Variation of the Permeability of Bacteriophage T4: Analysis by Use of a Protein-Specific Probe for the T4 Interior GARY A. GRIESS, SAEED A. KHAN, and PHILIP SERWER Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78284-7760
SYNOPSIS
The permeability of bacteriophage T4 and the change in T4 permeability caused by mutation to osmotic shock resistance are investigated here by quantification of the kinetics with which both a DNA-specific probe (ethidium) and a protein-specific probe [ 1,l'-bi (4-anilino) naphthalene-5,5'-di-sulfonic acid, or bis-ANS)] bind to T4. In the case of an osmotic shock-resistant mutant, T40s41, both ethidium and bis-ANS bind with first order kinetics. The first-order rate constant ( k * ) for both bis-ANS and ethidium is a function of anion type and concentration. Adenosine triphosphate, phosphate, bisulfite, sulfate, and acetate anions all reduce k* below the k* observed when chloride is the only anion. When chloride is the only anion a t 25"C, k* values for binding to T40s41 are orders of magnitude above k * values for binding to wild-type T 4 (T4wt). At 25"C, k* for T4wt is too small to measure, but k* for T4wt increases a t 50-55°C to values approaching those measured for T40s41, without inactivating T4wt, when chloride is the only anion; during heating, T4wt is stabilized by both ethidium and bis-ANS. Binding to T4wt is reversible a t 50-55"C, but not a t 25OC. Equilibrium binding of bis-ANS to T40s41 reveals 112 k 24 sites per T 4 capsid. Equilibrium binding of ethidium to T40s41 reveals both high- and low-affinity sites previously observed in the packaged DNA of other bacteriophages. The ATP-induced decrease in k* is not accompanied by a decrease in equilibrium binding. The following hypotheses are presented to explain the above data: ( a ) All detected bis-ANS binding sites on T 4 are interior to the outer surface of T4. ( b ) The value of k* for both bis-ANS and ethidium is controlled a t the p o r t ( s ) of passage through the outer shell of the T 4 capsid. ( c ) The anions present control k* values a t the port ( s )of entry, probably by controlling the size of this port. The effects on k* of phosphate explain the otherwise paradoxical observation [ P. J. McCall and V. A. Bloomfield (1976) Biopolymers 15, 2323-23361 that in a phosphate buffer the permeabilities of T4wt and T40s41 are the same.
INTRODUCTION Several double-stranded DNA bacteriophages-including $29, A, P22, T2, T 4 and T7-consist of a linear double-stranded DNA molecule packaged in a multiprotein capsid. The interstrand spacing of packaged DNA varies by no more than 4% among these bacteriophages (2.29-2.38 nm). Study of both the assembly of bacteriophage proteins to form a DNA-free capsid (procapsid) and the subsequent packaging of DNA by the procapsid has been conducted to understand the genetically controlled asBiopolymerh, VoI. : i l , 1 1 -21 (1991) C 1991 John U'iley & Sons, Inc.
CCC 0006-3525/91/010011-11$04.00
sembly that occurs in a comparatively simple model system (reviewed in Refs. 1-4). Quantification of the permeability of bacteriophage capsids is needed to understand mechanisms of ( a ) the assembly of procapsids and exclusion of host proteins, * ( b ) the loss of protein scaffolds during subsequent changes in the structure of procapsids,' and ( c ) entry of DNA into the ~ a p s i d .In ~ addition, this information is needed for understanding permeability-dependent destructive effects of freezing on cells; bacteriophage T 4 has been used as a model for this purpose."' For studies of permeability, the use of 7'4 and its relative, T2, is potentially advantageous because of the following: ( a ) The sensitivity of both wild-type T 4 (T4wt) and T2, but not other, unrelated, well11
12
GRIESS, KHAN, AND SERWER
studied bacteriophages, to sodium chloride-induced osmotic shock suggests that these bacteriophages are less permeable than other, well-studied bacteriophages. ( b ) Mutants of T4 resistant to osmotic shock have been ~ e l e c t e d . ~ T-o~determine directly whether resistance to osmotic shock is caused by an increase in capsid permeability, attempts have been made to quantify permeability by use of the kinetics of binding to T2 and T4 of DNA-reactive dyes.'.' In contradiction to expectations, a comparison of T4wt and an osmotic shock-resistant T4 mutant, T40s41, has revealed no difference in rate of binding to the DNA-reactive dye, proflavin.' Among possible explanations of this observation are either proflavin binding rates not limited by the permeability of T4"'' or permeability that varies with the buffer used. Sodium chloride-Tris buffer was used for osmotic shock, but a phosphate buffer was used to measure permeability. To obtain a more rigorous measurement of permeability, the binding rate of internal T4 components to both a protein-specific and a DNA-specific molecule (probe) should be measured. For this purpose we sought a proteinspecific probe that preferentially binds the T4 capsid protein that is not exposed a t the outer bacteriophage surface. Such a protein-specific probe would also be useful for quantifying the permeability of DNA-free bacteriophage capsids ( for an example, in the case of bacteriophage T7; see Ref. 111. In a previous study, l 2 unsuccessful attempts were made to use 1-anilino-naphthalene-8-sulfonate ( ANS) to probe the structure of bacteriophage T7. ANS had previously been used as a fluorescent probe for membrane alteration^'^ ; fluorescence enhancement occurs when ANS is transferred from an aqueous t o a more nonpolar environment (for example, either ethanol or hydrophobic sites in proteins).14 However, a dimer of ANS, bis-ANS, undergoes a greater fluorescence enhancement during this transfer and also binds the nucleotide-binding sites of several proteins 5-100 times as well as ANS (Ref. 14 and additional references therein). Thus, attempts were made to use bis-ANS as a proteinspecific probe for bacteriophage T4. These attempts were successful and, fortunately, no detected binding sites were on the outer surface of T4. In the present communication are described both the characterization of the binding of bis-ANS to bacteriophage T 4 and the use of bis-ANS, together with the DNAspecific probe, ethidium, to quantify permeability. The effects of several anions on the permeability of T 4 are described. These effects explain the contradictory findings of Ref. 8. Implications for the structure, function, and practical uses of T4 are discussed. 798
MATERIALS A N D METHODS Bacteriophages and Hosts
T4wt and T40s41, the latter selected for resistance to osmotic shock when diluted from solutions of sodium chloride, were received from Dr. W. B. Wood. The host for both T4wt and T40s41 was Escherichia coli BB/1. Bacteriophages were grown in M9 medium15 and purified by centrifugation in cesium chloride density gradients, by use of procedures previously described." Molar concentrations of bacteriophage were determined by use of absorbance a t 260 nm and a molar extinction coefficient of 2.68 nM-' cmP1.l6 Buffers and Reagents
The buffer used for storage and dilution of bacteriophages was a Tris/Mg buffer: 0.5M NaC1,O.OlM Tris Cl, pH 7.4, 0.001M MgC12. For assaying infective units, 100 pg/mL gelatin was included in this buffer. However, for measurement of bis-ANS fluorescence, the gelatin was not used, because of a background of gelatin-induced bis-ANS fluorescence enhancement. Phosphate buffer was the pH 7.6 buffer from Ref. 8. T o store DNA, NET buffer was used: O.1M NaC1, 0.01M Tris C1, 0.001M EDTA. Bis-ANS (molecular weight 670) was obtained from Molecular Probes, Inc. (Eugene, Oregon). Concentrations of bis-ANS were measured by use of absorbance a t 385 nm and a molar extinction coefficient of 16,790M-' cm-l.14 Preparation of D N A
To isolate DNA, purified bacteriophage were extracted with phenol, by use of procedures previously described.17 T o denature DNA in NET buffer, the DNA was boiled for 5 min and then rapidly chilled on ice. Kinetics of Binding and Dissociation: Measurement of Fluorescence
A Perkin-Elmer MPF-44A recording fluorescence spectrometer that had a thermostated cell holder was used for all measurements of fluorescence intensity ( I ) . For studies of the binding of bis-ANS, the wavelength of excitation was 400 nm. Bis-ANS has a peak emission a t 530 nm in water and 495 nm in ethanol; the peak intensity is 500 times greater in ethanol than it is in water.14 A solution of bis-
PERMEABILITY OF BACTERIOPHAGE T 4
ANS in ethanol was a standard for normalizing measurements of fluorescence enhancement. T o quantify ethidium-induced fluorescence enhancement after binding of ethidium to DNA, the above procedures were used at a 540-nm excitation and 600-nm emission wavelength (see also Ref. 1 0 ) . To measure the dependence on time ( t ) of the fluorescence at any given t ( I t ) ,a solution of bacteriophage was diluted by 100-2OOX into a solution of probe (either bis-ANS or ethidium) and I , was continuously monitored, a t the temperature indicated, by use of a strip chart recorder. T o quantify the kinetics of the binding of bis-ANS, It was processed by ( a ) assuming it proportional to the number of bis-ANS molecules bound, and ( b ) determining kinetic binding constants by use of the method of Kezdy and Swinbourne (reviewed in Ref. 1 8 ) . For each class of site with a detectably different firstorder rate constant ( k : , k: , . . . ), this method yields the rate constant and the corresponding equilibrium It ( I f ) .T h e method of Kezdy and Swinbourne has previously been used to detect two classes of site for the binding of ethidium to the packaged DNA of bacteriophage T7.l' In the case of first-order binding to a single class of sites, after subtraction of background from It and I f , a semilogarithmic plot of ( I f - I t ) / I fvs. t is linear and the slope yields the k*.8,'9 Calculations were made by use of a program written for the Apple IIe microcomputer. This program is available on request. After reaching equilibrium in the binding of either bis-ANS or ethidium to T4wt, dissociation was quantified by removing unassociated probe in less than 2 min, by passage through a PD-10 gel filtration column (Pharmacia) a t 25OC. Subsequently, It was monitored, as described above. Decrease in It revealed dissociation. T o test for changes in It caused by light-induced damage t o either the probe or bacteriophage T4, during measurement of It the cuvette was removed from the fluorimeter, shaken, and replaced. With a n exception described in the Results, no change in I , was detected by this procedure.
13
tha$ I f (background-subtracted) is proportional t o the concentration of bound dye. If Do 9 the molar bacteriophage concentration Po and if I,,, is I f a t saturation. then
A double reciprocal plot of 1 / I , vs l / D o yields Kd a s the slope divided by the intercept. The value of N is obtained by use of the relation
9 is the fluorescence enhancement per mole of bound bis-ANS. Although Kd does not depend on scaling factors, N depends on the scaling factor \k. T h e value of \k was determined by saturating a known concentration of bis-ANS with protein and determining the limiting fluorescence in protein excess (see also Ref. 20). Fluorescence units normalized to a reference standard (above) were used t o remove the dependence of \k on instrument settings. Because of error from light scattering, an excess of bacteriophage could not be used to determine \k. However, the emission spectrum for bis-ANS bound to T40s41 (Results) was indistinguishable from that of bis-ANS bound t o bovine serum albumin (not shown). Thus, according to data in Ref. 14, the assumption has been made that the quantum yield for bis-ANS bound to T40s41 is the same as that for bis-ANS bound to bovine serum albumin. Measurement of light Scattering
Light scattering was quantified by use of an excitation and emission wavelength of 450 nm, when applying the procedure used for quantifying fluorescence. Electron Microscopy
Binding at Equilibrium
For the binding a t equilibrium of ethidium, the saturation binding fraction B,, and the dissociation constant Kd were determined a t 25°C by use of Scatchard plots.20 For bis-ANS, both K d and the number N of binding sites per bacteriophage were determined a t 25°C by the following method2lP2*: Binding of bis-ANS was quantified as a function of the molar concentration Do of bis-ANS, by assuming
Electron microscopy of specimens negatively stained with uranyl acetate was performed by use of procedures previously described." Pulsed Field Agarose Gel Electrophoresis
Pulsed field agarose gel electrophoresis of mature T 4 DNA was performed by use of procedures described in Ref. 23.
14
GRIESS, KHAN, AND SERWER
RESULTS Quantification of the Binding of bis-ANS: Emission Spectrum
During preliminary studies, mixing of 1n M T40s41 with 1.9 pM bis-ANS revealed T40s41-induced Auorescence enhancement. To optimize quantification of the binding of bis-ANS, the emission spectrum of bis-ANS bound to T40s41 was determined. After equilibrating bis-ANS with T40s41, the emission spectrum (400-nm wavelength of excitation) had a maximum a t 495 nm (curve A in Figure 1) . In the studies described below, It was determined a t 500 nm. The background caused at 500 nm by both fluorescence of unbound bis-ANS (curve B in Figure 1) and light scattering from the bacteriophage (in the absence of bis-ANS; data not shown) were subtracted. The light scattering was decreased by a factor of 5 when DNA was extruded (details are in a subsequent section) from either T4wt or T40s41 by incubation a t 70°C. Thus, light scattering was used to determine whether or not DNA had been extruded during the experiments reported below. Unless otherwise indicated, in all cases, no detectable extrusion occurred. In addition, no loss in infectivity was found for either T4wt or T40s41 after incubation with bisANS or ethidium for 2-3 days a t 25°C. Immediately (within 10 s ) after adding either T4wt or T40s41 to a solution of bis-ANS, values of background-subtracted It did not differ significantly from zero. Values of It subsequently increased with time, as described in subsequent sections. In contrast, if either T4wt or T40s41 had been burst at 70"C, It was equal to I, in a time less than the time
"\,
,,/'
50
500
550
I
Emission Wavelength (nm)
Figure 1. Emission spectrum of bound bis-ANS. The emission spectrum excited by 400-nm light was determined for 1.9 p M bis-ANS after adding of the following: ( A ) 1.0 nM intact T40s41, ( B ) buffer, and ( C ) 1.0 n M T40s41
that had DNA expelledby incubation at 70°C for 10 min.
required for a measurement ( 10 s ) and I, was higher than that of intact T40s41. The emission spectrum of burst T40s41 (Figure 1,curve C ) also had a maximum a t 495 nm. In electron micrographs, the outer shell of the T 4 capsid was intact, but DNA was expelled, after incubation a t 70°C (not shown; see Ref. 3 ) . The purified DNA of T4wt ( 12.4 n M ) , either native or denatured, caused no detectable enhancement of the fluorescence of bis-ANS (not shown). Thus, all detected binding of bis-ANS is presumed to occur to the capsid of T40s41. That a t least one protein of T4 capsids has bisANS binding sites in the absence of packaged DNA is indicated by the presence of these sites after expulsion of DNA from the T4 capsid (data is presented in the last section of the Results). However, the relationship of binding sites in the intact bacteriophage to those present after expulsion of DNA is not known. By use of pulsed field agarose gel electrophoresis, the DNA of T40s41 is indistinguishable from the DNA of T4wt. That is, the length of T40s41 DNA is the same as the length of T4wt DNA & 2%. Buffers for Determination of I,
The I , vs t plot obtained for T40s41 varied dramatically with the buffer used. A plot of the It vs t obtained for the binding of ethidium to T40s41 was steeper in Tris/Mg buffer (Figure 2; Os, T / M ) than it was in the phosphate buffer previously used8,' to quantify the permeability (Figure 2; Os, P ) . The I, plot obtained for the binding of ethidium to T4wt in Tris/Mg buffer (Figure 2, WT, T / M ) was more than two orders of magnitude less steep than the plot for T40s41 in Tris/Mg buffer; no significant binding was observed for T4wt. This type of difference between T4wt and T40s41 is predicted on the basis of the known difference in sensitivity to osmotic shock, if binding rate is limited by permeIn contrast, T4wt bound detectable amounts of ethidium in phosphate buffer (Figure 2, WT, P ) . In phosphate buffer, the It vs t plot for T4wt was much closer to the It vs t plot for T40s41 (Figure 2, Os, P ) than it was in Tris/Mg buffer, although not as close as the comparable plot for proflavin in phosphate buffer.8 Thus, the contradiction discussed in the Introduction is caused, a t least in part, by sensitivity of binding rate to buffers. For T40s41, addition of 0.01 M Tris C1, pH 7.4 (i.e., a component of Tris/Mg buffer) to the phosphate buffer did not change the It observed for the phosphate buffer (not shown). However, addition of the complete Tris/Mg buffer to phosphate buffer caused
15
PERMEABILITY OF BACTERIOPHAGE T 4
understood. More detailed investigation of the effects of anions is described in subsequent sections:
Kinetics a s a Function of Probe Concentration
'
30 0
I 20
60
40
t
80
100
(min)
Figure 2. Fluorescence as a function of time: dependence on buffer. Values of It vs t were determined for both T4wt and T40s41 in different buffers containing 20 1 M ethidium bromide. The bacteriophage and buffer are indicated on the figure, by use of the following symbols: bacteriophage T4wt: WT; bacteriophage T40s41: 0 s ; Tris/ Mg buffer: T / M ; phosphate buffer: P.
Z, for T40s41 to become intermediate to the Its of
Before continuing with the analysis of effects on k* of common anions, effects on k* of the concentration of probe were determined. In accordance with the pattern observed for phosphate and ATP anions, as the concentration of (anionic ) bis-ANS increased, first-order binding kinetics were observed (not shown) and k* decreased (Figure 4; BA) . As the concentration of ethidium increased, first-order kinetics were also observed (not shown), but k* increased (Figure 4, E B ) . The steepness of the bisANS plot a t the lower concentrations of bis-ANS in Figure 4 introduces scatter when bis-ANS is used as the probe for determining the effect of ions on values of k*. Thus, most further studies of the effects on k* of anions were conducted by use of ethidium as the probe. T h e concentration of ethidium used, 5 pM, caused a n apparent increase of k* (Figure 4 ) equal t o 30%.
Kinetics a5 a Function of the Concentration of Several Anions
First-order kinetics were observed and the value of k* for the binding of ethidium to T40s41 decreased
+
the separate buffers (Figure 1, Os, P T / M ) . All further measurements of Zt for T40s41 in subsequent sections were made with Tris/Mg buffer that had the indicated additional components.
Analysis of the Kinetics of Binding of Ethidium and Bis-ANS T o quantify the permeability of T40s41, Zt vs t plots were obtained for both ethidium ( 5 p M ) and bisANS ( 5 p M ) , and these plots were analyzed by use of the Kezdy-Swinbourne procedure. Binding of both of these dyes occurred with kinetics that were indistinguishable from first order. This conclusion is also drawn from the observation of linearity in a semilogarithmic plot of (I,- I t )/I, vs. t (ethidium: Figure 3 , EB; bis-ANS: Figure 3, BA). To further test the effects of cations on kinetics, the experiment of Figure 3 was also performed by use of Tris Mg buffer that had 100 pM ATP. The ATP, like phosphate, caused a slowing of binding. For both 5 pM ethidium (Figure 3, EB-ATP) and 5 pM bis-ANS (Figure 3, BA-ATP), the kinetics remained first order, but k* was reduced. Oscillations in the plot for bis-ANS are reproducible, but are not
0
20
40
60
80
100
120
140
t (min)
Figure 3. Kinetics of binding for his-ANS and ethidium. Values of (I, - I,) /If are plotted semilogarithmically as a function of time for the binding of either 5 pM his-ANS ( B A ) or 5 p M ethidium ( E B ) to 1 nM T40s41. The effects of 100 p M ATP were determined for both bis-ANS (BAATP) and ethidium (EB-ATP).
16
*
.-
GRIESS, KHAN, AND SERWER
0.1
E v
4
0.0'
0
"
20
"
40
"
"
60
80
"
100
'
'
120
[DYE1 (PM)
Figure 4. Values of k* as a function of the concentration of either bis-ANS or ethidium. By use of the KezdySwinbourne procedure, It vs t was used to calculate k* at each of several concentrations of either bis-ANS (BA) or ethidium bromide ( E B ) . Values of k* are plotted as a function of the concentration of probe.
a s the concentration of several anions increased. In order of increasing effect on k*, these anions were acetate, bisulfite, phosphate, sulfate, and A T P (A, B, P, S , and ATP, respectively, in Figure 5 ) . A noncleavable A T P analogue, AMPPNP, had a n effect on k* indistinguishable from that of A T P in Figure 5 ( n o t shown). The following anions a t concentrations as high as 5 m M had no detectable effect on the k* for binding of ethidium to T40s41 in Tris/ Mg buffer: iodide, fluoride, and glutamate. Glutamate also had no effect a t 50 m M (not shown). Significant effects of anions in Figure 5 were detected a t concentrations as low as 5-10 pM ( t h e C1- concentration in Tris/Mg buffer is 500 m M) . A reproducible plateau in the plots of Figure 5 was observed a t anion concentrations of 10-50 pM for acetate, bisulfite, and phosphate. No anions other than those described here have been tested.
whether binding of bis-ANS and ethidium is controlled by such a barrier, the effect of temperature on the binding of bis-ANS and ethidium was determined semiquantitatively, by use of the following experiment. With 1 n M of T4wt was mixed 1.5 pM bis-ANS. Subsequently, the temperature was raised in steps and It was determined 15 min after temperature equilibration ( II5). The result was a comparatively steep rise in a t 50-55"C, followed by a plateau a t 55-65°C and then a second steep rise (Figure 6,115, BA) . Measurement of light scattering (Figure 6, LS, BA) revealed that 80-90% of the loss in light scattering occurred during the steep rise in 1 1 5 that occurred above 65°C. Thus, the T4wt appear not to have burst during the uptake of bis-ANS that occurred a t 50-55°C. In confirmation, infectivity of T4wt was not significantly decreased a t 55°C. When the measurement of light scattering in the experiment of Figure 6 was performed in the absence of bis-ANS, the drop in light scattering was shifted by 10°C toward a lower temperature (Figure 6, LS) . Thus, the bis-ANS stabilized T 4 to inactivation a t elevated temperature. The measurement of II5 made by use of bis-ANS in the experiment of Figure 6 was also made by use of ethidium. The result was the same as it was for
0.0
Some Effects of Temperature on the Binding to T4wt
Raising the temperature has previously been found t o both increase the penetration of cesium ions into T 4 before buoyant density centrifugation 24 and increase the dye-induced damage to packaged T 2 DNA.' One explanation for these observations is that raising temperature lowers a barrier to entry
(i.e., raises permeability). Thus, to further test
1
100
10
1000
[ANION] pM
Figure 5. Values of k* as a function of the type and concentration of anion. The k* for the binding of 5 p M ethidium to 1 n M T40s41 was determined as a function of the concentration of the following anions, added to Tris/ Mg buffer: adenosine triphosphate ( ATP), phosphate ( P ) , sulfate ( S ) , bisulfite ( B ) , and acetate ( A ) . The ratio of k* to the k* obtained without added anion [ k * ( O ) ] is
plotted.
PERMEABILITY OF BACTERIOPHAGE T 4
O L " 20
'
30
"
40
"
50
'
"
60
70
' " 1 60
90
T (QC)
Figure 6. Effects of temperature. Starting a t 25"C, the value of 1, 15 min after changing the temperature (115) of 5 fiM bis-ANS with 1 n M T4wt was determined for increments in temperature. In a separate experiment, performed by the same procedure, light scattering ( a t 90") was quantified, instead of 115, in the presence and absence of bis-ANS. The above procedure for measuring both Z15 and light scattering was also used for the fluorescence of 5 fiM ethidium mixed with 1 n M T4wt. The following are plotted as a function of temperature: Z15for bis-ANS (115, BA) , light scattering for bis-ANS (LS, BA) ,Z15for ethidium (115, EB) , light scattering for ethidium (LS, EB) , and light scattering without addition of probe (LS). In separate experiments, the direction of change in temperature was reversed a t either 55 or 75"C, during determination of ZI5for bis-ANS (dashed lines).
bis-ANS (Figure 6,115, E B ) . By the criterion of light scattering (Figure 6, LS, E B ) and measurement of infectivity ( n o t shown), the bursting of T4wt occurred during the steep rise in 115 above 65"C, as also found in the case of bis-ANS. The plot for light scattering in t h e presence of ethidium (Figure 6, LS, E B ) indicated stabilization of T4wt by the ethidium, as did the plot for light scattering in the presence of bis-ANS. A plateau was observed at 6065°C in the plot for light scattering in the presence of ethidium. Although the source of this plateau is not rigorously known, the likely source is incomplete stabilization of T4wt by ethidium, a t 60-65°C. All values of Z15,except those in the zone of either of the two transitions, are indistinguishable from the equilibrium values of It. T o determine whether the changes in bis-ANS ZI5at 55°C in Figure 6 were reversible, the experiment of Figure 6 was repeated for bis-ANS until the
17
temperature ramp reached 55°C. At 55"C, the ramp was reversed. However, the values of Z15did not reverse with the ramp; Z15 increased as temperature decreased (dashed line attached a t 55°C to the Z15, BA plot in Figure 6 ) . T h e same result was obtained for bis-ANS fluorescence when the temperature ramp of Figure 6 was reversed a t 75°C (dashed line attached a t 75°C to the ZI5, BA plot in Figure 6 ) . Reversal of the temperature ramp a t 55 and 75°C during binding of ethidium to T4wt also revealed irreversibility of binding; Z15 increased after the temperature ramp was reversed (data not shown). T h e increase in fluorescence that occurs during reversal of the temperature ramp (dashed lines in Figure 6 ) is, presumably, caused, a t least in part, by a n increase in fluorescence quantum yield usually associated with a decrease in t e m p e r a t ~ r e . Changes '~ in binding of probes could also contribute. The data of Figure 6 indicate a n increase in the k* of (intact) T4wt, when the temperature was raised from 25 to 50-55°C. In confirmation, the k* for binding of ethidium t o T4wt increased from less than 0.002 min-' a t 25°C to 0.053 min a t 55°C. The k* for binding of ethidium to T40s41 increased from 0.055 min-' a t 25°C to 0.64 min-' a t 55°C. Reversibility in the Absence of Probe
T o determine whether binding was reversible after removal of unassociated bis-ANS from T4wt, bisANS was incubated a t 55°C for 15 min with T4wt and the unassociated bis-ANS was then removed by use of molecular sieve chromatography a t 25"C, in less than 2 min. With the exception of a slight decrease in It caused by light-induced damage (see Materials and Methods), incubation of the isolated T4wt-bis-ANS complex a t 25°C did not detectably lower Zt (Figure 7, 25°C). However, incubation a t 55°C did lower It and, therefore, reverse binding (Figure 7, 55°C). The kinetics of reversal a t 55°C were first order (not shown). When the experiment of Figure 7 was performed by use of ethidium, irreversibility a t 25"C, but not 55"C, was also observed ( n o t shown). Equilibrium Binding: Effects of Bursting
In the case of ethidium, analysis of equilibrium binding revealed both high- and low-affinity binding sites in T40s41. Similar results have previously been obtained for bacteriophage T7*' and are not shown here. For the low-affinity ethidium binding sites of T40s41, the value of B,, and K d are in Table I.
18
GRIESS, KHAN, AND SERWER
90
:
0
"
"
"
"
'
I
"
'
...
.
.
.
I
,
... . .
,
.
25'C
8
3 0 ' ' ~ ' " ' " " ' ' L ' 0 10
,
'
'
'
'
"
'
20
'
'
'
'
. . I 30
t (min)
Figure 7. Dissociation of bis-ANS from T4wt. After equilibration of 5 pM bis-ANS with 1 n M T4wt a t 55"C, the unassociated bis-ANS was removed by molecular sieve chromatography at 25°C. Subsequently, It vs t was determined at either 25 or 55°C (indicated on the figure).
T o determine whether the ATP-induced reduction in k* caused a change in equilibrium binding of ethidium to T40s41, values of It were determined as a function of the concentration of ATP. For ATP concentrations between 0 and 100 pM, the value of I, for 5 pM ethidium was independent of the concentration of A T P (not shown), even though k* decreased (Figure 5). Values of Kd and B,, for the low-affinity sites (i.e., 93-97% of the total) were not significantly changed by the presence of 100 p M A T P (Table I ) .
T o obtain values of Kd and N for the binding of bis-ANS to T40s41, \kPo/Ifwasplotted as a function of l / D o . This plot was linear (Figure 8, intact); values of Kd and N are in Table I. When T40s41 was burst by incubation a t 70°C, the value of Kd decreased by a factor of 4.7 and N increased slightly (Figure 8, burst; Table I ) . T h e values of Kd and N for burst T4wt differed slightly from those for T40s41 (Table I ) . After bursting, the value of k* for both T4wt and T40s41 became too high t o measure.
Permeability and Structure
T o use values of k* for quantifying permeability, the following minimal conditions must be achieved: ( a ) the sites bound are not on the outer surface of the capsid, and ( b ) k* is controlled by events that occur during passage through the outer shell. Although condition ( a ) has been previously achieved by use of DNA-specific probes, tests for condition ( b ) have not previously been performed. Because, during use of a DNA-specific probe (proflavin) in phosphate buffer, T40s41 was previously8 found to have a k* indistinguishable from that of T4wt, the difference in osmotic shock susceptibility between T40s41 and T4wt was the basis for concluding that minimal condition ( b ) was not achieved for T4." However, this conclusion was based on the (latent) assumption that the permeability of T4wt and T40s41 in Tris-buffered 0.5-3.OM NaCl was the same as it was in the phosphate buffer used for Ref.
Table I Equilibrium and Kinetic Constants Ethidium Particle" (ATP Concentration) Intact T40s41 (0 pM) Intact T40s41 (100 pM) Burst T40s41 (0 pM) Burst T4wt (0 pM)
Bis-ANS'
k* (min-') 10%
Kd (pM)' f 15%
0.024 0.0042
11.9 12.1
-
-
0.050 0.054 -
-
-
-
*
Bape
-t 15%
k* (min-') 15%
Kd (PM) f 15%
N +- 20%
0.10 0.0058
4.68
112
-
-
d
1.00 0.58
186 120
*
d
The bacteriophage used. Burst particles were subjected to 70°C for 10 min; intact particles were not. The concentration of ATP present is indicated. Procedures for determining k*, Kd, and B., are in the Materials and Methods. For determining k * , the concentration of ethidium was 5 pM. ' Procedures for determining k*, Kd, and N are in the Materials and Methods. For determining k*, the concentration of bis-ANS was 5 p M . The value of k* was too high to measure. e Values for low-affinity sites are given.
PERMEABILITY OF BACTERIOPHAGE T4 60
50
40 0
0
30
. c
no
3 20
10
n ”
0
1
2
3
1I D ,
Figure 8. Equilibrium binding of bis-ANS. Double reciprocal plots of the equilibrium binding of bis-ANS are shown for intact bacteriophage T40s41 (indicated by “intact”), bacteriophage T40s41 from which DNA had been expelled by raising the temperature to 70°C for 10 min (indicated by “burst”).
8. This assumption from Ref. 10 has been shown here to be sufficiently incorrect t o invalidate the use of this assumption made in Ref. 10. T o more directly and rigorously determine whether k* is controlled by rate of passage through the outer shell of the capsid [i.e., condition ( b ) , above], in the present study, the k*s for a DNA reactive probe (ethidium) have been compared to k*s for a protein-reactive probe (bis-ANS) during binding to T40s41 in Tris/Mg buffer. Fortunately, bis-ANS did not react with T4wt a t 25°C. This latter observation is most simply interpreted by the assumption that, like ethidium, bis-ANS detectably binds only sites that are not on the outer surface of T4wt and that T4wt is impermeable to bis-ANS. T h a t this assumption is correct is supported by the following observations: ( a ) Qualitatively, anion-induced changes that occurred in the k* for binding of ethidium to T40s41 also occurred for the binding of bis-ANS to T40s41. ( b ) At 50-55°C in Tris/Mg buffer, (intact) T4wt bound and released both bisANS and ethidium; at 25°C T4wt neither bound nor released these probes. This latter observation is explained by a n elevated temperature-induced lowering of a permeability barrier. T h e data ( a and b of the previous paragraph) indicate that k* is controlled by events that occur
19
during the diffusion of either bis-ANS or ethidium toward binding sites. One possible site for control is the outer shell of the T40s41 capsid. However, a n alternative possible site for control of diffusion is the lattice of packaged DNA. If the latter site were correct, then the observed anion-induced decrease in the permeability of T40s41 should cause a decrease in DNA-DNA spacing. In the case of bacteriophage T7, decreasing DNA-DNA spacing by 2.0% causes both a 30% decrease in the equilibrium binding of ethidium to packaged DNA and a 3.1fold decrease in k*.’o,20In the case of T40s41, a n ATP-induced 10-fold decrease in k * produced no detectable change in equilibrium binding ( Results). Thus, assuming analogy of T40s41 and T7, the conclusion is drawn that, a t least in the presence of k * reducing anions, k* is not determined during passage through the lattice of packaged DNA. (Tris/Mg buffer was also used in the study of T7.) Th’is conclusion is in agreement with the observation that the k* for T40s41 (0.022) is 5-10-fold smaller than the k* for T 7 (0.117) a t 25°C in Tris/Mg buffer with 5 pg/mL ethidium; the k* for T 7 is controlled by diffusion through the packaged DNA.” T h a t is, based on observations made with T7, the k* of T40s41 is too low to be controlled by the density of packaged DNA. Additional, alternative possible sites for control of k* are the positions of the internal proteins and peptides that are surrounded by packaged DNA.25*26 Although a structural analysis of their positions has not been made,25,26internal proteins and peptides could, if present in sufficient amount, form a n internal, three-dimensional network of pores whose sieving-induced retardation of the diffusion of probes could explain a similarity of the responses of bisANS k* values to the responses of ethidium k* values, for T40s41. However, the total mass of this internal protein is less than 5% of the mass of the packaged DNA (calculated from data in Ref. 26). Thus, the increment in internal sieving that could be introduced by the internal proteins appears to be insignificant in comparison to that of the packaged DNA. Thus, the assumption will be made that all k*s for T40s41 are controlled by events that occur during passage through the outer shell of the T 4 capsid, i.e., that both conditions that are discussed above and that are necessary for the measurement of permeability exist in the case of T4. If so, then anions alter k* by altering the permeability of the outer shell of the capsid of T40s41. T h e measurement of k* is analogous to the measurement of ion fluxes during studies of channels in membranes. From the studies of ion channels, three
20
GRIESS, KHAN, AND SERWER
factors are known to influence k*: steric exclusion from the channel to be traversed, viscous retardation in the channel, and adsorption in the channel. The first two factors have been combined in the Renkin equation, 27 previously used in several studies of diffusion through pores (examples are in Refs. 27 and 28). Use of the Renkin equation is based on the assumption of negligible adsorption in the channel; this assumption cannot be made for ion channels.29 The Renkin equation is inconsistent with the finding that, for T40s41, the larger probe used here, bisANS, has a k* value greater than that of the smaller probe, ethidium, a t the lower concentrations of probe (Figure 4 ) . Thus, as in the case of ion channels, effects of adsorption cannot be ignored during studies of the permeability of T4. However, the observation that anions reduce k* for the binding of both a n anion (bis-ANS ) and a cation (ethidium) is best interpreted by assuming that k *-reducing anions exert their effect by narrowing a pore ( s ) in the outer shell of the T40s41 capsid. Further work will be needed t o make the transition from k* to the structure of T4. In any case, T4 appears to be a n excellent model for studying biological channels and their control. The anions most effective in decreasing the permeability of T40s41 (i.e., Figure 5 ) are strong polar kosmotropes in the Hofmeister series. The progressively less effective anions are progressively less k o s m o t r ~ p i cHowever, .~~ the effects on the permeability of T 4 occur a t concentrations 2-3 orders of magnitude smaller than concentrations needed for other physical effects of k o s m o t r ~ p e s . ~ ~ The current data reveal neither the nature of the bis-ANS binding sites nor the protein ( s ) that contains this site. Because this protein must extend internal t o the outer surface of the outer shell of the capsid, the candidate bis-ANS binding proteins are as follows: ( a ) T h e 10 proteins that form the outer shell of the T4 capsid-this outer shell consists of two icosahedral hemispheres separated by a cylindrical lattice of subunits. Included among proteins of the outer shell is the product of T 4 gene 24, located a t icosahedral vertices and the site of the Os41 mutation. ( b ) At least 3 internal proteins and some smaller pep tide^.^,^^,^^ Evolution and Function
Knowledge of the permeability that bacteriophage capsids have during morphogenesis is needed to help understand morphogenesis (Introduction). In the present study, data were obtained for intact bacteriophage T4. Therefore, conclusions concerning capsids in assembly pathways are necessarily indi-
rect. The primary conclusion from the data described here is that the permeability of the T 4 capsid can be controlled by anions, some of which are present in the T 4 host Escherichia coli. Because E. coli has a hydrated volume of approximately mL, of which 20% is solid that includes 0.6% acid soluble phosphates, 31 the intracellular concentration of acid soluble phosphate is a t least 1 m M and is probably higher. The intracellular concentration of glutamate is usually less than 50 mM.32Thus, control of permeability is exerted a t physiologically attained levels of phosphates, but not glutamate. Further work is needed to determine the permeability of the capsids that are in assembly pathways. T h e finding that no bis-ANS binding sites are on the outer surface of bacteriophage T 4 might be explained by evolutionary selection for some internal structure bound by bis-ANS. T h e following observations support the assumption that internal bisANS binding sites are present in unrelated bacteriophages and, therefore, undergo positive selection during evolution: ( a ) Although unrelated to both each other and to bacteriophage T4, bacteriophages P22 and T 7 also bind bis-ANS without any detectable rapid-binding phase. ( b ) Elevated temperatureinduced extrusion of DNA from P22 and T 7 increases bis-ANS binding and causes all detected sites to be rapid binding (data not shown). Implications for Use of T4 in Other Disciplines
By raising and lowering temperature, T4wt can be loaded with either bis-ANS, ethidium, or possibly other compounds. Because neither bis-ANS nor ethidium leaks out of T4wt a t either 25°C or lower temperature, T4wt should be useful as a transporting vehicle for a variety of (possibly toxic) compounds. Delivery would later be achieved by elevation of temperature. Because the basis of a t least some freeze-thaw induced damage to both T 4 and cells is osmotic shock that occurs during thawing, comparison of the properties of T4wt and T 4 osmotic shock-resistant mutants is used to develop improved procedures for recovery of viability after the freezing and thawing of cells and embryo^.^,^ Because of the dependence of k* on anion type and concentration, use of permeability t o explain effects of either osmotic shock or freeze-thaw must include the dependence demonstrated here of permeability on anion type and concentration. We thank Drs. Stanley P. Leibo and Paul M. Horowitz for helpful comments, E. Anthony Meyer for technical
PERMEABILITY OF BACTERIOPHAGE T4
assistance, and Linda C. Winchester for typing the manuscript. Support was received from the National Institutes of Health (GM-24365) , the National Science Foundation (DMB 90-03695), and the Robert A. Welch Foundation ( AQ-764).
REFERENCES 1. Murialdo, H. & Becker, A. (1978) Microbiol. Reu. 42,
529-576. 2. Earnshaw, W. & Casjens, S. (1980) Cell 21,319-331. 3. Black, L. W. (1989) Ann. Reu. Microbiol. 43, 267292. 4. Serwer, P. ( 1990) in Chromosomes: Eukaryotic, Prokaryotic and Viral, Vol. 111, Adolph, K. w., Ed., CRC Press, Boca Raton, FL, pp. 203-223. 5. Leibo, S. P. & Mazur, P. (1969) Virology 38,558566. 6. Leibo, S. P., Kellenberger, E., Kellenberger-van der Kamp, C., Frey, T. G. & Steinberg, C. M. (1979) J . Virol. 30, 327-338. 7. Brenner, S. & Barnett, L. (1959) Brookhauen Symp. Biol. 12, 86-94. 8. McCall, P. J. & Bloomfield, V. A. ( 1976 Biopolymers 15, 2323-2336. 9. Cummings, D. J. & Kozloff, L. M. (1962 J . Mol. Biol. 5 , 50-62. 10. Griess, G. A., Serwer, P., Kaushal, V. & Horowitz, P. (1986) Biopolymers 25, 1345-1357. 11. Serwer, P. (1980) J. Mol. Biol. 138,65-91. 12. Fidy, -J., Mauss, Y., Pataki, K., Chambron, J. & Ronto, Gy. (1983) Biophys. Struct. Mech. 10, 109-119. 13. Braun, V. & Oldmixon, E. (1979) J. Supramol. Struct. 10, 329-347. 14. Farris, F. J., Weber, G., Chiang, C. C. & Paul, I. C. - ( 1978) J. Am. Chem. SOC.100,4469-4474. 15. Kellenberger, E. & Skchaud, J . (1957) Virology 3,256274.
21
16. Bancroft, F. C. & Freifelder, D. (19701 J . Mol. Biol. 54,537-546. 17. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 18. Moore, J. W. & Pearson, R. G. (1981) Kinetics and Mechanism, John Wiley & Sons, New York. 19. Cantor, C. R. & Schimmel, P. R. (1980) Biophysical Chemistry, W. H. Freeman, San Francisco. 20. Griess, G. A., Serwer, P. & Horowitz, P. M. (1985) Biopolymers 24, 1635-1646. 21. Wang, J. L. & Edelman, G. M. (1971) J . Biol. Chem. 246,1185-1191. 22. Horowitz, P. M. & Criscimagna, N. L. (1985) Biochemistry 24, 2587-2593. 23. Lane, T., Serwer, P., Hayes, S. J. & Eiserling, F. ( 1990) Virology 174,472-478. 24. Mosig, G., Carnighan, J. R., Bibring, J. B., Cole, R., Bock, H.-G. 0. & Bock, S. (1972) J . Virol. 9, 857871. 25. Eiserling, F. A. ( 1983 ) in Bacteriophage T4, Mathews, C. K., Kutter, E. M., Mosig, G. & Berget, P. B., Eds., American Society for Microbiology, Washington, DC, pp. 11-24. 26. Yanagida, M., Suzuki, Y. & Toda, T. (1984) Adu. Biophys. 17, 97-146. 27. Renkin, E. (1954) J . Gen. Physiol. 38, 225-243. 28. Cannell, D. S. & Rondelez, F. ( 1980) Macromolecules 13, 1599-1602. 29. Jordan, P. C. (1987) Biophys. J . 51, 297-311. 30. Collins, K. D. & Washabaugh, M. W. (1985) Quart. Reu. Biophys. 18, 323-422. 31. Luria, S. E. (1960) in The Bacteria, Vol. I, Gunsalus, I. C. & Stanier, R. Y., Eds., Academic Press, New York, pp. 1-34. 32. Richey, B., Cayley, D. S., Mossing, M. C., Kolka, C., Anderson, C. F., Farrar, T. C. & Record, M. T., Jr. (1987) J . Biol. Chem. 262, 7157-7164. Received June 4, 1990 Accepted September 17, 1990
SYNOPSIS
The permeability of bacteriophage T4 and the change in T4 permeability caused by mutation to osmotic shock resistance are investigated here by quantification of the kinetics with which both a DNA-specific probe (ethidium) and a protein-specific probe [ 1,l'-bi (4-anilino) naphthalene-5,5'-di-sulfonic acid, or bis-ANS)] bind to T4. In the case of an osmotic shock-resistant mutant, T40s41, both ethidium and bis-ANS bind with first order kinetics. The first-order rate constant ( k * ) for both bis-ANS and ethidium is a function of anion type and concentration. Adenosine triphosphate, phosphate, bisulfite, sulfate, and acetate anions all reduce k* below the k* observed when chloride is the only anion. When chloride is the only anion a t 25"C, k* values for binding to T40s41 are orders of magnitude above k * values for binding to wild-type T 4 (T4wt). At 25"C, k* for T4wt is too small to measure, but k* for T4wt increases a t 50-55°C to values approaching those measured for T40s41, without inactivating T4wt, when chloride is the only anion; during heating, T4wt is stabilized by both ethidium and bis-ANS. Binding to T4wt is reversible a t 50-55"C, but not a t 25OC. Equilibrium binding of bis-ANS to T40s41 reveals 112 k 24 sites per T 4 capsid. Equilibrium binding of ethidium to T40s41 reveals both high- and low-affinity sites previously observed in the packaged DNA of other bacteriophages. The ATP-induced decrease in k* is not accompanied by a decrease in equilibrium binding. The following hypotheses are presented to explain the above data: ( a ) All detected bis-ANS binding sites on T 4 are interior to the outer surface of T4. ( b ) The value of k* for both bis-ANS and ethidium is controlled a t the p o r t ( s ) of passage through the outer shell of the T 4 capsid. ( c ) The anions present control k* values a t the port ( s )of entry, probably by controlling the size of this port. The effects on k* of phosphate explain the otherwise paradoxical observation [ P. J. McCall and V. A. Bloomfield (1976) Biopolymers 15, 2323-23361 that in a phosphate buffer the permeabilities of T4wt and T40s41 are the same.
INTRODUCTION Several double-stranded DNA bacteriophages-including $29, A, P22, T2, T 4 and T7-consist of a linear double-stranded DNA molecule packaged in a multiprotein capsid. The interstrand spacing of packaged DNA varies by no more than 4% among these bacteriophages (2.29-2.38 nm). Study of both the assembly of bacteriophage proteins to form a DNA-free capsid (procapsid) and the subsequent packaging of DNA by the procapsid has been conducted to understand the genetically controlled asBiopolymerh, VoI. : i l , 1 1 -21 (1991) C 1991 John U'iley & Sons, Inc.
CCC 0006-3525/91/010011-11$04.00
sembly that occurs in a comparatively simple model system (reviewed in Refs. 1-4). Quantification of the permeability of bacteriophage capsids is needed to understand mechanisms of ( a ) the assembly of procapsids and exclusion of host proteins, * ( b ) the loss of protein scaffolds during subsequent changes in the structure of procapsids,' and ( c ) entry of DNA into the ~ a p s i d .In ~ addition, this information is needed for understanding permeability-dependent destructive effects of freezing on cells; bacteriophage T 4 has been used as a model for this purpose."' For studies of permeability, the use of 7'4 and its relative, T2, is potentially advantageous because of the following: ( a ) The sensitivity of both wild-type T 4 (T4wt) and T2, but not other, unrelated, well11
12
GRIESS, KHAN, AND SERWER
studied bacteriophages, to sodium chloride-induced osmotic shock suggests that these bacteriophages are less permeable than other, well-studied bacteriophages. ( b ) Mutants of T4 resistant to osmotic shock have been ~ e l e c t e d . ~ T-o~determine directly whether resistance to osmotic shock is caused by an increase in capsid permeability, attempts have been made to quantify permeability by use of the kinetics of binding to T2 and T4 of DNA-reactive dyes.'.' In contradiction to expectations, a comparison of T4wt and an osmotic shock-resistant T4 mutant, T40s41, has revealed no difference in rate of binding to the DNA-reactive dye, proflavin.' Among possible explanations of this observation are either proflavin binding rates not limited by the permeability of T4"'' or permeability that varies with the buffer used. Sodium chloride-Tris buffer was used for osmotic shock, but a phosphate buffer was used to measure permeability. To obtain a more rigorous measurement of permeability, the binding rate of internal T4 components to both a protein-specific and a DNA-specific molecule (probe) should be measured. For this purpose we sought a proteinspecific probe that preferentially binds the T4 capsid protein that is not exposed a t the outer bacteriophage surface. Such a protein-specific probe would also be useful for quantifying the permeability of DNA-free bacteriophage capsids ( for an example, in the case of bacteriophage T7; see Ref. 111. In a previous study, l 2 unsuccessful attempts were made to use 1-anilino-naphthalene-8-sulfonate ( ANS) to probe the structure of bacteriophage T7. ANS had previously been used as a fluorescent probe for membrane alteration^'^ ; fluorescence enhancement occurs when ANS is transferred from an aqueous t o a more nonpolar environment (for example, either ethanol or hydrophobic sites in proteins).14 However, a dimer of ANS, bis-ANS, undergoes a greater fluorescence enhancement during this transfer and also binds the nucleotide-binding sites of several proteins 5-100 times as well as ANS (Ref. 14 and additional references therein). Thus, attempts were made to use bis-ANS as a proteinspecific probe for bacteriophage T4. These attempts were successful and, fortunately, no detected binding sites were on the outer surface of T4. In the present communication are described both the characterization of the binding of bis-ANS to bacteriophage T 4 and the use of bis-ANS, together with the DNAspecific probe, ethidium, to quantify permeability. The effects of several anions on the permeability of T 4 are described. These effects explain the contradictory findings of Ref. 8. Implications for the structure, function, and practical uses of T4 are discussed. 798
MATERIALS A N D METHODS Bacteriophages and Hosts
T4wt and T40s41, the latter selected for resistance to osmotic shock when diluted from solutions of sodium chloride, were received from Dr. W. B. Wood. The host for both T4wt and T40s41 was Escherichia coli BB/1. Bacteriophages were grown in M9 medium15 and purified by centrifugation in cesium chloride density gradients, by use of procedures previously described." Molar concentrations of bacteriophage were determined by use of absorbance a t 260 nm and a molar extinction coefficient of 2.68 nM-' cmP1.l6 Buffers and Reagents
The buffer used for storage and dilution of bacteriophages was a Tris/Mg buffer: 0.5M NaC1,O.OlM Tris Cl, pH 7.4, 0.001M MgC12. For assaying infective units, 100 pg/mL gelatin was included in this buffer. However, for measurement of bis-ANS fluorescence, the gelatin was not used, because of a background of gelatin-induced bis-ANS fluorescence enhancement. Phosphate buffer was the pH 7.6 buffer from Ref. 8. T o store DNA, NET buffer was used: O.1M NaC1, 0.01M Tris C1, 0.001M EDTA. Bis-ANS (molecular weight 670) was obtained from Molecular Probes, Inc. (Eugene, Oregon). Concentrations of bis-ANS were measured by use of absorbance a t 385 nm and a molar extinction coefficient of 16,790M-' cm-l.14 Preparation of D N A
To isolate DNA, purified bacteriophage were extracted with phenol, by use of procedures previously described.17 T o denature DNA in NET buffer, the DNA was boiled for 5 min and then rapidly chilled on ice. Kinetics of Binding and Dissociation: Measurement of Fluorescence
A Perkin-Elmer MPF-44A recording fluorescence spectrometer that had a thermostated cell holder was used for all measurements of fluorescence intensity ( I ) . For studies of the binding of bis-ANS, the wavelength of excitation was 400 nm. Bis-ANS has a peak emission a t 530 nm in water and 495 nm in ethanol; the peak intensity is 500 times greater in ethanol than it is in water.14 A solution of bis-
PERMEABILITY OF BACTERIOPHAGE T 4
ANS in ethanol was a standard for normalizing measurements of fluorescence enhancement. T o quantify ethidium-induced fluorescence enhancement after binding of ethidium to DNA, the above procedures were used at a 540-nm excitation and 600-nm emission wavelength (see also Ref. 1 0 ) . To measure the dependence on time ( t ) of the fluorescence at any given t ( I t ) ,a solution of bacteriophage was diluted by 100-2OOX into a solution of probe (either bis-ANS or ethidium) and I , was continuously monitored, a t the temperature indicated, by use of a strip chart recorder. T o quantify the kinetics of the binding of bis-ANS, It was processed by ( a ) assuming it proportional to the number of bis-ANS molecules bound, and ( b ) determining kinetic binding constants by use of the method of Kezdy and Swinbourne (reviewed in Ref. 1 8 ) . For each class of site with a detectably different firstorder rate constant ( k : , k: , . . . ), this method yields the rate constant and the corresponding equilibrium It ( I f ) .T h e method of Kezdy and Swinbourne has previously been used to detect two classes of site for the binding of ethidium to the packaged DNA of bacteriophage T7.l' In the case of first-order binding to a single class of sites, after subtraction of background from It and I f , a semilogarithmic plot of ( I f - I t ) / I fvs. t is linear and the slope yields the k*.8,'9 Calculations were made by use of a program written for the Apple IIe microcomputer. This program is available on request. After reaching equilibrium in the binding of either bis-ANS or ethidium to T4wt, dissociation was quantified by removing unassociated probe in less than 2 min, by passage through a PD-10 gel filtration column (Pharmacia) a t 25OC. Subsequently, It was monitored, as described above. Decrease in It revealed dissociation. T o test for changes in It caused by light-induced damage t o either the probe or bacteriophage T4, during measurement of It the cuvette was removed from the fluorimeter, shaken, and replaced. With a n exception described in the Results, no change in I , was detected by this procedure.
13
tha$ I f (background-subtracted) is proportional t o the concentration of bound dye. If Do 9 the molar bacteriophage concentration Po and if I,,, is I f a t saturation. then
A double reciprocal plot of 1 / I , vs l / D o yields Kd a s the slope divided by the intercept. The value of N is obtained by use of the relation
9 is the fluorescence enhancement per mole of bound bis-ANS. Although Kd does not depend on scaling factors, N depends on the scaling factor \k. T h e value of \k was determined by saturating a known concentration of bis-ANS with protein and determining the limiting fluorescence in protein excess (see also Ref. 20). Fluorescence units normalized to a reference standard (above) were used t o remove the dependence of \k on instrument settings. Because of error from light scattering, an excess of bacteriophage could not be used to determine \k. However, the emission spectrum for bis-ANS bound to T40s41 (Results) was indistinguishable from that of bis-ANS bound t o bovine serum albumin (not shown). Thus, according to data in Ref. 14, the assumption has been made that the quantum yield for bis-ANS bound to T40s41 is the same as that for bis-ANS bound to bovine serum albumin. Measurement of light Scattering
Light scattering was quantified by use of an excitation and emission wavelength of 450 nm, when applying the procedure used for quantifying fluorescence. Electron Microscopy
Binding at Equilibrium
For the binding a t equilibrium of ethidium, the saturation binding fraction B,, and the dissociation constant Kd were determined a t 25°C by use of Scatchard plots.20 For bis-ANS, both K d and the number N of binding sites per bacteriophage were determined a t 25°C by the following method2lP2*: Binding of bis-ANS was quantified as a function of the molar concentration Do of bis-ANS, by assuming
Electron microscopy of specimens negatively stained with uranyl acetate was performed by use of procedures previously described." Pulsed Field Agarose Gel Electrophoresis
Pulsed field agarose gel electrophoresis of mature T 4 DNA was performed by use of procedures described in Ref. 23.
14
GRIESS, KHAN, AND SERWER
RESULTS Quantification of the Binding of bis-ANS: Emission Spectrum
During preliminary studies, mixing of 1n M T40s41 with 1.9 pM bis-ANS revealed T40s41-induced Auorescence enhancement. To optimize quantification of the binding of bis-ANS, the emission spectrum of bis-ANS bound to T40s41 was determined. After equilibrating bis-ANS with T40s41, the emission spectrum (400-nm wavelength of excitation) had a maximum a t 495 nm (curve A in Figure 1) . In the studies described below, It was determined a t 500 nm. The background caused at 500 nm by both fluorescence of unbound bis-ANS (curve B in Figure 1) and light scattering from the bacteriophage (in the absence of bis-ANS; data not shown) were subtracted. The light scattering was decreased by a factor of 5 when DNA was extruded (details are in a subsequent section) from either T4wt or T40s41 by incubation a t 70°C. Thus, light scattering was used to determine whether or not DNA had been extruded during the experiments reported below. Unless otherwise indicated, in all cases, no detectable extrusion occurred. In addition, no loss in infectivity was found for either T4wt or T40s41 after incubation with bisANS or ethidium for 2-3 days a t 25°C. Immediately (within 10 s ) after adding either T4wt or T40s41 to a solution of bis-ANS, values of background-subtracted It did not differ significantly from zero. Values of It subsequently increased with time, as described in subsequent sections. In contrast, if either T4wt or T40s41 had been burst at 70"C, It was equal to I, in a time less than the time
"\,
,,/'
50
500
550
I
Emission Wavelength (nm)
Figure 1. Emission spectrum of bound bis-ANS. The emission spectrum excited by 400-nm light was determined for 1.9 p M bis-ANS after adding of the following: ( A ) 1.0 nM intact T40s41, ( B ) buffer, and ( C ) 1.0 n M T40s41
that had DNA expelledby incubation at 70°C for 10 min.
required for a measurement ( 10 s ) and I, was higher than that of intact T40s41. The emission spectrum of burst T40s41 (Figure 1,curve C ) also had a maximum a t 495 nm. In electron micrographs, the outer shell of the T 4 capsid was intact, but DNA was expelled, after incubation a t 70°C (not shown; see Ref. 3 ) . The purified DNA of T4wt ( 12.4 n M ) , either native or denatured, caused no detectable enhancement of the fluorescence of bis-ANS (not shown). Thus, all detected binding of bis-ANS is presumed to occur to the capsid of T40s41. That a t least one protein of T4 capsids has bisANS binding sites in the absence of packaged DNA is indicated by the presence of these sites after expulsion of DNA from the T4 capsid (data is presented in the last section of the Results). However, the relationship of binding sites in the intact bacteriophage to those present after expulsion of DNA is not known. By use of pulsed field agarose gel electrophoresis, the DNA of T40s41 is indistinguishable from the DNA of T4wt. That is, the length of T40s41 DNA is the same as the length of T4wt DNA & 2%. Buffers for Determination of I,
The I , vs t plot obtained for T40s41 varied dramatically with the buffer used. A plot of the It vs t obtained for the binding of ethidium to T40s41 was steeper in Tris/Mg buffer (Figure 2; Os, T / M ) than it was in the phosphate buffer previously used8,' to quantify the permeability (Figure 2; Os, P ) . The I, plot obtained for the binding of ethidium to T4wt in Tris/Mg buffer (Figure 2, WT, T / M ) was more than two orders of magnitude less steep than the plot for T40s41 in Tris/Mg buffer; no significant binding was observed for T4wt. This type of difference between T4wt and T40s41 is predicted on the basis of the known difference in sensitivity to osmotic shock, if binding rate is limited by permeIn contrast, T4wt bound detectable amounts of ethidium in phosphate buffer (Figure 2, WT, P ) . In phosphate buffer, the It vs t plot for T4wt was much closer to the It vs t plot for T40s41 (Figure 2, Os, P ) than it was in Tris/Mg buffer, although not as close as the comparable plot for proflavin in phosphate buffer.8 Thus, the contradiction discussed in the Introduction is caused, a t least in part, by sensitivity of binding rate to buffers. For T40s41, addition of 0.01 M Tris C1, pH 7.4 (i.e., a component of Tris/Mg buffer) to the phosphate buffer did not change the It observed for the phosphate buffer (not shown). However, addition of the complete Tris/Mg buffer to phosphate buffer caused
15
PERMEABILITY OF BACTERIOPHAGE T 4
understood. More detailed investigation of the effects of anions is described in subsequent sections:
Kinetics a s a Function of Probe Concentration
'
30 0
I 20
60
40
t
80
100
(min)
Figure 2. Fluorescence as a function of time: dependence on buffer. Values of It vs t were determined for both T4wt and T40s41 in different buffers containing 20 1 M ethidium bromide. The bacteriophage and buffer are indicated on the figure, by use of the following symbols: bacteriophage T4wt: WT; bacteriophage T40s41: 0 s ; Tris/ Mg buffer: T / M ; phosphate buffer: P.
Z, for T40s41 to become intermediate to the Its of
Before continuing with the analysis of effects on k* of common anions, effects on k* of the concentration of probe were determined. In accordance with the pattern observed for phosphate and ATP anions, as the concentration of (anionic ) bis-ANS increased, first-order binding kinetics were observed (not shown) and k* decreased (Figure 4; BA) . As the concentration of ethidium increased, first-order kinetics were also observed (not shown), but k* increased (Figure 4, E B ) . The steepness of the bisANS plot a t the lower concentrations of bis-ANS in Figure 4 introduces scatter when bis-ANS is used as the probe for determining the effect of ions on values of k*. Thus, most further studies of the effects on k* of anions were conducted by use of ethidium as the probe. T h e concentration of ethidium used, 5 pM, caused a n apparent increase of k* (Figure 4 ) equal t o 30%.
Kinetics a5 a Function of the Concentration of Several Anions
First-order kinetics were observed and the value of k* for the binding of ethidium to T40s41 decreased
+
the separate buffers (Figure 1, Os, P T / M ) . All further measurements of Zt for T40s41 in subsequent sections were made with Tris/Mg buffer that had the indicated additional components.
Analysis of the Kinetics of Binding of Ethidium and Bis-ANS T o quantify the permeability of T40s41, Zt vs t plots were obtained for both ethidium ( 5 p M ) and bisANS ( 5 p M ) , and these plots were analyzed by use of the Kezdy-Swinbourne procedure. Binding of both of these dyes occurred with kinetics that were indistinguishable from first order. This conclusion is also drawn from the observation of linearity in a semilogarithmic plot of (I,- I t )/I, vs. t (ethidium: Figure 3 , EB; bis-ANS: Figure 3, BA). To further test the effects of cations on kinetics, the experiment of Figure 3 was also performed by use of Tris Mg buffer that had 100 pM ATP. The ATP, like phosphate, caused a slowing of binding. For both 5 pM ethidium (Figure 3, EB-ATP) and 5 pM bis-ANS (Figure 3, BA-ATP), the kinetics remained first order, but k* was reduced. Oscillations in the plot for bis-ANS are reproducible, but are not
0
20
40
60
80
100
120
140
t (min)
Figure 3. Kinetics of binding for his-ANS and ethidium. Values of (I, - I,) /If are plotted semilogarithmically as a function of time for the binding of either 5 pM his-ANS ( B A ) or 5 p M ethidium ( E B ) to 1 nM T40s41. The effects of 100 p M ATP were determined for both bis-ANS (BAATP) and ethidium (EB-ATP).
16
*
.-
GRIESS, KHAN, AND SERWER
0.1
E v
4
0.0'
0
"
20
"
40
"
"
60
80
"
100
'
'
120
[DYE1 (PM)
Figure 4. Values of k* as a function of the concentration of either bis-ANS or ethidium. By use of the KezdySwinbourne procedure, It vs t was used to calculate k* at each of several concentrations of either bis-ANS (BA) or ethidium bromide ( E B ) . Values of k* are plotted as a function of the concentration of probe.
a s the concentration of several anions increased. In order of increasing effect on k*, these anions were acetate, bisulfite, phosphate, sulfate, and A T P (A, B, P, S , and ATP, respectively, in Figure 5 ) . A noncleavable A T P analogue, AMPPNP, had a n effect on k* indistinguishable from that of A T P in Figure 5 ( n o t shown). The following anions a t concentrations as high as 5 m M had no detectable effect on the k* for binding of ethidium to T40s41 in Tris/ Mg buffer: iodide, fluoride, and glutamate. Glutamate also had no effect a t 50 m M (not shown). Significant effects of anions in Figure 5 were detected a t concentrations as low as 5-10 pM ( t h e C1- concentration in Tris/Mg buffer is 500 m M) . A reproducible plateau in the plots of Figure 5 was observed a t anion concentrations of 10-50 pM for acetate, bisulfite, and phosphate. No anions other than those described here have been tested.
whether binding of bis-ANS and ethidium is controlled by such a barrier, the effect of temperature on the binding of bis-ANS and ethidium was determined semiquantitatively, by use of the following experiment. With 1 n M of T4wt was mixed 1.5 pM bis-ANS. Subsequently, the temperature was raised in steps and It was determined 15 min after temperature equilibration ( II5). The result was a comparatively steep rise in a t 50-55"C, followed by a plateau a t 55-65°C and then a second steep rise (Figure 6,115, BA) . Measurement of light scattering (Figure 6, LS, BA) revealed that 80-90% of the loss in light scattering occurred during the steep rise in 1 1 5 that occurred above 65°C. Thus, the T4wt appear not to have burst during the uptake of bis-ANS that occurred a t 50-55°C. In confirmation, infectivity of T4wt was not significantly decreased a t 55°C. When the measurement of light scattering in the experiment of Figure 6 was performed in the absence of bis-ANS, the drop in light scattering was shifted by 10°C toward a lower temperature (Figure 6, LS) . Thus, the bis-ANS stabilized T 4 to inactivation a t elevated temperature. The measurement of II5 made by use of bis-ANS in the experiment of Figure 6 was also made by use of ethidium. The result was the same as it was for
0.0
Some Effects of Temperature on the Binding to T4wt
Raising the temperature has previously been found t o both increase the penetration of cesium ions into T 4 before buoyant density centrifugation 24 and increase the dye-induced damage to packaged T 2 DNA.' One explanation for these observations is that raising temperature lowers a barrier to entry
(i.e., raises permeability). Thus, to further test
1
100
10
1000
[ANION] pM
Figure 5. Values of k* as a function of the type and concentration of anion. The k* for the binding of 5 p M ethidium to 1 n M T40s41 was determined as a function of the concentration of the following anions, added to Tris/ Mg buffer: adenosine triphosphate ( ATP), phosphate ( P ) , sulfate ( S ) , bisulfite ( B ) , and acetate ( A ) . The ratio of k* to the k* obtained without added anion [ k * ( O ) ] is
plotted.
PERMEABILITY OF BACTERIOPHAGE T 4
O L " 20
'
30
"
40
"
50
'
"
60
70
' " 1 60
90
T (QC)
Figure 6. Effects of temperature. Starting a t 25"C, the value of 1, 15 min after changing the temperature (115) of 5 fiM bis-ANS with 1 n M T4wt was determined for increments in temperature. In a separate experiment, performed by the same procedure, light scattering ( a t 90") was quantified, instead of 115, in the presence and absence of bis-ANS. The above procedure for measuring both Z15 and light scattering was also used for the fluorescence of 5 fiM ethidium mixed with 1 n M T4wt. The following are plotted as a function of temperature: Z15for bis-ANS (115, BA) , light scattering for bis-ANS (LS, BA) ,Z15for ethidium (115, EB) , light scattering for ethidium (LS, EB) , and light scattering without addition of probe (LS). In separate experiments, the direction of change in temperature was reversed a t either 55 or 75"C, during determination of ZI5for bis-ANS (dashed lines).
bis-ANS (Figure 6,115, E B ) . By the criterion of light scattering (Figure 6, LS, E B ) and measurement of infectivity ( n o t shown), the bursting of T4wt occurred during the steep rise in 115 above 65"C, as also found in the case of bis-ANS. The plot for light scattering in t h e presence of ethidium (Figure 6, LS, E B ) indicated stabilization of T4wt by the ethidium, as did the plot for light scattering in the presence of bis-ANS. A plateau was observed at 6065°C in the plot for light scattering in the presence of ethidium. Although the source of this plateau is not rigorously known, the likely source is incomplete stabilization of T4wt by ethidium, a t 60-65°C. All values of Z15,except those in the zone of either of the two transitions, are indistinguishable from the equilibrium values of It. T o determine whether the changes in bis-ANS ZI5at 55°C in Figure 6 were reversible, the experiment of Figure 6 was repeated for bis-ANS until the
17
temperature ramp reached 55°C. At 55"C, the ramp was reversed. However, the values of Z15did not reverse with the ramp; Z15 increased as temperature decreased (dashed line attached a t 55°C to the Z15, BA plot in Figure 6 ) . T h e same result was obtained for bis-ANS fluorescence when the temperature ramp of Figure 6 was reversed a t 75°C (dashed line attached a t 75°C to the ZI5, BA plot in Figure 6 ) . Reversal of the temperature ramp a t 55 and 75°C during binding of ethidium to T4wt also revealed irreversibility of binding; Z15 increased after the temperature ramp was reversed (data not shown). T h e increase in fluorescence that occurs during reversal of the temperature ramp (dashed lines in Figure 6 ) is, presumably, caused, a t least in part, by a n increase in fluorescence quantum yield usually associated with a decrease in t e m p e r a t ~ r e . Changes '~ in binding of probes could also contribute. The data of Figure 6 indicate a n increase in the k* of (intact) T4wt, when the temperature was raised from 25 to 50-55°C. In confirmation, the k* for binding of ethidium t o T4wt increased from less than 0.002 min-' a t 25°C to 0.053 min a t 55°C. The k* for binding of ethidium to T40s41 increased from 0.055 min-' a t 25°C to 0.64 min-' a t 55°C. Reversibility in the Absence of Probe
T o determine whether binding was reversible after removal of unassociated bis-ANS from T4wt, bisANS was incubated a t 55°C for 15 min with T4wt and the unassociated bis-ANS was then removed by use of molecular sieve chromatography a t 25"C, in less than 2 min. With the exception of a slight decrease in It caused by light-induced damage (see Materials and Methods), incubation of the isolated T4wt-bis-ANS complex a t 25°C did not detectably lower Zt (Figure 7, 25°C). However, incubation a t 55°C did lower It and, therefore, reverse binding (Figure 7, 55°C). The kinetics of reversal a t 55°C were first order (not shown). When the experiment of Figure 7 was performed by use of ethidium, irreversibility a t 25"C, but not 55"C, was also observed ( n o t shown). Equilibrium Binding: Effects of Bursting
In the case of ethidium, analysis of equilibrium binding revealed both high- and low-affinity binding sites in T40s41. Similar results have previously been obtained for bacteriophage T7*' and are not shown here. For the low-affinity ethidium binding sites of T40s41, the value of B,, and K d are in Table I.
18
GRIESS, KHAN, AND SERWER
90
:
0
"
"
"
"
'
I
"
'
...
.
.
.
I
,
... . .
,
.
25'C
8
3 0 ' ' ~ ' " ' " " ' ' L ' 0 10
,
'
'
'
'
"
'
20
'
'
'
'
. . I 30
t (min)
Figure 7. Dissociation of bis-ANS from T4wt. After equilibration of 5 pM bis-ANS with 1 n M T4wt a t 55"C, the unassociated bis-ANS was removed by molecular sieve chromatography at 25°C. Subsequently, It vs t was determined at either 25 or 55°C (indicated on the figure).
T o determine whether the ATP-induced reduction in k* caused a change in equilibrium binding of ethidium to T40s41, values of It were determined as a function of the concentration of ATP. For ATP concentrations between 0 and 100 pM, the value of I, for 5 pM ethidium was independent of the concentration of A T P (not shown), even though k* decreased (Figure 5). Values of Kd and B,, for the low-affinity sites (i.e., 93-97% of the total) were not significantly changed by the presence of 100 p M A T P (Table I ) .
T o obtain values of Kd and N for the binding of bis-ANS to T40s41, \kPo/Ifwasplotted as a function of l / D o . This plot was linear (Figure 8, intact); values of Kd and N are in Table I. When T40s41 was burst by incubation a t 70°C, the value of Kd decreased by a factor of 4.7 and N increased slightly (Figure 8, burst; Table I ) . T h e values of Kd and N for burst T4wt differed slightly from those for T40s41 (Table I ) . After bursting, the value of k* for both T4wt and T40s41 became too high t o measure.
Permeability and Structure
T o use values of k* for quantifying permeability, the following minimal conditions must be achieved: ( a ) the sites bound are not on the outer surface of the capsid, and ( b ) k* is controlled by events that occur during passage through the outer shell. Although condition ( a ) has been previously achieved by use of DNA-specific probes, tests for condition ( b ) have not previously been performed. Because, during use of a DNA-specific probe (proflavin) in phosphate buffer, T40s41 was previously8 found to have a k* indistinguishable from that of T4wt, the difference in osmotic shock susceptibility between T40s41 and T4wt was the basis for concluding that minimal condition ( b ) was not achieved for T4." However, this conclusion was based on the (latent) assumption that the permeability of T4wt and T40s41 in Tris-buffered 0.5-3.OM NaCl was the same as it was in the phosphate buffer used for Ref.
Table I Equilibrium and Kinetic Constants Ethidium Particle" (ATP Concentration) Intact T40s41 (0 pM) Intact T40s41 (100 pM) Burst T40s41 (0 pM) Burst T4wt (0 pM)
Bis-ANS'
k* (min-') 10%
Kd (pM)' f 15%
0.024 0.0042
11.9 12.1
-
-
0.050 0.054 -
-
-
-
*
Bape
-t 15%
k* (min-') 15%
Kd (PM) f 15%
N +- 20%
0.10 0.0058
4.68
112
-
-
d
1.00 0.58
186 120
*
d
The bacteriophage used. Burst particles were subjected to 70°C for 10 min; intact particles were not. The concentration of ATP present is indicated. Procedures for determining k*, Kd, and B., are in the Materials and Methods. For determining k * , the concentration of ethidium was 5 pM. ' Procedures for determining k*, Kd, and N are in the Materials and Methods. For determining k*, the concentration of bis-ANS was 5 p M . The value of k* was too high to measure. e Values for low-affinity sites are given.
PERMEABILITY OF BACTERIOPHAGE T4 60
50
40 0
0
30
. c
no
3 20
10
n ”
0
1
2
3
1I D ,
Figure 8. Equilibrium binding of bis-ANS. Double reciprocal plots of the equilibrium binding of bis-ANS are shown for intact bacteriophage T40s41 (indicated by “intact”), bacteriophage T40s41 from which DNA had been expelled by raising the temperature to 70°C for 10 min (indicated by “burst”).
8. This assumption from Ref. 10 has been shown here to be sufficiently incorrect t o invalidate the use of this assumption made in Ref. 10. T o more directly and rigorously determine whether k* is controlled by rate of passage through the outer shell of the capsid [i.e., condition ( b ) , above], in the present study, the k*s for a DNA reactive probe (ethidium) have been compared to k*s for a protein-reactive probe (bis-ANS) during binding to T40s41 in Tris/Mg buffer. Fortunately, bis-ANS did not react with T4wt a t 25°C. This latter observation is most simply interpreted by the assumption that, like ethidium, bis-ANS detectably binds only sites that are not on the outer surface of T4wt and that T4wt is impermeable to bis-ANS. T h a t this assumption is correct is supported by the following observations: ( a ) Qualitatively, anion-induced changes that occurred in the k* for binding of ethidium to T40s41 also occurred for the binding of bis-ANS to T40s41. ( b ) At 50-55°C in Tris/Mg buffer, (intact) T4wt bound and released both bisANS and ethidium; at 25°C T4wt neither bound nor released these probes. This latter observation is explained by a n elevated temperature-induced lowering of a permeability barrier. T h e data ( a and b of the previous paragraph) indicate that k* is controlled by events that occur
19
during the diffusion of either bis-ANS or ethidium toward binding sites. One possible site for control is the outer shell of the T40s41 capsid. However, a n alternative possible site for control of diffusion is the lattice of packaged DNA. If the latter site were correct, then the observed anion-induced decrease in the permeability of T40s41 should cause a decrease in DNA-DNA spacing. In the case of bacteriophage T7, decreasing DNA-DNA spacing by 2.0% causes both a 30% decrease in the equilibrium binding of ethidium to packaged DNA and a 3.1fold decrease in k*.’o,20In the case of T40s41, a n ATP-induced 10-fold decrease in k * produced no detectable change in equilibrium binding ( Results). Thus, assuming analogy of T40s41 and T7, the conclusion is drawn that, a t least in the presence of k * reducing anions, k* is not determined during passage through the lattice of packaged DNA. (Tris/Mg buffer was also used in the study of T7.) Th’is conclusion is in agreement with the observation that the k* for T40s41 (0.022) is 5-10-fold smaller than the k* for T 7 (0.117) a t 25°C in Tris/Mg buffer with 5 pg/mL ethidium; the k* for T 7 is controlled by diffusion through the packaged DNA.” T h a t is, based on observations made with T7, the k* of T40s41 is too low to be controlled by the density of packaged DNA. Additional, alternative possible sites for control of k* are the positions of the internal proteins and peptides that are surrounded by packaged DNA.25*26 Although a structural analysis of their positions has not been made,25,26internal proteins and peptides could, if present in sufficient amount, form a n internal, three-dimensional network of pores whose sieving-induced retardation of the diffusion of probes could explain a similarity of the responses of bisANS k* values to the responses of ethidium k* values, for T40s41. However, the total mass of this internal protein is less than 5% of the mass of the packaged DNA (calculated from data in Ref. 26). Thus, the increment in internal sieving that could be introduced by the internal proteins appears to be insignificant in comparison to that of the packaged DNA. Thus, the assumption will be made that all k*s for T40s41 are controlled by events that occur during passage through the outer shell of the T 4 capsid, i.e., that both conditions that are discussed above and that are necessary for the measurement of permeability exist in the case of T4. If so, then anions alter k* by altering the permeability of the outer shell of the capsid of T40s41. T h e measurement of k* is analogous to the measurement of ion fluxes during studies of channels in membranes. From the studies of ion channels, three
20
GRIESS, KHAN, AND SERWER
factors are known to influence k*: steric exclusion from the channel to be traversed, viscous retardation in the channel, and adsorption in the channel. The first two factors have been combined in the Renkin equation, 27 previously used in several studies of diffusion through pores (examples are in Refs. 27 and 28). Use of the Renkin equation is based on the assumption of negligible adsorption in the channel; this assumption cannot be made for ion channels.29 The Renkin equation is inconsistent with the finding that, for T40s41, the larger probe used here, bisANS, has a k* value greater than that of the smaller probe, ethidium, a t the lower concentrations of probe (Figure 4 ) . Thus, as in the case of ion channels, effects of adsorption cannot be ignored during studies of the permeability of T4. However, the observation that anions reduce k* for the binding of both a n anion (bis-ANS ) and a cation (ethidium) is best interpreted by assuming that k *-reducing anions exert their effect by narrowing a pore ( s ) in the outer shell of the T40s41 capsid. Further work will be needed t o make the transition from k* to the structure of T4. In any case, T4 appears to be a n excellent model for studying biological channels and their control. The anions most effective in decreasing the permeability of T40s41 (i.e., Figure 5 ) are strong polar kosmotropes in the Hofmeister series. The progressively less effective anions are progressively less k o s m o t r ~ p i cHowever, .~~ the effects on the permeability of T 4 occur a t concentrations 2-3 orders of magnitude smaller than concentrations needed for other physical effects of k o s m o t r ~ p e s . ~ ~ The current data reveal neither the nature of the bis-ANS binding sites nor the protein ( s ) that contains this site. Because this protein must extend internal t o the outer surface of the outer shell of the capsid, the candidate bis-ANS binding proteins are as follows: ( a ) T h e 10 proteins that form the outer shell of the T4 capsid-this outer shell consists of two icosahedral hemispheres separated by a cylindrical lattice of subunits. Included among proteins of the outer shell is the product of T 4 gene 24, located a t icosahedral vertices and the site of the Os41 mutation. ( b ) At least 3 internal proteins and some smaller pep tide^.^,^^,^^ Evolution and Function
Knowledge of the permeability that bacteriophage capsids have during morphogenesis is needed to help understand morphogenesis (Introduction). In the present study, data were obtained for intact bacteriophage T4. Therefore, conclusions concerning capsids in assembly pathways are necessarily indi-
rect. The primary conclusion from the data described here is that the permeability of the T 4 capsid can be controlled by anions, some of which are present in the T 4 host Escherichia coli. Because E. coli has a hydrated volume of approximately mL, of which 20% is solid that includes 0.6% acid soluble phosphates, 31 the intracellular concentration of acid soluble phosphate is a t least 1 m M and is probably higher. The intracellular concentration of glutamate is usually less than 50 mM.32Thus, control of permeability is exerted a t physiologically attained levels of phosphates, but not glutamate. Further work is needed to determine the permeability of the capsids that are in assembly pathways. T h e finding that no bis-ANS binding sites are on the outer surface of bacteriophage T 4 might be explained by evolutionary selection for some internal structure bound by bis-ANS. T h e following observations support the assumption that internal bisANS binding sites are present in unrelated bacteriophages and, therefore, undergo positive selection during evolution: ( a ) Although unrelated to both each other and to bacteriophage T4, bacteriophages P22 and T 7 also bind bis-ANS without any detectable rapid-binding phase. ( b ) Elevated temperatureinduced extrusion of DNA from P22 and T 7 increases bis-ANS binding and causes all detected sites to be rapid binding (data not shown). Implications for Use of T4 in Other Disciplines
By raising and lowering temperature, T4wt can be loaded with either bis-ANS, ethidium, or possibly other compounds. Because neither bis-ANS nor ethidium leaks out of T4wt a t either 25°C or lower temperature, T4wt should be useful as a transporting vehicle for a variety of (possibly toxic) compounds. Delivery would later be achieved by elevation of temperature. Because the basis of a t least some freeze-thaw induced damage to both T 4 and cells is osmotic shock that occurs during thawing, comparison of the properties of T4wt and T 4 osmotic shock-resistant mutants is used to develop improved procedures for recovery of viability after the freezing and thawing of cells and embryo^.^,^ Because of the dependence of k* on anion type and concentration, use of permeability t o explain effects of either osmotic shock or freeze-thaw must include the dependence demonstrated here of permeability on anion type and concentration. We thank Drs. Stanley P. Leibo and Paul M. Horowitz for helpful comments, E. Anthony Meyer for technical
PERMEABILITY OF BACTERIOPHAGE T4
assistance, and Linda C. Winchester for typing the manuscript. Support was received from the National Institutes of Health (GM-24365) , the National Science Foundation (DMB 90-03695), and the Robert A. Welch Foundation ( AQ-764).
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529-576. 2. Earnshaw, W. & Casjens, S. (1980) Cell 21,319-331. 3. Black, L. W. (1989) Ann. Reu. Microbiol. 43, 267292. 4. Serwer, P. ( 1990) in Chromosomes: Eukaryotic, Prokaryotic and Viral, Vol. 111, Adolph, K. w., Ed., CRC Press, Boca Raton, FL, pp. 203-223. 5. Leibo, S. P. & Mazur, P. (1969) Virology 38,558566. 6. Leibo, S. P., Kellenberger, E., Kellenberger-van der Kamp, C., Frey, T. G. & Steinberg, C. M. (1979) J . Virol. 30, 327-338. 7. Brenner, S. & Barnett, L. (1959) Brookhauen Symp. Biol. 12, 86-94. 8. McCall, P. J. & Bloomfield, V. A. ( 1976 Biopolymers 15, 2323-2336. 9. Cummings, D. J. & Kozloff, L. M. (1962 J . Mol. Biol. 5 , 50-62. 10. Griess, G. A., Serwer, P., Kaushal, V. & Horowitz, P. (1986) Biopolymers 25, 1345-1357. 11. Serwer, P. (1980) J. Mol. Biol. 138,65-91. 12. Fidy, -J., Mauss, Y., Pataki, K., Chambron, J. & Ronto, Gy. (1983) Biophys. Struct. Mech. 10, 109-119. 13. Braun, V. & Oldmixon, E. (1979) J. Supramol. Struct. 10, 329-347. 14. Farris, F. J., Weber, G., Chiang, C. C. & Paul, I. C. - ( 1978) J. Am. Chem. SOC.100,4469-4474. 15. Kellenberger, E. & Skchaud, J . (1957) Virology 3,256274.
21
16. Bancroft, F. C. & Freifelder, D. (19701 J . Mol. Biol. 54,537-546. 17. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 18. Moore, J. W. & Pearson, R. G. (1981) Kinetics and Mechanism, John Wiley & Sons, New York. 19. Cantor, C. R. & Schimmel, P. R. (1980) Biophysical Chemistry, W. H. Freeman, San Francisco. 20. Griess, G. A., Serwer, P. & Horowitz, P. M. (1985) Biopolymers 24, 1635-1646. 21. Wang, J. L. & Edelman, G. M. (1971) J . Biol. Chem. 246,1185-1191. 22. Horowitz, P. M. & Criscimagna, N. L. (1985) Biochemistry 24, 2587-2593. 23. Lane, T., Serwer, P., Hayes, S. J. & Eiserling, F. ( 1990) Virology 174,472-478. 24. Mosig, G., Carnighan, J. R., Bibring, J. B., Cole, R., Bock, H.-G. 0. & Bock, S. (1972) J . Virol. 9, 857871. 25. Eiserling, F. A. ( 1983 ) in Bacteriophage T4, Mathews, C. K., Kutter, E. M., Mosig, G. & Berget, P. B., Eds., American Society for Microbiology, Washington, DC, pp. 11-24. 26. Yanagida, M., Suzuki, Y. & Toda, T. (1984) Adu. Biophys. 17, 97-146. 27. Renkin, E. (1954) J . Gen. Physiol. 38, 225-243. 28. Cannell, D. S. & Rondelez, F. ( 1980) Macromolecules 13, 1599-1602. 29. Jordan, P. C. (1987) Biophys. J . 51, 297-311. 30. Collins, K. D. & Washabaugh, M. W. (1985) Quart. Reu. Biophys. 18, 323-422. 31. Luria, S. E. (1960) in The Bacteria, Vol. I, Gunsalus, I. C. & Stanier, R. Y., Eds., Academic Press, New York, pp. 1-34. 32. Richey, B., Cayley, D. S., Mossing, M. C., Kolka, C., Anderson, C. F., Farrar, T. C. & Record, M. T., Jr. (1987) J . Biol. Chem. 262, 7157-7164. Received June 4, 1990 Accepted September 17, 1990
