9, No. 1, 2008
plex, we probably have same interaction between donor (CH2,COH,CH) groups of the collagen and acceptor phosphate groups of the DNA. Furthermore, some chemical shifts in the region from 800 to 1200 cm-1 point to rearrangement of the quasistructural water of collagen hydration. Also, according to theoretical predictions, the salmon sperm DNA could interact with 15 collagen molecules. So the DNA segments (the segments’ length is proposed to be similar to collagen length 300 nm) could be packed in collagen molecules and form a fiberlike complex (Figure 2), and the complex is wrapped in unusual aggregates of the collagen. The aggregates could have different geometry from native collagen fibers. However, (determination) clarification of collagen–DNA complex nanostructure was not possible from AFM measurements but was shown that spontaneously formed collagen–DNA complexes were inserted in collagen aggregates in collagen–DNA films. Acknowledgment. Financial support from NATO Collaborative Linkage Grant number PDD (CP)-(CBP.EAP.CLG 982215) is gratefully aknowledged.
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