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Compaction of DNA Induced by Like-Charge Protein: Opposite Salt-Effect against the Polymer-Salt-Induced Condensation with Neutral Polymer Kenichi Yoshikawa,*,† Seiko Hirota,† Naoko Makita,‡ and Yuko Yoshikawa§ †
Department of Physics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan, ‡Faculty of Environmental and Information Sciences, Yokkaichi University, Yokkaichi 512-8512, Japan, and §Laboratory of Environmental Biotechnology, Research Organization of Science and Engineering, Ritsumeikan University, Kusatsu 525-8577, Japan
ABSTRACT We observed the collapsing transition of a DNA molecule in a crowding environment with a water-soluble negatively charged protein, albumin. We performed fluorescence microscopic observation to monitor the change in the conformation of individual giant DNA molecules. It is found that DNA molecules exhibit compact conformation above critical concentrations of the negatively charged protein. We interpret this transition in terms of depletion interaction, segregation of different polymers driven by excluded volume interaction. Interestingly, coexisting salt causes retardation on the collapsing transition, being opposite to the well-known phenomenon of DNA compaction in a crowding solution of neutral polymer, polymer-salt-induced-condensation of DNA (psicondensation). The possible biological significance of the transition of higherorder structure on DNA in a concentrated protein solution is discussed in relation to the highly crowded conditions in living cells. SECTION Biophysical Chemistry
As a protein, we used bovine serum albumin (BSA), which exhibits -18 net charges with a molecular weight of 66000 g/mol.13 It was reported that crowding the solution with BSA facilitates the formation of amyloid fibril.14 Occurrence of segregation on an aqueous solution of rod-shaped tobacco mosaic virus in the presence of BSA was observed, which was discussed in terms of a mutually exclusive interaction.15 It is also reported that BSA promotes the DNA condensation induced by HU, a histone-like protein.16 Thus, it is of interest to examine the depletion effect of BSA on the higher-order structure of DNA, in relation to the crowding effect on genomic DNA in living cells. Figure 1 shows typical fluorescence microscopic images of single DNA molecules at different concentrations of BSA in a 150 mM NaCl solution. At 1% BSA, DNA molecules assume an elongated coil conformation that exhibits significant intrachain Brownian motion (Figure 1A). When the BSA concentration is high enough, as in Figure 1C, DNA molecules show a tightly compact state which is characterized as a bright optical dot with relatively large translational Brownian motion. At intermediate concentrations of BSA, DNA molecules take partially folded structures in which the elongated coil part and compact part coexist, as shown in Figure 1B.
A
ll living cells on Earth maintain their vital state under crowded conditions with a rich variety of macromolecules. It has been a long-standing question how a cell can perform many important processes, such as RNA transcription and DNA replication, in such a crowded environment.1-3 In fact, the effect of molecular crowding remains a subject of considerable research.4-8 In general, we can consider two different types of interaction between different solvable polymers, attractive and exclusive. When polymers are mutually attractive, coacervation occurs, accompanied by segregation between polymer-rich condensed and dispersed phases. On the other hand, through exclusive interaction, two polymers can segregate into different phases. A well-known example of such phase segregation on DNA is polymer-salt-induced (psi) condensation or Ψ-condensation.9-12 When a water-soluble neutral polymer such as polyethylene glycol (PEG) is added to a DNA solution, DNA molecules undergo condensation into a dense phase that is depleted by PEG molecules. Ψ-condensation has been regarded as an in vitro model of a crowded environment. However, in the actual intracellular environment, most proteins are electronically charged. Therefore, it would be worthwhile to examine the effect of charged proteins on the conformation of DNA. Although the interaction of DNA with cationic proteins, such as histone proteins, has been extensively studied in the past, there does not appear to have been any study on the effect of negatively charged protein on the conformation of DNA. In the present study, we examined the crowding effect of negatively charged protein on DNA.
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Received Date: May 4, 2010 Accepted Date: May 20, 2010 Published on Web Date: May 24, 2010
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Figure 1. Fluorescence images of single T4 DNA molecules in 150 mM NaCl solutions at different concentrations of BSA (left panel), quasi three-dimensional expression of their fluorescent intensity (middle panel), and schematic representations of the conformation of DNA molecules (right panel). BSA concentrations are (A) 1, (B) 5, and (C) 15% in weight per volume (w/v). The apparent sizes in the fluorescence images are larger than the actual size of DNA due to a blurring effect, which is on the order of 0.2-0.3 μm.
Figure 2A summarizes the distributions of the long-axis length of DNA molecules at different concentrations of BSA, and Figure 2B shows the conformations of DNA schematically. At 1% BSA, all DNA molecules exist as an elongated coil with a mean long-axis length of around 3 μm. With an increase in the BSA concentration, a partially folded conformation appears, where the long-axis length is smaller than that of coiled DNA. At 20% BSA, all of the DNA molecules exist in the folded compact state. Figure 2C shows a phase diagram of the conformational state of DNA molecules as a function of the BSA and NaCl concentrations. Overall, DNA molecules collapse to a smaller size with an increase in the BSA concentration. It is to be noted that NaCl retards the transition from the elongated state to the compact state. This retarding effect of salt is opposite that in the case of Ψ-condensation, that is, salt promotes the condensation or precipitation of DNA. In the present study, we have shown that a negatively charged protein can induce a large conformational transition in the higher-order structure of DNA molecules. To the best of our knowledge, this is the first observation of the depletion of DNA by a negatively charged protein. This depletion is attributable to the exclusive interaction, or steric repulsion, of globular protein. It is expected that DNA molecules would be excluded from the sea of BSA when the total excluded volume of BSA occupies the whole solution volume. This depletion interaction is depicted schematically in Figure 3. This scheme indicates that the formation of the compact state in DNA causes an increase in the free volume of solution
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Figure 2. (A) Distributions of the long-axis length, L, of T4 DNA molecules in a 150 mM NaCl solution at various concentrations of BSA, (a) 1, (b) 5, (c) 10, (d) 15, and (e) 20%. Histograms correspond to DNA in the coil (white bars), partially compact (gray bars), and fully compact (black bars) states. (B) Schematic representation of the change in the higher-order structure of DNA with an increase in the BSA concentration. Regions I and III are the extreme states in which all DNA molecules exhibit the elongated coil and fully compact conformations, respectively. Region II shows the intermediate state, which includes intrachain segregated conformation, as depicted schematically. Partially compacted DNA molecules only exist in region II. (C) Phase diagram of the conformation of DNA as a function of the NaCl and BSA concentrations.
and that this effect decreases the steric repulsion between BSA molecules in the crowded condition. If we consider the mechanism of the depletion interaction as presented in Figure 3, we must next (1) examine whether the experimentally observed critical BSA concentration for inducing the DNA transition corresponds to the overlapping concentration c* of BSA molecules and (2) clarify the reason for the retardation effect of salt on DNA compaction.
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BSA molecule is ∼700 nm3, and the radius R is calculated to be 5.6 nm. On the other hand, the literatures give values of 3.4-3.5 nm for the hydrodynamic radius RH of BSA based on measurements with light scattering.12 We confirmed the validity of this value of RH through the measurement of dynamic light scattering (data not shown). It has been well established that in a good solvent, the gyration radius Rg of a polymer chain is larger than its hydrodynamic radius RH; in an ideal chain, Rg = (3/2)RH..18 For a real polymer in a good solvent, we can expect that Rg is 20-40% larger than RH.11 Clearly, the effective excluded volume of a globular polymer should be interpreted in terms of Rg since RH is a measure of viscous friction and, in a good solvent, should be smaller than the actual extent of the polymer. We may assume that most of the charged segments in a BSA molecule should adapt a rather swelled conformation. Thus, we can expect the radius of the excluded volume of BSA to be somewhat larger that RH, being around 4.5 nm. In addition, we have to consider the effect of electronic repulsion between BSA and DNA. For the characteristic length of Columbic repulsion, we adopt the Debye length λ rffiffiffiffiffiffiffiffiffiffiffiffiffiffi εkB T ð2Þ λ ¼ ZNA e2 I where ε is dielectric constant, kB is the Boltzmann constant, T is the absolute temperature, Z is the ionic valency, NA is Avogadro's number, e is the unit electronic charge, and I is ionic strength. We can evaluate that the Debye length is ∼1 nm at 100 mM in a solution of a 1:1 electrolyte such as NaCl. From the above discussion, we can estimate the radius of the excluded interaction of BSA to be around 5.5 nm at 100 mM salt concentration, which corresponds to the expected value of R = 5.6 nm deduced from the experimental observation. We can now address the retarding effect of salt on DNA compaction. As is clearly shown in eq 2 regarding Columbic repulsive interaction, the Debye length λ is highly dependent on the ionic strength or salt concentration. If we consider a 1 mM salt solution, the Debye length λ becomes on the order of 10 nm. This suggests that there is a significant increase in the effect of the depletion volume of BSAwith a decrease in the salt concentration. Thus, the retarding effect of salt on DNA compaction can be attributed to a decrease in the excluded volume due to the weakening of Columbic repulsion with salt. It is known that in the nucleus, the concentration of macromolecules is on the order of 100-200 mg/mL.6 In the present study, it become clear that BSA at the concentrations of 10-20%, that is, 100-200 mg/mL, caused the significant change on the higher-order structure of DNA, suggesting the important effect of crowding environment on the genomic DNA molecules in living cells. In relation to our observation, by use of Monte Carlo simulation, de Vries studied19 the change of a pairwise interaction between rod-like segments as the model of oligomeric DNA (shorter than 100 base pairs) with the increase of the concentration of negatively charged proteins. It was concluded that, at lower ionic conditions than physiological salt concentration, negatively charged proteins may change the interaction between the like-charge segments to be attractive, where the term of “DNAcondensation”
Figure 3. (A) Schematic representation of the excluded volume interaction between DNA and BSA. The effective radius of the excluded volume of BSA can be represented as the sum of the radius of gyration, Rg, and the Debye length, λ. (B) Depletion effect on DNA by charged globular proteins. Left: elongated coil DNA in a crowded environment with BSA. Right: free space for crowded BSA molecule increases accompanying the compaction of DNA.
The overlap concentration c* is the state in which the sum of the excluded volumes is the same as the total volume V of the solution, as indicated in eq 1 VM ð1Þ c ¼ 4πR3 NA 3 where M is the molecular weight of BSA (M = 66000), R is the radius of the self-avoiding volume of a single BSA molecule, and NA is Avogadro's number.17 The experimental observation given in Figure 2 indicates that the critical concentration of BSA for inducing the full compaction of DNA is around 15%, that is, c = 0.15 g/cm3. We can expect that this concentration corresponds to the overlap concentration c* by considering that the amount of DNA is negligibly small compared to that of BSA. Thus, the excluded volume of a
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was used. Thus, de Vries correctly predicted the weakening of the depletion interaction by salt, despite the ambiguous usage of the term of DNA condensation. During the last couple of decades, it has been clarified20 that compaction of long DNA, above the size of several tens of kilo base pairs, is interpreted in terms of first-order phase transition and that the transition point is much different from the critical condition of switching on the pairwise interaction between repulsion and attraction. Nevertheless, to fully explain the experimentally observed results given in Figure 3, it may be necessary to take into account various effects on free energy, including the translational entropy of the counterions12 and the conformational entropy of DNA, in addition to the depletion interaction.
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EXPERIMENTAL SECTION We used bacteriophage T4 DNA (165.6 kbp) (Nippon Gene, Toyama, Japan). To visualize individual DNA molecules, a fluorescent dye, YOYO-1 (quinolinium,1,10 -[1,3-propanediylbis[(dimethylimino)-3,1-propanediyl]]bis[4-[(3-methyl-2(3H)benzoxazolylidene)methyl]]-tetraiodide) (Molecular Probes Inc., Eugene, OR, U.S.A.), was used for DNA staining. BSA solution was obtained from Sigma-Aldrich, Japan (Tokyo, Japan), as an aqueous solution (35% (w/v) in 0.85% NaCl). DNA-dye stock solution was allowed to equilibrate for at least 12 h at 4 °C. DNA samples were prepared by diluting the DNAdye stock solution with BSA solution at various concentrations of NaCl and allowed to equilibrate for at least 1 day at 4 °C. The final concentrations of DNA and YOYO-1 were 250 nM in base pairs and 25 nM, respectively. The direct measurement of the conformation of single DNA molecules was performed by fluorescence microscopy with a Nikon Eclipse TE2000-U microscope (Nikon Corp., Tokyo) equipped with a 100 oilimmersed objective. Real-time fluorescence images were recorded on DVD through a highly sensitive Hamamatsu EBCCD camera and an image processor (DVS-20, Hamamatsu Photonics, Hamamatsu, Japan). The recorded fluorescence images were analyzed using Image J software (NIH). The longaxis length, defined as the longest distance in the outline of the DNA image (Figure 1), was measured on 50 randomly chosen DNA molecules under each condition. Due to the relatively low time resolution and high sensitivity of the camera, Brownian motion leads to a blurring effect on the order of 0.2-0.3 μm, as is schematically shown in Figure 1.21
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AUTHOR INFORMATION
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Corresponding Author: *To whom correspondence should be addressed. E-mail: yoshikaw@ scphys.kyoto-u.ac.jp.
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ACKNOWLEDGMENT This study was supported in part by a
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Grant-in-Aid for Scientific Research (18GS0421) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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