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Reply to Comments on Complex Pattern Formation by Cowpea Mosaic Virus Nanoparticles
In the paper Comments on Complex Pattern Formation by Cowpea Mosaic Virus Nanoparticles by G. S. Watson,1 it is claimed that pattern formation was observed in a buffer solution in the absence of the virus, using identical chemical and experimental conditions as used in our study. We present here all our experimental results in detail showing unambiguously that the buffer solution as prepared by us does not form patterned lines in the absence of the virus in the solution. We show that the presence of patterned lines in the buffer solution in the absence of virus particles is strongly dependent on the method of buffer preparation. Even though we do not explicitly mention them in our paper,2 we had carried out a series of control experiments to ascertain that the lines are not an artifact but are indeed due to the self-assembly of cowpea mosaic virus (CPMV) particles. The buffer used in our case is a mixture of 100 mM potassium phosphate with 10 mM Tris at pH 8.0 in the ratio 1:99. We looked at a dried drop of this buffer solution in the absence of CPMV and found no evidence of line formation. An optical micrograph and an AFM scan of such a control experiment are shown in Figure 1. The buffer is found to bead up and dry as small droplets on the mica surface. In fact, control experiments carried out at 10 times higher phosphate concentration showed similar results with no line formation (see Figure 2). We have seen exactly the same results when the solution without virus is dried on the acid-treated mica; we see crosses only in the presence of virus particles. In fact, the patterns are not observed when the virus proteins are denatured, again confirming that intact capsids are necessary to form stable lines. Intrigued by this discrepancy between Watson’s and our observations, we decided to deliberately change the recipe for the Tris pH 8.0 buffer. In all our experiments we have prepared Tris buffer starting with Tris-base in the following way:3 We started with a solution of Trisbase (MW ) 121.1 g/mol, Sigma). 0.304 g of it was mixed with 200 mL of RO water. Measured pH under this condition was 8.8. 200 µL of 2.4 M HCl was added to decrease the pH to 8.0. The volume was completed to 250 mL with RO water and then filter sterilized. As mentioned above, this buffer does not show any lines on freshly cleaved (Figure 3a) and acid-treated mica as well as on acid-cleaned glass. As an alternate method we decided to start with Tris-HCl and bring up the pH by adding KOH. The recipe used is as follows: We started with a solution of Tris-HCl (MW ) 157.6 g/mol, Sigma) by mixing 0.400 g of Tris-HCl with 200 mL of RO water. At this point, the measured pH was 4.8, and to this solution was added 3.4 mL of 500 mM KOH to increase the pH to 8.0. The volume was completed to 250 mL with RO water and then filter sterilized. To our surprise, when this buffer was used to * To whom correspondence should be addressed. E-mail: ratna@ nrl.navy.mil. † Naval Research Laboratory. ‡ George Mason University Summer Intern. § The Scripps Research Institute. (1) Watson, G. S. Langmuir 2003, 19, 486. (2) Fang, J.; Soto, C. M.; Lin, T.; Johnson, J. E.; Ratna, B. Complex Pattern Formation by Cowpea Mosaic Virus Nanoparticles. Langmuir 2002, 18, 308-310. (3) Current Protocols in Molecular Biology; John Wiley and Sons Inc.: New York, 1997; Vol. 1, p A.2.6.
Figure 1. (a) Optical micrograph of a drop of 1 mM phosphate in 10 mM Tris buffer dried on freshly cleaved mica. (b) AFM image of the same sample.
Figure 2. 2 µL drop of 10 mM potassium phosphate buffer in 10 mM Tris pH 8.0.
dilute 100 mM phosphate buffer2 to a final concentration of 1 mM, we observed that a 2 µL drop of this mixture dried on a mica substrate showed lines as shown in Figure 3b. However, these lines are not stable with time whereas the lines formed by the virus particles are very stable and last for months. Figure 3c is an optical micrograph of the same sample as that in Figure 3b but taken after 7 days and shows that some of the lines had disappeared. This study clearly shows that the final outcome depends on the methodology used for preparing the buffer. However, since we do not know the recipe used by Watson, we cannot speculate on the reason for his observations. In addition to these control experiments, we have studied the pattern formation in fluorescent-labeled virus samples which clearly show that the virus is intimately associated with the lines. We covalently attached Alexa Fluor 488 (Molecular Probes) with a maleimide end group to bind the thiol terminus of cysteines in a CPMV mutant. The drying drop experiment was conducted on a mixture of 25 wt % of this fluorescent-labeled virus with 75% of
10.1021/la0265934 CCC: $25.00 © 2003 American Chemical Society Published on Web 12/14/2002
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Comments
Figure 4. Fluorescence micrograph of 25% fluorescent-labeled, cysteine-modified CPMV mutant mixed with 75% of wild-type CPMV.
Figure 3. (a) Drying of a 2 µL drop of 1 mM potassium phosphate in 10 mM Tris pH 8.0 as prepared by us (protocol from ref 1) showing lack of pattern formation. (b) Lines formation observed on drying of a 2 µL of 1 mM potassium phosphate in 10 mM Tris pH 8.0 prepared starting from Tris-HCl. (c) Optical micrograph of the same region as in (b) after 7 days.
wild-type CPMV at a final concentration of 0.15 mg/mL. A microphotograph taken under a fluorescence microscope shown in Figure 4 clearly indicates the presence of virus particles in the pattern. We also labeled the RNA inside the wild-type virus using SYTO 13 (Molecular Probes), a dye that fluoresces only when it is bound to RNA. Again, the line and cross formations were observed when 2 µL of 0.15 mg/mL of STYO 13-stained virus (10 mM tris pH 8.0 buffer) was dried on freshly cleaved mica as well as acid-cleaned mica, respectively. Figure 5a shows the crosses observed on the acid-treated mica. A corresponding control experiment carried out with just the presence of the SYTO 13 in the buffer solution does not show either lines or fluorescence, as expected. In summary, our results show that the Tris buffer used in our experiments does not exhibit pattern formation, as demonstrated by all the control experiments. The line
Figure 5. Fluorescence micrographs of (a) wild-type CPMV stained with SYTO 13 dried on acid-treated mica and (b) SYTO 13 in 10 mM Tris buffer solution in the absence of CPMV. Scale bars ) 50 µm.
patterns, observed only in the presence of intact virus capsids, are very stable with time. The lines formed by salt-containing buffers are unstable and disappear after a few days. Thus, our data unambiguously confirm that the orthogonal line formation observed on drying a droplet of CPMV-containing solution is indeed due to the selfassembly of the virus. B. R. Ratna,*,† C. M. Soto,† L. Danner,‡ A. S. Blum,† J. Fang,† T. Lin,§ and J. E. Johnson§
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, D.C. 20375-5438; and Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037 Received September 23, 2002 LA0265934
