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The cation binding associated with structural transitions in bromegrass mosaic virus
VIROLOGY
81,
419-432
The Cation
(1977)
Binding
PIERRE Laboratoire
de Virologie,
Associated with Structural Bromegrass Mosaic Virus
PFEIFFER Institut
AND
ANTHONY
de Biologic Moldculuire 67084 Strasbourg, Accepted
May
Transitions
in
C. H. DURHAM’
et Cellulaire France
du CNRS,
15 rue Descartes,
1,1977
Hydrogen ion titration curves are described for bromegrass mosaic virus under various conditions of temperature, ionic strength, and Ca2+, Mg2+, or polyamine concentration, with particular reference to the hysteresis region near neutral pH. The curves are correlated with the sedimentation properties of the virus. There are two relatively nonspecific cation-binding sites per protein subunit, present in the virion but not in the protein or RNA alone, which control the virion’s swelling. The swelling is reversible in the presence of enough cations; without them, swollen BMV can contract to an incorrect structure with RNA partially exposed. We interpret these results qualitatively and structurally in terms of binding sites formed in a region of high electronegativity contributed by RNA phosphates and protein carboxylates. INTRODUCTION
is the ion whose binding is most easily measured (Durham and Hendry, 1977). Here we report the hydrogen ion titration curves of BMV in the presence and absence of K+, Mg2+, Ca2+, and polyamine ions. Thanks to the large quantities of BMV that can be prepared easily, we have been able to observe the hysteresis region of the curves, corresponding to the swelling transition of the virus, in detail. The results are most easily interpreted in terms of simple electrostatic interactions at the RNA-protein interface.
Bromegrass mosaic virus (BMV) is a small spherical three-component plant virus which has been extensively studied (for reviews see Bancroft, 1970; Lane, 1974). It swells near neutral pH in the absence of multivalent cations (Incardona and Kaesberg, 1964; Incardona et al., 19731, and the RNA then moves towards the particle surface (Jacrot et al., 1976). Swollen BMV is sensitive to nucleases (Brakke, 1963; Kassanis and Lebeurier, 1969) and proteases (Agrawal and Tremaine, 1972; Pfeiffer and Hirth, 1975). The closely related cowpea chlorotic mottle virus (CCMV) shows many similar features and has been studied in some detail (Johnson et al., 1973; Adolph and Butler, 1974; Adolph, 1975a and b; Jacrot, 1975). Most, perhaps all, plant viruses possess distinctive sites for binding cations (Durham et al., 1977). Durham (submitted for publication) has suggested that these sites function in viva by transporting Ca2+ ions across membranes to provide free energy for simultaneous virus penetration and decapsidation. However, experimentally H+ I Author dressed.
to whom
reprint
requests
should
MATERIALS
AND
METHODS
BMV (the common strain derived initially by Dr. Lane of the University of Wisconsin) was multiplied in barley plants, cv. Rika, and purified by an improved version of the method of Bockstahler and Kaesberg (1962). Fresh leaves were collected 12 days after inoculation and minced, and their juice was squeezed out. The residue was rehydrated with one-half volume of distilled water and again squeezed out. All the juice was pooled. After standing overnight at pH 4.8 in the cold room, to precipitate the host plant proteins, and after low speed centrifuga-
be ad419
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0042-6822
420
PFEIFFER
AND
tion, the clear supernatant fluid was made and 6% in polyethylene glycol 20,000. The virus precipitate was collected by a 30-min centrifugation at 10,000 rpm and submitted to several cycles of high and low speed centrifugation. Resuspending virus pellets in distilled water rather than in buffer helped to eliminate contaminating plant membranes. BMV protein and RNA were prepared simultaneously by degradation of the virus in 1 M CaClz at pH 6.7 (Herzog et al., 1976). Samples of virus, protein, or RNA, at concentrations of 3 to 4 g/liter, were prepared for titration by dialysis against about 20 mM sodium EDTA, pH 5, followed by several days of dialysis against many changes of 50 mM KCl, adjusted to pH 5 with HCl, followed by addition of solid KC1 to the required KC1 concentration. (Any polyvalent cations firmly enough bound to resist this treatment would probably have had no effect on the subsequent experiments.) Titration results are expressed as protons per protein subunit, after correction for titration of the solvent, using the following extinction coefficients: virus, E .$$“A% = 5.08; RNA, E .&~~~t; = 23.0; and protein, E 1280 g/liter “In = 0.76; and taking the protein subunit weight as 20,300, the overall RNA content of the virus as 21.4%, and the number of protein subunits per virion as 180 (Lane, 1974). Titrations were performed under CO,free damp air, with the same apparatus and techniques as used by Durham and Hendry (1977), but in a thermostatically controlled vessel rather than in an airconditioned room. Titrants were 10 mM KOH and HCl, adjusted to the ionic strength of the sample with KCl. Five experimental details are worth emphasizing. The 10 n&f titrants were a compromise; stronger titrants would have reduced the precision of volume measurements and caused local denaturation of the virus, whereas weaker titrants would have increased the correction necessary for titration of solvent alone. To reduce CO, pickup, all samples were kept at or below pH 5 before titration, and KOH titrants were carefully protected from atmospheric
0.1 it4 in NaCl
DURHAM
CO,. A major source of error was slow titrant leakage past the three-way stopcock of our autoburets; for the slow titrations it was sometimes necessary to measure this rate of leakage and correct for it in the data handling. The third digit on the pH meter was very valuable for observing pH changes with time. It was essential to take great care to dialyze away all EDTA before beginning titrations. Analytical ultracentrifugation of undiluted samples taken directly from the titration vessel was performed by G. de Marcillac, using a Beckman Spinco Model E ultracentrifuge equipped with Schlieren optics, running at 35,600 rpm and 20 to 25 with bar angle 50 to 60”. Native BMV at pH 5 was often used as an internal standard for viscosity and density correction. Polyacrylamide-gel electrophoreses were performed as described by Loening (1967) on cylindrical 2.8% gels for RNA and according to Laemmli (1970) on slab gels for protein analysis. RNA gels were scanned at 260-nm wavelength in a Beckman Acta III recording spectrophotometer equipped with a gel-scanning attachment. Chemicals were of analytical grade, purchased mainly from Merck. Polyamine hydrochlorides, ribonuclease A, and chymotrypsin were purchased from Sigma. RESULTS
The titration curve of BMV at 22” in 50 m&f KC1 (Fig. 1A) is the basic standard of comparison for all conditions studied here. It is essentially the same as the curve that Durham et al. (1977) found for their independently propagated strain in their preliminary study of BMV titration. Figures 1B to E show how the curve changed if temperature, ionic strength, or divalent ion concentrations were changed. Although there were small changes elsewhere, it was the hysteresis region between pH 6.0 and 7.2, where the virus conformation changes, which we investigated in detail. Observing the time-dependent change of pH in the hysteresis region presented certain technical problems. To obtain consistent measurements there, we used an automatic device to command the autoburet to
421
EMV CATION BINDING
I
4
I
1
5
I
1
6
I
I
PH
7
I
I
8
I
1
FIG. 1. Titration curves of BMV under the following conditions: (A) In 50 mM KC1 at 22”; (B) as for A, but at 0”; (C) as for A, but in 300 mM KCl; (D) as for A, plus 1 mM CaCl,; (E) as for A, plus 1 mM MgCl*; (F) as for A, plus 0.5 mM spermine. The vertical axis is marked at intervals of one proton per subunit. The vertical positioning of the curves is arbitrary.
deliver 20 ~1 of titrant (equivalent to about 0.3 protons per subunit) regularly every 4 min. At this slow rate of titration, the experimental problems of buret leakage, titrant strength determination, and virus denaturation often accumulated to a discrepancy of up to half a proton per subunit between the beginning and end of a titration. On the other hand, pH measurements were probably dependable to + 0.02
pH unit or better. In general, therefore, before drawing significant conclusions we accumulated a whole array of titration curves under related conditions, ofien obtaining the curves with several different batches of virus, The first variables studied were temperature and ionic strength provided by KCl. Figure 2 shows an array of hysteresis curves (i.e., the ascending branch of the
422
PFEIFFER KCI
AND
DURHAM
concentration
30
P”
FIG. 2. How the size and shape of the titration hysteresis loop vary with ionic strength and temperature. Each individual curve represents the difference between the ascending and the descending branch of the titration curve of BMV between pH 6 and 7.2 under each set of conditions. The number in each frame is the pH of maximum hysteresis. -
titration curve subtracted from the descending branch) obtained between 0 and 30” and between 25 and 600 n-&f KCI. From these results, and from the original complete titration curves (not shown here), we draw these conclusions: (1) The size of the hysteresis loop, in terms of both height and total area, decreases slightly with increasing temperature or ionic strength but never completely disappears in the range studied. (2) The maximum number of protons per subunit titrating cooperatively in the hysteresis region is very close to two. Other protons also titrate more normally in this region. (3) Both protons titrate quite steeply and closely together on the ascending branch, at a pH that slowly decreases as ionic strength increases. (4) On the descending branch, one proton titrates relatively consistently near pH 6.3. The second proton titrates near there at high ionic strengths, but at low ionic strengths its titration is deferred until about pH 4.5. (5) The nonclosure of the hysteresis loop is more pronounced at lower ionic strengths and lower temperatures. (6) The pH al-
ways changes with time towards the points of maximum steepness in the titration curves, which are not the same on the ascending and descending branches. Therefore, the hysteresis loop would not close up completely if the titrations were performed still more slowly. Because divalent ions affect the structure and stability of BMV (Brakke, 1963, 1971; Incardona et al., 1973; Jacrot et al., 1976), we next titrated BMV at 22” in 50 n&Z KCI, to which various amounts of CaCI, or MgC12 had been added at acid pH. Figures 1D and E show complete titration curves in the presence of 1 mM Ca2+ and and Fig. 3 shows an Mg2+, respectively, array of the hysteresis curves obtained as the divalent ion concentration was varied. From all the Ca2+ and Mg2’ curves, we conclude that: (1) Both divalent cations efficiently close up the hysteresis loop and make the titration curves reversible. (2) Both cations displace one proton at concentrations below 1 m&I (which is equivalent to about 8 ions per protein subunit, or 0.5 ions per RNA phosphate). (3) Higher con-
BMV
CATION
BINDING
423
Other polycations worth investigating for their effect on titration curves are the polyamines (Bachrach, 19731, since they are often found associated with purified viruses. Their chain lengths and numbers of charges can be varied to investigate the structural requirements for binding. Structural changes in southern bean mosaic virus (unrelated to BMV) have previously been shown to depend on polyamines as well as on divalent metal cations (Hsu et al., 1976). As shown in Fig. lF, addition of polyaFIG. 3. How the size and shape of the titration mines to BMV efficiently closed up the hysteresis loop of BMV vary with concentration of hysteresis loop and made the curves reCaCl, (upper) and MgCl, (lower) added to 50 mM versible. Compared with Ca2+, and to a KC1 at 22”. The number in each frame is the pH of lesser extent Mg2+, polyamines were less maximum hysteresis. effective at displacing the protons involved in the hysteresis but much more effective at shifting the hysteresis to lower pH. Figure 4 shows how the positions of maximum steepness of the up and the down branches of the titration curves were shifted by added polyamines. Notice that spermine, with four positive charges, was more effective than spermidine, with three, which was in turn far more effective than the two diamines. An estimate of the affinity of the polyamines for BMV can be gained from the fact that alterations to the titration curve of BMV dialyzed against excess spermine first became noticeable at 0.1 mM concentration at pH 5. The changes caused by polyamines happened rapidly 60 65 and could be reversed by dialysis against PH 50 m&f KC1 at pH 5. Amantadine, a monoFIG. 4. Effects of four polyamines on the posiamine used as an antiviral drug (Parkes, tions of the steepest parts of the ascending (right) 19741,had no noticeable effect at a concenand the descending (left) branches of the titration tration of 14 mM. curves of BMV in 50 mM KCl. Solid squares, spermAs Durham et al. (1977) observed, BMV ine; open squares, spermidine; triangles, 1,4-diamiprecipitated at low pH and ionic strength, nobutane; circles, 1,5-diaminopentane. The vertical if it had once been swollen, in a way that scale expresses the ratio of positive charges provided correlated with the nonclosure of the hysby polyamines to negative charges on the virus teresis loop and varied slightly from batch RNA. Notice that both the vertical and horizontal scales are logarithmic. to batch of the virus. This did not happen if high concentrations of KC1 or low concentrations of Ca2+displace the second pro- centrations of polycations were present throughout the titration. Addition of a ton, thus effacing the hysteresis loop while not greatly shifting its position. (4) Higher larger amount of salt to precipitated BMV concentrations of Mg’+ tend to shifi the (below pH 5.2 in 50 mit4 KC11 could clarify position of the hysteresis loop before they the solution, For this “rescue,” spermine displace the second proton. (5) MgzC at 40 was effective at 1 mM concentration, while mM begins to break down the virus at high as much as 20 mM Ca2+or 170 mM K+ was needed to achieve the same results. PH.
424
PFEIFFER
AND
Precipitation after titration at low ionic strength is unlikely to have been due to a chemical change, since analytical gel-electrophoresis patterns of BMV RNA and protein (not shown) revealed no significant changes during the titration. On the other hand, ribonuclease A added to a precipitated virus suspension clarified it in seconds. Figure 5 shows how the various “rescue” agents affected the sedimentation behavior of BMV that had undergone a cycle of titration. Two classes of particles were generally formed: a fast component sedimenting around 80 S, very similar to native BMV, and a slower species sedimenting at about 70 S, which presumably corresponds to swollen, improperly refolded BMV. It is, however, difficult to judge whether RNA strands dangle outside both types of particles, thus allowing them to aggregate; this point is being studied now. Next, other changes in hydrodynamic properties were correlated with potentiometric titration by analytical ultracentrifugation. A gallery of Schlieren patterns summarizing the essential features is given in Fig. 6. The hysteresis loop does indeed correspond with a swelling of the virion, whose sedimentation coefficient dropped from 82 to about 65-72 S (depending on the batch) in 50 mM KC1 when the pH was raised. Moreover, part of the virus broke down at this low ionic strength, again to an extent that varied from batch
3OOmM KCI + Ribonudea*
+ Ca++ + spermine
FIG. 5. The “rescue” of BMV after precipitation on titration back to pH 5. (a) Lower trace, treated with 20 @g/ml of ribonuclease; upper trace, with the KC1 concentration increased to 300 mM; (b) lower trace, with 1 mM spermine added; upper trace, with 20 mhf Ca2+ added.
DURHAM
to batch. The components formed by breakdown are likely to be the same as those found at higher pH by Brakke (1971), which arose through dissociation of those particles containing one copy of each of the 0.3 and 0.7 million-dalton RNAs. At high pH in 50 n-&f KCI, BMV proved to be exceedingly sensitive to ribonuclease and broke down into what electron microscopy revealed to be the small T = 1 shells already described by Kassanis and Lebeurier (1969). Native BMV at pH 5 was fully resistant to ribonuclease, but BMV, returned to pH 5 after a cycle of titration in 50 n-&f KCl, was not; this indicates improper refolding of the virus with the RNA in an unnatural position, but the number and nature of the cleavage sites are still unknown. The second row of Schlieren patterns in Fig. 6 dramatically illustrates how the presence of divalent cations rendered the structural change fully reversible; this is further documented by the complete resistance to ribonuclease of virus back-titrated to pH 5. Finally, the third row shows how moderate concentrations of monovalent cations stablized BMV at high pH, thus reducing the extent of swelling and rendering the action of ribonuclease much more limited than at low ionic strength; again, BMV apparently refolded well upon return to low pH, but some of the particles were then still ribonuclease sensitive. When multivalent cations were added during the titration, their action depended very much upon the point reached in the titration. Figure 7 shows how BMV just about to swell was compacted back into the native form by addition of Ca*+ ions. The addition of spermine under the same conditions had the paradoxical effect of compacting the few particles that were already swollen (converting 72 to 76 S) but of simultaneously swelling those particles that were still compact (converting 82 to 76 S). This behavior was, however, to be expected from the way in which spermine had shifted the hysteresis to lower pH values. The Schlieren patterns in Fig. 8 show how adding divalent cations or moderately high concentrations of monovalent cations
BMV
CATION
425
BINDING
50mM KCI + Ca++ -J
km
L 83
300mM KCI 3 FIG. 6. The effects of monovalent and divalent ions on the reversibility of structural changes in BMV and on the susceptibility of the virus to pancreatic ribonuclease. Top row: BMV titrated in 50 n&f KC1 to various pHs: (a) Lower trace, the original BMV at pH 5; upper trace, titrated to pH 8 (note the partial disassembly); (b) the same as (a) but with 20 pg/ml of ribonuclease added; (c) lower trace, the original BMV at pH 5; upper trace, after titration back to pH 5.5 (note the partial compacting); (d) the same as (cl but with 20 pglml ribonuclease added. Middle row: Showing the stabilizing and protecting effect of 20 mM Ca2+ (upper) and 1 mM spermine (lower) ions on BMV during titration: (a) Titrated to pH 8; (b) the same, but with 20 pg/ml of ribonuclease added (showing the complete resistance to nuclease digestion); (cl after titration back to pH 5.5 (note the return to the S value of native BMV); (d) the same as (c), but with 20 pg/ml of ribonuclease added. Bottom row: Showing the effect of increased ionic strength on compactness of the virus at high pH, and on the reversibility of the conformational changes: (a) Lower trace, BMV at pH 5 in 300 mM KCl; upper trace, the same at pH 8 (note the lesser extent of swelling and increased homogeneity compared with 50 miW KCl; (b) the same as (a), but with 20 @g/ml ribonuclease added; (cl lower trace, the original BMV at pH 5 in 300 m&f KCl; upper trace, after titration back to pH 5.5; (d) the same as (cl, but with 20 pg/ml ribonuclease added.
during a titration affected the homogeneity of the virus and its resistance to ribonuclease. There it can be seen how spermine, in contrast with Ca2+, was unable to render swollen BMV fully resistant to ribonuclease. Both cations, added after the return to pH 5.5 in 50 mM KCl, made BMV only partially resistant to ribonuclease. This confirms that some of the RNA loops, once trapped in an unnatural position, can
no longer be pulled back to their proper positions. Interestingly, the addition of 300 nGI4 KC1 to BMV was much more effective in rendering the virus resistant to ribonuclease when added at pH 8, where the virus was swollen, than at pH 5. This was a general trend in the action of stabilizing agents: Much less was needed at pH 8 to facilitate return to normal, than at pH 5.5 to rescue the virus. Presumably at the
426
PFEIFFER
lower pH the compact permits the necessary sition so easily. Figure 9 shows the displaceable from BMV or Mg2+, as a function liter samples of BMV
structure no longer readjustment of ponumber of protons by addition of Ca*+ of pH. Five-milliwere titrated to a
i Spermine
&I++
FIG. 7. The effect of addition of cations on BMV about to swell. Lower traces, BMV at pH 6.49 on the ascending branch; upper trace left, with about 1.8 mM spermine added; upper trace right, with about 37 mM CaCIZ added.
50mM
KCI
AND
Spermine
added
DURHAM
given pH on the up curve or on the down curve; 100 ~1 of 100 m&I CaCl, or MgCl, were then added, and the amount of alkali needed to restore the pH to that value was noted. Then 200 ~1 of 1 M CaCl, or MgCl, were added and the amount of alkali needed again noted. These amounts correspond to about 16 and 340 cations per protein subunit, or about 1 and 19 cations per RNA phosphate respectively. From these results we deduce: (1) Only the two hysteresis protons are significantly displaced by CaZ+ or Mg2+; (2) the two sites differ significantly in their affinities for each cation tested; (3) the difference in affmity between Ca2+ and Mg*+ for any one site is very slight; and (4) the cation-binding sites are only partially restored when the virus refolds improperly in 50 mM KCl. Besides stabilizing the virus structure and making the titrations more reversible, high concentrations of monovalent cations could also displace protons. Therefore, we determined proton displacement figures by Ca++added
250 mM KCI added
FIG. 8. The effect of stabilizing agents added during titration. (No effect was seen on the original BMV at pH 5, so the patterns are not shown.) The effects of added cations were essentially instantaneous, but the photographs were obtained at least 0.5 hr after cation addition. Upper row: Lower traces, BMV titrated to pH 8 in 50 mM KC1 before the cations were added; upper traces, BMV titrated back to pH 5.5 before the cations were added. (a) No added cations; (b) 1 mM spermine added; (c) 20 mM CaCl, added; (d) KC1 concentration raised to 300 mM. Lower row: The same samples as in the upper row, but with 20 pglml of ribonuclease added 15 min after addition of the stabilizing agent.
BMV
CATION
FIG. 9. The numbers of protons displaceable by Mg’+ (upper) and Ca2+ (lower) from BMV in 50 mM KC1 at 22”, as a function of pH. Solid circles, cations added when the virus was on the ascending branch of the titration curve; open circles, virus on descending branch. Upper two lines in each figure, final divalent ion concentration about 37 m&f; lower two lines, about 1.8 mM. Reactions were complete in a matter of seconds.
Mg2+ and Ca2+, at a constant pH of 6.10, but at increasing KC1 concentration. Table 1 shows that raising the KC1 concentration above 50 mM displaced up to one proton per subunit from BMV. At the same time, the number of protons displaceable by Mg2+ and Ca2+ decreased, but the total number of protons displaceable remained remarkably constant.
Next the protein and the RNA of the virus were titrated separately. Figure 10A shows the titration curve for BMV-RNA in 50 n&f KCl. It is smooth, slowly varying, without hysteresis, and almost flat between pH 6 and 7. Addition of 40 mM Ca*+ to the RNA flattened the titration curve even more and displaced the equivalent of 0.3 proton per protein subunit in the virus, between pH 6 and 7. The integrity of the RNA and protein used for these curves was better than 95%, as judged by their analytical gel-electrophoresis patterns. Figure 10B shows the titration curve of BMV protein in 500 mM KC1 at 22”. The curve is relatively flat between pH 6 and 8, and its hysteresis loop occurs in the vicinity of pH 5.8 in 500 mM KCl, where capsids begin to form from dissociated protein (Pfeiffer and Hirth, 1974a). Raising the ionic strength displaced the hysteresis towards higher pH, agreeing with the observation of Pfeiffer and Hirth (1974b) that capsids form at higher pH as the ionic strength increases. Moderate concentrations of divalent cations had no effect on the titration of BMV protein. At 0” the hysteresis in the protein titration was shifted to slightly lower pH (not shown here). The sum of the titration curves of BMV RNA and protein is shown in Fig. 1OC.
TABLE PROTON
Final KC1 concentration (n&f)
H+ released bv added K”+
DISPLACEMENTV’
PRODUCED
H+ released
1 BY K+,
0.40 0.64 0.81 0.91 0.97 1.13
Mg*+,
AND
CaZ+ Total added
by added -
M&l,
50 100 200 300 500 800 1500
427
BINDING
CaCl,
1.8 mM
31 mM
1.8 mM
37 mM
0.60 0.29 0.09 0.05 0.02 0.02 0.02
1.29 0.97 0.64 0.45 0.35 0.31 0.25
0.84 0.35 0.25 0.18 0.05 0.05 0.04
1.77 1.28 0.86 0.78 0.51 0.51
0.42 Mean
H+ released K+ followed
by bv
31 mM Mg*+
37 mM
1.29 1.37 1.28 1.25 1.26 1.28 1.38
1.77 1.68 1.50 1.49 1.42 1.48 1.55
1.30
1.55
CaZ+
” Five milliliters of 3.2 g/liter BMV suspension in 50 m&f KC1 at 22” were titrated to pH 6.10, and sufficient solid KC1 was then added to produce the required ionic strength. The proton release induced by K+ was measured as the KOH uptake needed to restore pH 6.10; then 100 gl of 100 m&f MgCl, or CaClz were added, and pH 6.10 again restored; finally 200 ~1 of 1.0 M MgCl, or CaClz were added, and the pH again restored. All figures are corrected for the control alkali uptake observed with 5 ml of 50 mM KC1 dialysate.
428
PFEIFFER
AND
There are several differences between this and the titration curve of the virus, even after allowance for the inevitable buildup of inaccuracies in quantitation (i.e., on the vertical scale) inherent in such a comparison. Evidently some of the buffering capacity of RNA below pH 6, and of protein above pH 8, is pushed further away from neutrality when the components combine. This is qualitatively as would be expected when some RNA phosphates and protein lysine side chains are involved in charge pairs. However, the interesting point is the relative slopes in the hysteresis region near pH 6.5. Neither protein nor RNA alone, nor their sum, buffers as much or as cooperatively as virus; it is their combination in complete particles that generates specific cation binding sites. Finally, Fig. 10D shows the curves of one sample of BMV titrated up and down in pH twice, in 50 mikf KC1 at 22”. Notice that the two descending branches are very similar, but the two ascending branches are quite different, another demonstration that BMV refolded at low ionic strength differs from BMV in its initial state.
DURHAM
low concentrations of CaC12, MgC12, or polyamine hydrochlorides, permit BMV to regain its original state more easily. Multivalent ions have two effects on the hysteresis loop: They both suppress it and displace it to lower pH. Ca2+ ions tend to suppress more than displace; polyamines tend to displace more than suppress; Mg2+ is intermediate. The two sites differ clearly in their affinity for cations. From the data in Fig. 8 showing that 1.8 mi%f Caz+ or Mg2+ displaces one proton at pH 5, from a site with a pK, near 7.0 and assuming noncooperative titration in a fixed structure, following the simple Hender-
DISCUSSION
BMV contains several cation-binding sites per protein subunit, present only when both RNA and protein are correctly assembled into the virion. None of our evidence establishes exactly how many sites there are, but the most likely integral number per protein subunit is two. Both sites titrate together cooperatively at about pH 6.7 on the ascending branch of the curve. Their true pKH, if it could be observed uncomplicated by structural changes in the virion, would therefore be a little higher than this, perhaps about 7.0. Both sites have lower pK, values in the swollen virion, certainly below 6, and probably much lower still. When both sites bind H+, Ca*+, or Mg2+ ions, the virus is compact, but as the ions are released the virion swells. Restoring cations makes the virion contract again, but not necessarily by a direct reversal of its swelling pathway. At low ionic strength it does not regain its original conformation. High concentrations of KCl, or
L
II
I
5
6
I
L
7
6
-I
PH
FIG. 10. (A) Titration curve of 0.9 g/liter of BMV RNA in 50 mM KCI. Lower curve, without added Ca*+ ions; upper curve, with 40 mM CaCl* added at low pH; the relative vertical positioning of these two RNA curves is meaningful. These results are expressed as if the RNA were in its usual proportion in the virion, and the vertical scale is marked at intervals of one proton per protein subunit. (B) Titration curve of 4.5 g/liter of BMV protein in 500 mM KCl, starting from capsids at low pH. (C) The sum of the titration curves of BMV RNA and protein (the curve without Ca*+ from A and the ascending branch from B). (D) Titration of a single sample of 2 g/liter of BMV through two successive titration cycles (the first marked “1” and the second marked “2”). All four branches were arbitrarily aligned together at pH 8.0. (E, inset) The difference between the ascending and descending branches of the protein titration curve B.
BMV
CATION
BINDING
429
son-Hasselbach type of theory set out by dii of the two ions. At the other extreme, Durham and Hendry (1977), one may de- mathematical descriptions of enhanced duce a very approximate pKca and pK,, cation binding can be derived for generalfigure of 4.5 for one site. The correspondized regions of electronegativity, without ing figure for the second site is probably any precise structural assumptions (Mauabout 3.5. rel and Douzou, 1976). We propose that the observed behavior The simplest possible assumption capaof the virus can be explained as a conseble of explaining our data is that carboxylquence of simple electrostatic interactions ate and phosphate groups directly contribat the RNA-protein interface. Suppose ute some of the nearest neighbor atoms of that there are two carboxylate groups on the cations. The different cations could each BMV protein subunit, positioned so then take up slightly different positions. as to be close to RNA phosphate groups in Protons would probably bind more closely the compact form of the virion. The high to the carboxylates, Ca2+ a little less onelocal concentration of electronegativity sidedly, Mg2+ more towards the RNA, and raises the pK, of the carboxylate groups polyamines quite definitely on the RNA. and creates sites with significant affinity According to this view, Fig. 11 shows diafor metal cations. When the carboxylates grammatically the changes in charge durare free of attached cations, and negaing one hysteresis cycle. On the ascending tively charged, they repel the phosphates branch of the curve the virus is compact, and tend to make the virus swell. Once the with higher pKss, and has to go higher pH virus capsid is swollen the RNA can adjust to swell; on the descending branch, the its position. If the pH is lowered, or multiswollen virus has lower pK, values, and valent cations are added, the virion is has to go to lower pH to contract. pulled back into a compact state. It is possible that the RNA phosphates In this model, protein subunits are to a do not directly approach the metal cations. first approximation rigid bodies, while the Jacrot (1975) and his colleagues in CamRNA is more flexible. Small ions interpose bridge have suggested that cations bind to an electrostatic shield between charged CCMV at sites well away from the RNA, groups. KC1 therefore acts as a kind of so that the phosphate negative charges act nonspecific lubricant. CaCI, and MgCl, do only indirectly by stabilizing the protein the same at much lower concentrations, shell. In fact, the most recent studies on because Ca2+ and Mg2+ bind preferentially tobacco mosaic virus (Stubbs et al., 1977; to the most crucial positions. (Ionic Durham, et al., 1977; D. A. Hendry and A. strength is not a valid parameter for comC. H. Durham, paper in preparation) sugparing ions of different valences in this gest that cations bind to that virus in a type of interaction.) Polyamines bind cage of carboxylates near the axis of the mainly to RNA, neutralizing its charges, virus, with the RNA phosphates contributfavoring the stability of double-stranded ing negative charges nearby, perhaps in a regions (Bachrach, 19731, and pulling it charge-relay system, but without actually into a more compact form away from the approaching the cations directly. A similar protein. situation involving localized conformaThere are various precedents to guide tional changes in the protein might be true thinking about the structures of the catfor BMV too. ion-binding sites in BMV. X-ray crystalConcerning the hysteresis, one may ask lography generally shows that Ca*+ ions why the transition between partially proare bound by proteins inside cages of six or tonated swollen and partially protonated seven precisely positioned electronegative unswollen virus is forbidden; why is the oxygen atoms (Kretsinger and Nelson, apparent activation energy for the confor1976). However, the binding affinities of mational change a function of the total Ca2+ and Mg2+ are low compared with energy change for the reaction? We interthose in proteins like troponin and are also pret this phenomenon as a consequence of very similar despite the very different rathe large number of protein subunits that
430
PFEIFFER
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DURHAM
Low pti - aboutto contract
High
pli - swollen
t Forbidden transition I
Low pli - contmcted
virus
High
pti - about
b
well
FIG. 11. A diagram of the postulated structural changes in BMV as it traverses the hysteresis loop of its titration curve, starting from bottom left. Only the crucial negative charges on two protein subunits and a little of the RNA are shown. All other interactions, such as the attractive protein-RNA ionic links, are ignored for simplicity. The geometrical arrangement is only one among many possibilities.
have to change positions. In order for the change to proceed, a minimum number of subunits has to move and become a nucleus before the movement can propagate around the whole particle. This nucleus cannot build up while the free energy for an individual transition is too small. Thus, the hysteresis in the virus, which retains a constant molecular weight, is essentially analogous with the hysteresis involved in protein aggregation. In the hysteresis of BMV protein alone, we argue that the changes in pK, are probably fairly typical of any polymerizing protein and not directly relevant to the virus swelling. The improperly refolded state of all or some of the particles, at low pH after a titration at low ionic strength, presumably has some RNA strands caught at the surfaces of the contracted particles. This negatively charged RNA then cross-links the virus particles, whose surfaces are positively charged at this pH (Rice and Horst, 1972). A similar precipitation occurs when RNA is added to a suspension of capsids under these conditions (Pfeiffer, unpub-
lished results). Ribonuclease presumably resolubilizes the particles by cleaving the exposed RNA. Salts added to “rescue” the virus presumably suck the RNA back in, by neutralizing charges and at the same time lubricating the necessary relative movement of charges. BMV structural changes have also been investigated by Zulauf (personal communication of a draft paper), using intensity fluctuation spectroscopy. He found that the diffusion coefficient of the virus changed rapidly during swelling but that on contraction. there was a first fast step followed by a much slower annealing process. There are several ways in which the present understanding needs to be developed further: How do the three types of particle differ in cation binding and swelling? Which parts of the RNA stick out from swollen or incorrectly refolded virus? Which are the rate-determining steps in the hysteresis? How does the charge on BMV vary with solution conditions? Does BMV protein contain side-chain carboxyl-
BMV
CATION
ates close to the probable RNA-binding regions, as do for example ribosomal proteins (Ehresmann et al., 1976)? We believe that too much attention is currently given to the geometrical features that are peculiar to each virus, and not enough to the basic electrostatic interactions that are common to all polyelectrolytes. When two charged macromolecules come together in solution, numerous counterions are released or bound. In the case of opposite charges brought together by an RNA-protein interaction, Record et al. (1976) pointed out that the major net free energy change is the entropy of counterion release; there is thus a danger of confusing this with hydrophobic interactions. The present paper emphasizes the significance of repulsive interactions between like charges; their strength is critically dependent on the ionic content of the medium. We have not, however, stressed the well-known effects of ions in the medium in reducing electrostatic attractions and in changing the structure of water. Two of our results suggest that BMV probably does not disassemble in the cytoplasm of its host cell. First, BMV RNA and protein do not physically separate at pH 7 and moderate ionic strength, in the absence of multivalent cations. Second, BMV would bind substantial amounts of Mg’+ and polyamines, even if not Ca4*, from the concentrations of these ions typically prevailing in cytoplasm. One could postulate that some component of host cell cytoplasm actively pulls the RNA out of the virion, but we think it is more likely that BMV disassembles in the act of crossing a membrane (Durham, paper submitted for publication). Free energy to drive the process would come from the dilution of Ca2+ ions transported into cytoplasm. Bonds directly or indirectly involving protein carboxylates, metal ions, and phosphates are quite widespread in biology. Examples occur in actin-ATP (StrzeDrabikowski, lecka-Golaszewska and 1967), tubulin-GTP (Arai and Kaziro, 1976), thymidylate synthetase (Whitfield et al., 1976), many nucleic acid-handling and -degrading enzymes (e.g., Poulos and
431
BINDING
Price, 1974), phospholipase A2 (Van DamMieras et al., 1975), prothrombin (Furie et al., 1976), and all enzymes using ATP-Mg. It follows that small changes in intracellular divalent ion concentrations are capable of producing profound shifts in the pattern of cellular metabolism. The significance of this fact for virus replication, as well as for assembly and disassembly, is explained elsewhere (Durham, 1977; and paper submitted for publication). REFERENCES ADOLPH, K. W. (1975a). Conformational features of cowpea chlorotic mottle virus. J. Gen. Viral. 28, 137-145. ADOLPH, K. W. (1975b). Structural transitions of cowpea chlorotic mottle virus. J. Gen. Virol. 28, 147-154. ADOLPH, K. W., and BUTLER, P. J. G. (1974). Studies on the assembly of a spherical plant virus. I. States of aggregation of the isolated protein. J. Mol. Biol. 88, 327-341. AGRAWAL, H. O., and TREMAINE, J. H. (1972). Proteins of cowpea chlorotic mottle, broad bean mottle and brome mosaic viruses. Virology 47,8-20. ARAI, T., and KAZIRO, Y. (1976). Effect of guanine nucleotides on the assembly of brain microtubules: Ability of the C’-guanylyl imidodiphosphate to replace GTP in promoting the polymerization of microtubules in vitro. Biochen. Biophys. Res. Commun. 69, 369-376. BACHRACH, U. (1973). “Function of Naturally Occurring Polyamines.” Academic Press, New York. BANCROFT, J. B. (1970). The self-assembly of spherical plant viruses. Aduan. Virus Res. 16,99-134. BOCKSTAHLER, L. E., and KAESBERG, P. (1962). The molecular weight and other biophysical properties of bromegrass mosaic virus. Biophys. J. 2, l-9. BRAKKE, M. K. (1963). Stabilization of brome mosaic virus by magnesium and calcium. Virology 19, 367-374. BRAKKE, M. K. (1971). Degradation of brome mosaic virus and tobacco mosaic virus in bentonite. Virology 46, 575-585. DURHAM, A. C. H. (1977). Do viruses use calcium ions to shut off host cell functions? Nature (London) 267, 375. DURHAM, A. C. H., and HENDRY, D. A. (1977). Cation binding by tobacco mosaic virus. Virology 77, 510-519. DURHAM, A. C. H. HENDRY, D. A., and VON WECHMAR, M. B. (1977). Does calcium ion binding control plant virus disassembly? Virology 77, 524-533. DURHAM, A. C. H., VOGEL, D., and DE MARCILLAC, G. (1977). Hydrogen ion titration curves of tobacco
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mosaic virus protein polymers. Eur. J. Biochem., in press. EHRESMANN, B., REINBOLT, J., BACKENDORF, C., TRITSCH, D., and EBEL, J. P. (1976). Studies of the binding sites ofE.scherichia coli ribosomal protein S7 with 16s RNA by ultraviolet irradiation. FEBS Lett. 67, 316-319. FURIE, B. C., MANN, K. G., and FURIE, B. (1976). Substitution of lanthanide ions for calcium ions in the activation of bovine prothrombin by activated factor X. High affinity metal-binding sites of prothrombin and the derivatives of prothrombin activation. J. Biol. Chem. 251, 3235-3241. HERZOG, M., PFEIFFER, P., and HIRTH, L. (1976). Pathways of degradation of brome mosaic virus particles as influenced by their RNA content. Virology 69, 394-407. Hsu, C. H., SEHGAL, 0. P., and PICKETT, E. E. (1976). Stabilizing effect of divalent metal ions on virions of southern bean mosaic virus. Virology 69, 587-595. INCARDONA, N. L., and KAESBERG, P. (1964). A pHinduced structural change in bromegrass mosaic virus. Biophys. J. 4, 11-21. INCARDONA, N. L., MCKEE, S., and FLANEGAN, J. B. (1973). Noncovalent interactions in viruses: Characterization of their role in the pH and thermally induced conformational changes in bromegrass mosaic virus. Virology 53, 204-214. JACROT, B. (1975). Studies on the assembly of a spherical plant virus. II. The mechanism of protein aggregation and virus swelling. J. Mol. Biol. 95, 433-446. JACROT, B., PFEIFFER, P., and WITZ, J. (1976). The structure of a spherical plant virus (bromegrass mosaic virus) established by neutron scattering. Phil. Trans. Roy. Sot. Land. B 276, 109-112. JOHNSON, M. W., WAGNER, G. W., and BANCROFT, J. B. (19731. A titrimetric and electrophoretic study of cowpea chlorotic mottle virus and its protein. J. Gen. Virol. 19, 263-273. KASSANIS, B., and LEBEURIER, G. (1969). The behaviour of tomato bushy stunt virus and brome mosaic virus at different temperatures in vivo and in vitro. J. Gen. Virol. 4, 385-395. KRETSINGER, R. H., and NELSON, D. J. (1976). Calcium in biological systems. Coo&. Chem. Rev. 18, 29-124. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685.
DURHAM LANE, L. C. (1974). The bromoviruses. Aduan. Virus Res. 19, 151-220. LOENING, U. E. (1967). The fractionation of highmolecular-weight ribonucleic acid by polyacrylamide-gel electrophoresis. Biochem. J. 102, 251257. MAUREL, P., and Douzou, P. (1976). Catalytic implications of electrostatic potentials: The lytic activity of lysozyme as a model. J. Mol. Biol. 102, 253264. PARKES, D. (1974). Amantadine. Aduan. Drug Res. 8, 11-81. PFEIFFER, P., and HIRTH, L. (1974a). Formation of artificial top component from brome mosaic virus at high salt concentrations. Virology 58, 362-368. PFEIFFER, P., and HIRTH, L. (1974b). Aggregation states of brome mosaic virus protein. Virology 61, 160-167. PFEIFFER, P., and HIRTH, L. (1975). The effect of conformational changes in brome mosaic virus upon its sensitivity to trypsin, chymotrypsin and ribonuclease. FEBS Lett. 56, 144-148. POULOS, T. L., and PRICE, P. A. (1974). The involvement of serine and carboxyl groups in the activity of bovine pancreatic deoxyribonuclease A. J. Biol. Chem. 249, 1453-1457. RECORD, M. T., LOHMAN, T. M., and DE HASETH, P. (1976). Ion effects on ligand-nucleic acid interactions. J. Mol. Biol. 107, 145-158. RICE, R. H., and HORST, J. (1972). Isoelectric focusing of viruses in polyacrylamide. Virology 49,602604. STRZELECKA-GOLASZEWSKA, H., and DRABIKOWSKI, W. (1967). Correlation between the binding of calcium and ATP by G-actin. Acta Biochim. Pol. 14, 195-208. STUBBS, G. J., WARREN, S., and HOLMES, K. C. (1977). The structure of the RNA and the RNA binding site in tobacco mosaic virus as revealed by a 4 A map calculated from X-ray libre diagrams. Nature (London) 267, 216-221. VAN DAM-MIERAS, M. C. E., SLOTBOOM, A. J., PIETERSON, W. A., and DE HAAS, G. H. (19751. The interaction of phospholipase A2 with micellar interfaces. The role of the N-terminal region. Biochemistry 14, 5387-5394. WHITFIELD, J. F., MACMANLJS, J. P., RIXON, R. H., BOYNTON, A. L., YOUDALE, T., and SWIERENGA, S. (1976). The positive control of proliferation by the interplay of calcium ions and cyclic nucleotides. A review. In Vitro 12, l-17.
81,
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The Cation
(1977)
Binding
PIERRE Laboratoire
de Virologie,
Associated with Structural Bromegrass Mosaic Virus
PFEIFFER Institut
AND
ANTHONY
de Biologic Moldculuire 67084 Strasbourg, Accepted
May
Transitions
in
C. H. DURHAM’
et Cellulaire France
du CNRS,
15 rue Descartes,
1,1977
Hydrogen ion titration curves are described for bromegrass mosaic virus under various conditions of temperature, ionic strength, and Ca2+, Mg2+, or polyamine concentration, with particular reference to the hysteresis region near neutral pH. The curves are correlated with the sedimentation properties of the virus. There are two relatively nonspecific cation-binding sites per protein subunit, present in the virion but not in the protein or RNA alone, which control the virion’s swelling. The swelling is reversible in the presence of enough cations; without them, swollen BMV can contract to an incorrect structure with RNA partially exposed. We interpret these results qualitatively and structurally in terms of binding sites formed in a region of high electronegativity contributed by RNA phosphates and protein carboxylates. INTRODUCTION
is the ion whose binding is most easily measured (Durham and Hendry, 1977). Here we report the hydrogen ion titration curves of BMV in the presence and absence of K+, Mg2+, Ca2+, and polyamine ions. Thanks to the large quantities of BMV that can be prepared easily, we have been able to observe the hysteresis region of the curves, corresponding to the swelling transition of the virus, in detail. The results are most easily interpreted in terms of simple electrostatic interactions at the RNA-protein interface.
Bromegrass mosaic virus (BMV) is a small spherical three-component plant virus which has been extensively studied (for reviews see Bancroft, 1970; Lane, 1974). It swells near neutral pH in the absence of multivalent cations (Incardona and Kaesberg, 1964; Incardona et al., 19731, and the RNA then moves towards the particle surface (Jacrot et al., 1976). Swollen BMV is sensitive to nucleases (Brakke, 1963; Kassanis and Lebeurier, 1969) and proteases (Agrawal and Tremaine, 1972; Pfeiffer and Hirth, 1975). The closely related cowpea chlorotic mottle virus (CCMV) shows many similar features and has been studied in some detail (Johnson et al., 1973; Adolph and Butler, 1974; Adolph, 1975a and b; Jacrot, 1975). Most, perhaps all, plant viruses possess distinctive sites for binding cations (Durham et al., 1977). Durham (submitted for publication) has suggested that these sites function in viva by transporting Ca2+ ions across membranes to provide free energy for simultaneous virus penetration and decapsidation. However, experimentally H+ I Author dressed.
to whom
reprint
requests
should
MATERIALS
AND
METHODS
BMV (the common strain derived initially by Dr. Lane of the University of Wisconsin) was multiplied in barley plants, cv. Rika, and purified by an improved version of the method of Bockstahler and Kaesberg (1962). Fresh leaves were collected 12 days after inoculation and minced, and their juice was squeezed out. The residue was rehydrated with one-half volume of distilled water and again squeezed out. All the juice was pooled. After standing overnight at pH 4.8 in the cold room, to precipitate the host plant proteins, and after low speed centrifuga-
be ad419
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0042-6822
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PFEIFFER
AND
tion, the clear supernatant fluid was made and 6% in polyethylene glycol 20,000. The virus precipitate was collected by a 30-min centrifugation at 10,000 rpm and submitted to several cycles of high and low speed centrifugation. Resuspending virus pellets in distilled water rather than in buffer helped to eliminate contaminating plant membranes. BMV protein and RNA were prepared simultaneously by degradation of the virus in 1 M CaClz at pH 6.7 (Herzog et al., 1976). Samples of virus, protein, or RNA, at concentrations of 3 to 4 g/liter, were prepared for titration by dialysis against about 20 mM sodium EDTA, pH 5, followed by several days of dialysis against many changes of 50 mM KCl, adjusted to pH 5 with HCl, followed by addition of solid KC1 to the required KC1 concentration. (Any polyvalent cations firmly enough bound to resist this treatment would probably have had no effect on the subsequent experiments.) Titration results are expressed as protons per protein subunit, after correction for titration of the solvent, using the following extinction coefficients: virus, E .$$“A% = 5.08; RNA, E .&~~~t; = 23.0; and protein, E 1280 g/liter “In = 0.76; and taking the protein subunit weight as 20,300, the overall RNA content of the virus as 21.4%, and the number of protein subunits per virion as 180 (Lane, 1974). Titrations were performed under CO,free damp air, with the same apparatus and techniques as used by Durham and Hendry (1977), but in a thermostatically controlled vessel rather than in an airconditioned room. Titrants were 10 mM KOH and HCl, adjusted to the ionic strength of the sample with KCl. Five experimental details are worth emphasizing. The 10 n&f titrants were a compromise; stronger titrants would have reduced the precision of volume measurements and caused local denaturation of the virus, whereas weaker titrants would have increased the correction necessary for titration of solvent alone. To reduce CO, pickup, all samples were kept at or below pH 5 before titration, and KOH titrants were carefully protected from atmospheric
0.1 it4 in NaCl
DURHAM
CO,. A major source of error was slow titrant leakage past the three-way stopcock of our autoburets; for the slow titrations it was sometimes necessary to measure this rate of leakage and correct for it in the data handling. The third digit on the pH meter was very valuable for observing pH changes with time. It was essential to take great care to dialyze away all EDTA before beginning titrations. Analytical ultracentrifugation of undiluted samples taken directly from the titration vessel was performed by G. de Marcillac, using a Beckman Spinco Model E ultracentrifuge equipped with Schlieren optics, running at 35,600 rpm and 20 to 25 with bar angle 50 to 60”. Native BMV at pH 5 was often used as an internal standard for viscosity and density correction. Polyacrylamide-gel electrophoreses were performed as described by Loening (1967) on cylindrical 2.8% gels for RNA and according to Laemmli (1970) on slab gels for protein analysis. RNA gels were scanned at 260-nm wavelength in a Beckman Acta III recording spectrophotometer equipped with a gel-scanning attachment. Chemicals were of analytical grade, purchased mainly from Merck. Polyamine hydrochlorides, ribonuclease A, and chymotrypsin were purchased from Sigma. RESULTS
The titration curve of BMV at 22” in 50 m&f KC1 (Fig. 1A) is the basic standard of comparison for all conditions studied here. It is essentially the same as the curve that Durham et al. (1977) found for their independently propagated strain in their preliminary study of BMV titration. Figures 1B to E show how the curve changed if temperature, ionic strength, or divalent ion concentrations were changed. Although there were small changes elsewhere, it was the hysteresis region between pH 6.0 and 7.2, where the virus conformation changes, which we investigated in detail. Observing the time-dependent change of pH in the hysteresis region presented certain technical problems. To obtain consistent measurements there, we used an automatic device to command the autoburet to
421
EMV CATION BINDING
I
4
I
1
5
I
1
6
I
I
PH
7
I
I
8
I
1
FIG. 1. Titration curves of BMV under the following conditions: (A) In 50 mM KC1 at 22”; (B) as for A, but at 0”; (C) as for A, but in 300 mM KCl; (D) as for A, plus 1 mM CaCl,; (E) as for A, plus 1 mM MgCl*; (F) as for A, plus 0.5 mM spermine. The vertical axis is marked at intervals of one proton per subunit. The vertical positioning of the curves is arbitrary.
deliver 20 ~1 of titrant (equivalent to about 0.3 protons per subunit) regularly every 4 min. At this slow rate of titration, the experimental problems of buret leakage, titrant strength determination, and virus denaturation often accumulated to a discrepancy of up to half a proton per subunit between the beginning and end of a titration. On the other hand, pH measurements were probably dependable to + 0.02
pH unit or better. In general, therefore, before drawing significant conclusions we accumulated a whole array of titration curves under related conditions, ofien obtaining the curves with several different batches of virus, The first variables studied were temperature and ionic strength provided by KCl. Figure 2 shows an array of hysteresis curves (i.e., the ascending branch of the
422
PFEIFFER KCI
AND
DURHAM
concentration
30
P”
FIG. 2. How the size and shape of the titration hysteresis loop vary with ionic strength and temperature. Each individual curve represents the difference between the ascending and the descending branch of the titration curve of BMV between pH 6 and 7.2 under each set of conditions. The number in each frame is the pH of maximum hysteresis. -
titration curve subtracted from the descending branch) obtained between 0 and 30” and between 25 and 600 n-&f KCI. From these results, and from the original complete titration curves (not shown here), we draw these conclusions: (1) The size of the hysteresis loop, in terms of both height and total area, decreases slightly with increasing temperature or ionic strength but never completely disappears in the range studied. (2) The maximum number of protons per subunit titrating cooperatively in the hysteresis region is very close to two. Other protons also titrate more normally in this region. (3) Both protons titrate quite steeply and closely together on the ascending branch, at a pH that slowly decreases as ionic strength increases. (4) On the descending branch, one proton titrates relatively consistently near pH 6.3. The second proton titrates near there at high ionic strengths, but at low ionic strengths its titration is deferred until about pH 4.5. (5) The nonclosure of the hysteresis loop is more pronounced at lower ionic strengths and lower temperatures. (6) The pH al-
ways changes with time towards the points of maximum steepness in the titration curves, which are not the same on the ascending and descending branches. Therefore, the hysteresis loop would not close up completely if the titrations were performed still more slowly. Because divalent ions affect the structure and stability of BMV (Brakke, 1963, 1971; Incardona et al., 1973; Jacrot et al., 1976), we next titrated BMV at 22” in 50 n&Z KCI, to which various amounts of CaCI, or MgC12 had been added at acid pH. Figures 1D and E show complete titration curves in the presence of 1 mM Ca2+ and and Fig. 3 shows an Mg2+, respectively, array of the hysteresis curves obtained as the divalent ion concentration was varied. From all the Ca2+ and Mg2’ curves, we conclude that: (1) Both divalent cations efficiently close up the hysteresis loop and make the titration curves reversible. (2) Both cations displace one proton at concentrations below 1 m&I (which is equivalent to about 8 ions per protein subunit, or 0.5 ions per RNA phosphate). (3) Higher con-
BMV
CATION
BINDING
423
Other polycations worth investigating for their effect on titration curves are the polyamines (Bachrach, 19731, since they are often found associated with purified viruses. Their chain lengths and numbers of charges can be varied to investigate the structural requirements for binding. Structural changes in southern bean mosaic virus (unrelated to BMV) have previously been shown to depend on polyamines as well as on divalent metal cations (Hsu et al., 1976). As shown in Fig. lF, addition of polyaFIG. 3. How the size and shape of the titration mines to BMV efficiently closed up the hysteresis loop of BMV vary with concentration of hysteresis loop and made the curves reCaCl, (upper) and MgCl, (lower) added to 50 mM versible. Compared with Ca2+, and to a KC1 at 22”. The number in each frame is the pH of lesser extent Mg2+, polyamines were less maximum hysteresis. effective at displacing the protons involved in the hysteresis but much more effective at shifting the hysteresis to lower pH. Figure 4 shows how the positions of maximum steepness of the up and the down branches of the titration curves were shifted by added polyamines. Notice that spermine, with four positive charges, was more effective than spermidine, with three, which was in turn far more effective than the two diamines. An estimate of the affinity of the polyamines for BMV can be gained from the fact that alterations to the titration curve of BMV dialyzed against excess spermine first became noticeable at 0.1 mM concentration at pH 5. The changes caused by polyamines happened rapidly 60 65 and could be reversed by dialysis against PH 50 m&f KC1 at pH 5. Amantadine, a monoFIG. 4. Effects of four polyamines on the posiamine used as an antiviral drug (Parkes, tions of the steepest parts of the ascending (right) 19741,had no noticeable effect at a concenand the descending (left) branches of the titration tration of 14 mM. curves of BMV in 50 mM KCl. Solid squares, spermAs Durham et al. (1977) observed, BMV ine; open squares, spermidine; triangles, 1,4-diamiprecipitated at low pH and ionic strength, nobutane; circles, 1,5-diaminopentane. The vertical if it had once been swollen, in a way that scale expresses the ratio of positive charges provided correlated with the nonclosure of the hysby polyamines to negative charges on the virus teresis loop and varied slightly from batch RNA. Notice that both the vertical and horizontal scales are logarithmic. to batch of the virus. This did not happen if high concentrations of KC1 or low concentrations of Ca2+displace the second pro- centrations of polycations were present throughout the titration. Addition of a ton, thus effacing the hysteresis loop while not greatly shifting its position. (4) Higher larger amount of salt to precipitated BMV concentrations of Mg’+ tend to shifi the (below pH 5.2 in 50 mit4 KC11 could clarify position of the hysteresis loop before they the solution, For this “rescue,” spermine displace the second proton. (5) MgzC at 40 was effective at 1 mM concentration, while mM begins to break down the virus at high as much as 20 mM Ca2+or 170 mM K+ was needed to achieve the same results. PH.
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Precipitation after titration at low ionic strength is unlikely to have been due to a chemical change, since analytical gel-electrophoresis patterns of BMV RNA and protein (not shown) revealed no significant changes during the titration. On the other hand, ribonuclease A added to a precipitated virus suspension clarified it in seconds. Figure 5 shows how the various “rescue” agents affected the sedimentation behavior of BMV that had undergone a cycle of titration. Two classes of particles were generally formed: a fast component sedimenting around 80 S, very similar to native BMV, and a slower species sedimenting at about 70 S, which presumably corresponds to swollen, improperly refolded BMV. It is, however, difficult to judge whether RNA strands dangle outside both types of particles, thus allowing them to aggregate; this point is being studied now. Next, other changes in hydrodynamic properties were correlated with potentiometric titration by analytical ultracentrifugation. A gallery of Schlieren patterns summarizing the essential features is given in Fig. 6. The hysteresis loop does indeed correspond with a swelling of the virion, whose sedimentation coefficient dropped from 82 to about 65-72 S (depending on the batch) in 50 mM KC1 when the pH was raised. Moreover, part of the virus broke down at this low ionic strength, again to an extent that varied from batch
3OOmM KCI + Ribonudea*
+ Ca++ + spermine
FIG. 5. The “rescue” of BMV after precipitation on titration back to pH 5. (a) Lower trace, treated with 20 @g/ml of ribonuclease; upper trace, with the KC1 concentration increased to 300 mM; (b) lower trace, with 1 mM spermine added; upper trace, with 20 mhf Ca2+ added.
DURHAM
to batch. The components formed by breakdown are likely to be the same as those found at higher pH by Brakke (1971), which arose through dissociation of those particles containing one copy of each of the 0.3 and 0.7 million-dalton RNAs. At high pH in 50 n-&f KCI, BMV proved to be exceedingly sensitive to ribonuclease and broke down into what electron microscopy revealed to be the small T = 1 shells already described by Kassanis and Lebeurier (1969). Native BMV at pH 5 was fully resistant to ribonuclease, but BMV, returned to pH 5 after a cycle of titration in 50 n-&f KCl, was not; this indicates improper refolding of the virus with the RNA in an unnatural position, but the number and nature of the cleavage sites are still unknown. The second row of Schlieren patterns in Fig. 6 dramatically illustrates how the presence of divalent cations rendered the structural change fully reversible; this is further documented by the complete resistance to ribonuclease of virus back-titrated to pH 5. Finally, the third row shows how moderate concentrations of monovalent cations stablized BMV at high pH, thus reducing the extent of swelling and rendering the action of ribonuclease much more limited than at low ionic strength; again, BMV apparently refolded well upon return to low pH, but some of the particles were then still ribonuclease sensitive. When multivalent cations were added during the titration, their action depended very much upon the point reached in the titration. Figure 7 shows how BMV just about to swell was compacted back into the native form by addition of Ca*+ ions. The addition of spermine under the same conditions had the paradoxical effect of compacting the few particles that were already swollen (converting 72 to 76 S) but of simultaneously swelling those particles that were still compact (converting 82 to 76 S). This behavior was, however, to be expected from the way in which spermine had shifted the hysteresis to lower pH values. The Schlieren patterns in Fig. 8 show how adding divalent cations or moderately high concentrations of monovalent cations
BMV
CATION
425
BINDING
50mM KCI + Ca++ -J
km
L 83
300mM KCI 3 FIG. 6. The effects of monovalent and divalent ions on the reversibility of structural changes in BMV and on the susceptibility of the virus to pancreatic ribonuclease. Top row: BMV titrated in 50 n&f KC1 to various pHs: (a) Lower trace, the original BMV at pH 5; upper trace, titrated to pH 8 (note the partial disassembly); (b) the same as (a) but with 20 pg/ml of ribonuclease added; (c) lower trace, the original BMV at pH 5; upper trace, after titration back to pH 5.5 (note the partial compacting); (d) the same as (cl but with 20 pglml ribonuclease added. Middle row: Showing the stabilizing and protecting effect of 20 mM Ca2+ (upper) and 1 mM spermine (lower) ions on BMV during titration: (a) Titrated to pH 8; (b) the same, but with 20 pg/ml of ribonuclease added (showing the complete resistance to nuclease digestion); (cl after titration back to pH 5.5 (note the return to the S value of native BMV); (d) the same as (c), but with 20 pg/ml of ribonuclease added. Bottom row: Showing the effect of increased ionic strength on compactness of the virus at high pH, and on the reversibility of the conformational changes: (a) Lower trace, BMV at pH 5 in 300 mM KCl; upper trace, the same at pH 8 (note the lesser extent of swelling and increased homogeneity compared with 50 miW KCl; (b) the same as (a), but with 20 @g/ml ribonuclease added; (cl lower trace, the original BMV at pH 5 in 300 m&f KCl; upper trace, after titration back to pH 5.5; (d) the same as (cl, but with 20 pg/ml ribonuclease added.
during a titration affected the homogeneity of the virus and its resistance to ribonuclease. There it can be seen how spermine, in contrast with Ca2+, was unable to render swollen BMV fully resistant to ribonuclease. Both cations, added after the return to pH 5.5 in 50 mM KCl, made BMV only partially resistant to ribonuclease. This confirms that some of the RNA loops, once trapped in an unnatural position, can
no longer be pulled back to their proper positions. Interestingly, the addition of 300 nGI4 KC1 to BMV was much more effective in rendering the virus resistant to ribonuclease when added at pH 8, where the virus was swollen, than at pH 5. This was a general trend in the action of stabilizing agents: Much less was needed at pH 8 to facilitate return to normal, than at pH 5.5 to rescue the virus. Presumably at the
426
PFEIFFER
lower pH the compact permits the necessary sition so easily. Figure 9 shows the displaceable from BMV or Mg2+, as a function liter samples of BMV
structure no longer readjustment of ponumber of protons by addition of Ca*+ of pH. Five-milliwere titrated to a
i Spermine
&I++
FIG. 7. The effect of addition of cations on BMV about to swell. Lower traces, BMV at pH 6.49 on the ascending branch; upper trace left, with about 1.8 mM spermine added; upper trace right, with about 37 mM CaCIZ added.
50mM
KCI
AND
Spermine
added
DURHAM
given pH on the up curve or on the down curve; 100 ~1 of 100 m&I CaCl, or MgCl, were then added, and the amount of alkali needed to restore the pH to that value was noted. Then 200 ~1 of 1 M CaCl, or MgCl, were added and the amount of alkali needed again noted. These amounts correspond to about 16 and 340 cations per protein subunit, or about 1 and 19 cations per RNA phosphate respectively. From these results we deduce: (1) Only the two hysteresis protons are significantly displaced by CaZ+ or Mg2+; (2) the two sites differ significantly in their affinities for each cation tested; (3) the difference in affmity between Ca2+ and Mg*+ for any one site is very slight; and (4) the cation-binding sites are only partially restored when the virus refolds improperly in 50 mM KCl. Besides stabilizing the virus structure and making the titrations more reversible, high concentrations of monovalent cations could also displace protons. Therefore, we determined proton displacement figures by Ca++added
250 mM KCI added
FIG. 8. The effect of stabilizing agents added during titration. (No effect was seen on the original BMV at pH 5, so the patterns are not shown.) The effects of added cations were essentially instantaneous, but the photographs were obtained at least 0.5 hr after cation addition. Upper row: Lower traces, BMV titrated to pH 8 in 50 mM KC1 before the cations were added; upper traces, BMV titrated back to pH 5.5 before the cations were added. (a) No added cations; (b) 1 mM spermine added; (c) 20 mM CaCl, added; (d) KC1 concentration raised to 300 mM. Lower row: The same samples as in the upper row, but with 20 pglml of ribonuclease added 15 min after addition of the stabilizing agent.
BMV
CATION
FIG. 9. The numbers of protons displaceable by Mg’+ (upper) and Ca2+ (lower) from BMV in 50 mM KC1 at 22”, as a function of pH. Solid circles, cations added when the virus was on the ascending branch of the titration curve; open circles, virus on descending branch. Upper two lines in each figure, final divalent ion concentration about 37 m&f; lower two lines, about 1.8 mM. Reactions were complete in a matter of seconds.
Mg2+ and Ca2+, at a constant pH of 6.10, but at increasing KC1 concentration. Table 1 shows that raising the KC1 concentration above 50 mM displaced up to one proton per subunit from BMV. At the same time, the number of protons displaceable by Mg2+ and Ca2+ decreased, but the total number of protons displaceable remained remarkably constant.
Next the protein and the RNA of the virus were titrated separately. Figure 10A shows the titration curve for BMV-RNA in 50 n&f KCl. It is smooth, slowly varying, without hysteresis, and almost flat between pH 6 and 7. Addition of 40 mM Ca*+ to the RNA flattened the titration curve even more and displaced the equivalent of 0.3 proton per protein subunit in the virus, between pH 6 and 7. The integrity of the RNA and protein used for these curves was better than 95%, as judged by their analytical gel-electrophoresis patterns. Figure 10B shows the titration curve of BMV protein in 500 mM KC1 at 22”. The curve is relatively flat between pH 6 and 8, and its hysteresis loop occurs in the vicinity of pH 5.8 in 500 mM KCl, where capsids begin to form from dissociated protein (Pfeiffer and Hirth, 1974a). Raising the ionic strength displaced the hysteresis towards higher pH, agreeing with the observation of Pfeiffer and Hirth (1974b) that capsids form at higher pH as the ionic strength increases. Moderate concentrations of divalent cations had no effect on the titration of BMV protein. At 0” the hysteresis in the protein titration was shifted to slightly lower pH (not shown here). The sum of the titration curves of BMV RNA and protein is shown in Fig. 1OC.
TABLE PROTON
Final KC1 concentration (n&f)
H+ released bv added K”+
DISPLACEMENTV’
PRODUCED
H+ released
1 BY K+,
0.40 0.64 0.81 0.91 0.97 1.13
Mg*+,
AND
CaZ+ Total added
by added -
M&l,
50 100 200 300 500 800 1500
427
BINDING
CaCl,
1.8 mM
31 mM
1.8 mM
37 mM
0.60 0.29 0.09 0.05 0.02 0.02 0.02
1.29 0.97 0.64 0.45 0.35 0.31 0.25
0.84 0.35 0.25 0.18 0.05 0.05 0.04
1.77 1.28 0.86 0.78 0.51 0.51
0.42 Mean
H+ released K+ followed
by bv
31 mM Mg*+
37 mM
1.29 1.37 1.28 1.25 1.26 1.28 1.38
1.77 1.68 1.50 1.49 1.42 1.48 1.55
1.30
1.55
CaZ+
” Five milliliters of 3.2 g/liter BMV suspension in 50 m&f KC1 at 22” were titrated to pH 6.10, and sufficient solid KC1 was then added to produce the required ionic strength. The proton release induced by K+ was measured as the KOH uptake needed to restore pH 6.10; then 100 gl of 100 m&f MgCl, or CaClz were added, and pH 6.10 again restored; finally 200 ~1 of 1.0 M MgCl, or CaClz were added, and the pH again restored. All figures are corrected for the control alkali uptake observed with 5 ml of 50 mM KC1 dialysate.
428
PFEIFFER
AND
There are several differences between this and the titration curve of the virus, even after allowance for the inevitable buildup of inaccuracies in quantitation (i.e., on the vertical scale) inherent in such a comparison. Evidently some of the buffering capacity of RNA below pH 6, and of protein above pH 8, is pushed further away from neutrality when the components combine. This is qualitatively as would be expected when some RNA phosphates and protein lysine side chains are involved in charge pairs. However, the interesting point is the relative slopes in the hysteresis region near pH 6.5. Neither protein nor RNA alone, nor their sum, buffers as much or as cooperatively as virus; it is their combination in complete particles that generates specific cation binding sites. Finally, Fig. 10D shows the curves of one sample of BMV titrated up and down in pH twice, in 50 mikf KC1 at 22”. Notice that the two descending branches are very similar, but the two ascending branches are quite different, another demonstration that BMV refolded at low ionic strength differs from BMV in its initial state.
DURHAM
low concentrations of CaC12, MgC12, or polyamine hydrochlorides, permit BMV to regain its original state more easily. Multivalent ions have two effects on the hysteresis loop: They both suppress it and displace it to lower pH. Ca2+ ions tend to suppress more than displace; polyamines tend to displace more than suppress; Mg2+ is intermediate. The two sites differ clearly in their affinity for cations. From the data in Fig. 8 showing that 1.8 mi%f Caz+ or Mg2+ displaces one proton at pH 5, from a site with a pK, near 7.0 and assuming noncooperative titration in a fixed structure, following the simple Hender-
DISCUSSION
BMV contains several cation-binding sites per protein subunit, present only when both RNA and protein are correctly assembled into the virion. None of our evidence establishes exactly how many sites there are, but the most likely integral number per protein subunit is two. Both sites titrate together cooperatively at about pH 6.7 on the ascending branch of the curve. Their true pKH, if it could be observed uncomplicated by structural changes in the virion, would therefore be a little higher than this, perhaps about 7.0. Both sites have lower pK, values in the swollen virion, certainly below 6, and probably much lower still. When both sites bind H+, Ca*+, or Mg2+ ions, the virus is compact, but as the ions are released the virion swells. Restoring cations makes the virion contract again, but not necessarily by a direct reversal of its swelling pathway. At low ionic strength it does not regain its original conformation. High concentrations of KCl, or
L
II
I
5
6
I
L
7
6
-I
PH
FIG. 10. (A) Titration curve of 0.9 g/liter of BMV RNA in 50 mM KCI. Lower curve, without added Ca*+ ions; upper curve, with 40 mM CaCl* added at low pH; the relative vertical positioning of these two RNA curves is meaningful. These results are expressed as if the RNA were in its usual proportion in the virion, and the vertical scale is marked at intervals of one proton per protein subunit. (B) Titration curve of 4.5 g/liter of BMV protein in 500 mM KCl, starting from capsids at low pH. (C) The sum of the titration curves of BMV RNA and protein (the curve without Ca*+ from A and the ascending branch from B). (D) Titration of a single sample of 2 g/liter of BMV through two successive titration cycles (the first marked “1” and the second marked “2”). All four branches were arbitrarily aligned together at pH 8.0. (E, inset) The difference between the ascending and descending branches of the protein titration curve B.
BMV
CATION
BINDING
429
son-Hasselbach type of theory set out by dii of the two ions. At the other extreme, Durham and Hendry (1977), one may de- mathematical descriptions of enhanced duce a very approximate pKca and pK,, cation binding can be derived for generalfigure of 4.5 for one site. The correspondized regions of electronegativity, without ing figure for the second site is probably any precise structural assumptions (Mauabout 3.5. rel and Douzou, 1976). We propose that the observed behavior The simplest possible assumption capaof the virus can be explained as a conseble of explaining our data is that carboxylquence of simple electrostatic interactions ate and phosphate groups directly contribat the RNA-protein interface. Suppose ute some of the nearest neighbor atoms of that there are two carboxylate groups on the cations. The different cations could each BMV protein subunit, positioned so then take up slightly different positions. as to be close to RNA phosphate groups in Protons would probably bind more closely the compact form of the virion. The high to the carboxylates, Ca2+ a little less onelocal concentration of electronegativity sidedly, Mg2+ more towards the RNA, and raises the pK, of the carboxylate groups polyamines quite definitely on the RNA. and creates sites with significant affinity According to this view, Fig. 11 shows diafor metal cations. When the carboxylates grammatically the changes in charge durare free of attached cations, and negaing one hysteresis cycle. On the ascending tively charged, they repel the phosphates branch of the curve the virus is compact, and tend to make the virus swell. Once the with higher pKss, and has to go higher pH virus capsid is swollen the RNA can adjust to swell; on the descending branch, the its position. If the pH is lowered, or multiswollen virus has lower pK, values, and valent cations are added, the virion is has to go to lower pH to contract. pulled back into a compact state. It is possible that the RNA phosphates In this model, protein subunits are to a do not directly approach the metal cations. first approximation rigid bodies, while the Jacrot (1975) and his colleagues in CamRNA is more flexible. Small ions interpose bridge have suggested that cations bind to an electrostatic shield between charged CCMV at sites well away from the RNA, groups. KC1 therefore acts as a kind of so that the phosphate negative charges act nonspecific lubricant. CaCI, and MgCl, do only indirectly by stabilizing the protein the same at much lower concentrations, shell. In fact, the most recent studies on because Ca2+ and Mg2+ bind preferentially tobacco mosaic virus (Stubbs et al., 1977; to the most crucial positions. (Ionic Durham, et al., 1977; D. A. Hendry and A. strength is not a valid parameter for comC. H. Durham, paper in preparation) sugparing ions of different valences in this gest that cations bind to that virus in a type of interaction.) Polyamines bind cage of carboxylates near the axis of the mainly to RNA, neutralizing its charges, virus, with the RNA phosphates contributfavoring the stability of double-stranded ing negative charges nearby, perhaps in a regions (Bachrach, 19731, and pulling it charge-relay system, but without actually into a more compact form away from the approaching the cations directly. A similar protein. situation involving localized conformaThere are various precedents to guide tional changes in the protein might be true thinking about the structures of the catfor BMV too. ion-binding sites in BMV. X-ray crystalConcerning the hysteresis, one may ask lography generally shows that Ca*+ ions why the transition between partially proare bound by proteins inside cages of six or tonated swollen and partially protonated seven precisely positioned electronegative unswollen virus is forbidden; why is the oxygen atoms (Kretsinger and Nelson, apparent activation energy for the confor1976). However, the binding affinities of mational change a function of the total Ca2+ and Mg2+ are low compared with energy change for the reaction? We interthose in proteins like troponin and are also pret this phenomenon as a consequence of very similar despite the very different rathe large number of protein subunits that
430
PFEIFFER
AND
DURHAM
Low pti - aboutto contract
High
pli - swollen
t Forbidden transition I
Low pli - contmcted
virus
High
pti - about
b
well
FIG. 11. A diagram of the postulated structural changes in BMV as it traverses the hysteresis loop of its titration curve, starting from bottom left. Only the crucial negative charges on two protein subunits and a little of the RNA are shown. All other interactions, such as the attractive protein-RNA ionic links, are ignored for simplicity. The geometrical arrangement is only one among many possibilities.
have to change positions. In order for the change to proceed, a minimum number of subunits has to move and become a nucleus before the movement can propagate around the whole particle. This nucleus cannot build up while the free energy for an individual transition is too small. Thus, the hysteresis in the virus, which retains a constant molecular weight, is essentially analogous with the hysteresis involved in protein aggregation. In the hysteresis of BMV protein alone, we argue that the changes in pK, are probably fairly typical of any polymerizing protein and not directly relevant to the virus swelling. The improperly refolded state of all or some of the particles, at low pH after a titration at low ionic strength, presumably has some RNA strands caught at the surfaces of the contracted particles. This negatively charged RNA then cross-links the virus particles, whose surfaces are positively charged at this pH (Rice and Horst, 1972). A similar precipitation occurs when RNA is added to a suspension of capsids under these conditions (Pfeiffer, unpub-
lished results). Ribonuclease presumably resolubilizes the particles by cleaving the exposed RNA. Salts added to “rescue” the virus presumably suck the RNA back in, by neutralizing charges and at the same time lubricating the necessary relative movement of charges. BMV structural changes have also been investigated by Zulauf (personal communication of a draft paper), using intensity fluctuation spectroscopy. He found that the diffusion coefficient of the virus changed rapidly during swelling but that on contraction. there was a first fast step followed by a much slower annealing process. There are several ways in which the present understanding needs to be developed further: How do the three types of particle differ in cation binding and swelling? Which parts of the RNA stick out from swollen or incorrectly refolded virus? Which are the rate-determining steps in the hysteresis? How does the charge on BMV vary with solution conditions? Does BMV protein contain side-chain carboxyl-
BMV
CATION
ates close to the probable RNA-binding regions, as do for example ribosomal proteins (Ehresmann et al., 1976)? We believe that too much attention is currently given to the geometrical features that are peculiar to each virus, and not enough to the basic electrostatic interactions that are common to all polyelectrolytes. When two charged macromolecules come together in solution, numerous counterions are released or bound. In the case of opposite charges brought together by an RNA-protein interaction, Record et al. (1976) pointed out that the major net free energy change is the entropy of counterion release; there is thus a danger of confusing this with hydrophobic interactions. The present paper emphasizes the significance of repulsive interactions between like charges; their strength is critically dependent on the ionic content of the medium. We have not, however, stressed the well-known effects of ions in the medium in reducing electrostatic attractions and in changing the structure of water. Two of our results suggest that BMV probably does not disassemble in the cytoplasm of its host cell. First, BMV RNA and protein do not physically separate at pH 7 and moderate ionic strength, in the absence of multivalent cations. Second, BMV would bind substantial amounts of Mg’+ and polyamines, even if not Ca4*, from the concentrations of these ions typically prevailing in cytoplasm. One could postulate that some component of host cell cytoplasm actively pulls the RNA out of the virion, but we think it is more likely that BMV disassembles in the act of crossing a membrane (Durham, paper submitted for publication). Free energy to drive the process would come from the dilution of Ca2+ ions transported into cytoplasm. Bonds directly or indirectly involving protein carboxylates, metal ions, and phosphates are quite widespread in biology. Examples occur in actin-ATP (StrzeDrabikowski, lecka-Golaszewska and 1967), tubulin-GTP (Arai and Kaziro, 1976), thymidylate synthetase (Whitfield et al., 1976), many nucleic acid-handling and -degrading enzymes (e.g., Poulos and
431
BINDING
Price, 1974), phospholipase A2 (Van DamMieras et al., 1975), prothrombin (Furie et al., 1976), and all enzymes using ATP-Mg. It follows that small changes in intracellular divalent ion concentrations are capable of producing profound shifts in the pattern of cellular metabolism. The significance of this fact for virus replication, as well as for assembly and disassembly, is explained elsewhere (Durham, 1977; and paper submitted for publication). REFERENCES ADOLPH, K. W. (1975a). Conformational features of cowpea chlorotic mottle virus. J. Gen. Viral. 28, 137-145. ADOLPH, K. W. (1975b). Structural transitions of cowpea chlorotic mottle virus. J. Gen. Virol. 28, 147-154. ADOLPH, K. W., and BUTLER, P. J. G. (1974). Studies on the assembly of a spherical plant virus. I. States of aggregation of the isolated protein. J. Mol. Biol. 88, 327-341. AGRAWAL, H. O., and TREMAINE, J. H. (1972). Proteins of cowpea chlorotic mottle, broad bean mottle and brome mosaic viruses. Virology 47,8-20. ARAI, T., and KAZIRO, Y. (1976). Effect of guanine nucleotides on the assembly of brain microtubules: Ability of the C’-guanylyl imidodiphosphate to replace GTP in promoting the polymerization of microtubules in vitro. Biochen. Biophys. Res. Commun. 69, 369-376. BACHRACH, U. (1973). “Function of Naturally Occurring Polyamines.” Academic Press, New York. BANCROFT, J. B. (1970). The self-assembly of spherical plant viruses. Aduan. Virus Res. 16,99-134. BOCKSTAHLER, L. E., and KAESBERG, P. (1962). The molecular weight and other biophysical properties of bromegrass mosaic virus. Biophys. J. 2, l-9. BRAKKE, M. K. (1963). Stabilization of brome mosaic virus by magnesium and calcium. Virology 19, 367-374. BRAKKE, M. K. (1971). Degradation of brome mosaic virus and tobacco mosaic virus in bentonite. Virology 46, 575-585. DURHAM, A. C. H. (1977). Do viruses use calcium ions to shut off host cell functions? Nature (London) 267, 375. DURHAM, A. C. H., and HENDRY, D. A. (1977). Cation binding by tobacco mosaic virus. Virology 77, 510-519. DURHAM, A. C. H. HENDRY, D. A., and VON WECHMAR, M. B. (1977). Does calcium ion binding control plant virus disassembly? Virology 77, 524-533. DURHAM, A. C. H., VOGEL, D., and DE MARCILLAC, G. (1977). Hydrogen ion titration curves of tobacco
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DURHAM LANE, L. C. (1974). The bromoviruses. Aduan. Virus Res. 19, 151-220. LOENING, U. E. (1967). The fractionation of highmolecular-weight ribonucleic acid by polyacrylamide-gel electrophoresis. Biochem. J. 102, 251257. MAUREL, P., and Douzou, P. (1976). Catalytic implications of electrostatic potentials: The lytic activity of lysozyme as a model. J. Mol. Biol. 102, 253264. PARKES, D. (1974). Amantadine. Aduan. Drug Res. 8, 11-81. PFEIFFER, P., and HIRTH, L. (1974a). Formation of artificial top component from brome mosaic virus at high salt concentrations. Virology 58, 362-368. PFEIFFER, P., and HIRTH, L. (1974b). Aggregation states of brome mosaic virus protein. Virology 61, 160-167. PFEIFFER, P., and HIRTH, L. (1975). The effect of conformational changes in brome mosaic virus upon its sensitivity to trypsin, chymotrypsin and ribonuclease. FEBS Lett. 56, 144-148. POULOS, T. L., and PRICE, P. A. (1974). The involvement of serine and carboxyl groups in the activity of bovine pancreatic deoxyribonuclease A. J. Biol. Chem. 249, 1453-1457. RECORD, M. T., LOHMAN, T. M., and DE HASETH, P. (1976). Ion effects on ligand-nucleic acid interactions. J. Mol. Biol. 107, 145-158. RICE, R. H., and HORST, J. (1972). Isoelectric focusing of viruses in polyacrylamide. Virology 49,602604. STRZELECKA-GOLASZEWSKA, H., and DRABIKOWSKI, W. (1967). Correlation between the binding of calcium and ATP by G-actin. Acta Biochim. Pol. 14, 195-208. STUBBS, G. J., WARREN, S., and HOLMES, K. C. (1977). The structure of the RNA and the RNA binding site in tobacco mosaic virus as revealed by a 4 A map calculated from X-ray libre diagrams. Nature (London) 267, 216-221. VAN DAM-MIERAS, M. C. E., SLOTBOOM, A. J., PIETERSON, W. A., and DE HAAS, G. H. (19751. The interaction of phospholipase A2 with micellar interfaces. The role of the N-terminal region. Biochemistry 14, 5387-5394. WHITFIELD, J. F., MACMANLJS, J. P., RIXON, R. H., BOYNTON, A. L., YOUDALE, T., and SWIERENGA, S. (1976). The positive control of proliferation by the interplay of calcium ions and cyclic nucleotides. A review. In Vitro 12, l-17.