d. Mol. Biol. (1975) 98, 479-484
Structure Studies on Styrene-treated lmmunoglobulin Crystals R. S ~
~ D G. ZALOGX
Department of Biochemistry 8tare University of New York at 8tony Broo~ Etony Broot~, N.Y. 11794, U.8.A.
(Re,dyed I A ~ l 1975) DOBt immunoglobnlln crystals ordinarily arc highly radiation sensitive and give a limited X-ray diffraction pattern to about 6 A resolution, thereby preventing structure analysis at high resolution. Crystals grown in the presence of monomers of styrene last very much longer in the X-ray beam and give a diffTaction pattern extending to about 4 ,~ resolution. This paper describes the experimental conditions we have used for obtaining optimum results. A difference electron density map calculated between the treated and untreated crystals shows no significant features indicating that styrene treatment does not cause any major conformational change in the protein. I t is also possible to prepare heavy-atem derivatives with styrene-treated crystals as seen from a difference Patterson map in the case of a mercury derivative. We speculate that monomers of styrene trapped in the crystal lattice are polymerized by the X-rays and reduce the molecular motion and scavenge the free radicals formed in the crystal lattice.
1. I n t r o d u c t i o n
The major problem preventing a high resolution structure analysis of the D 0 B t immunoglobulln molecule has been its poor diffraction pattern and its extreme sensitivity to X-radiation (Sarma et al., 1971a). I n a recent report (Zaloga & Sarma, 1974) we described some preliminary results of experiments using monomers of styrene in preventing the radiation damage and extending the diffraction pattern from the above crystals. Since then we have extended these studies to understand more fully t h e exact nature of the protection provided b y polystyrene. As a prerequisite to the regular use of styrene in the structure analysis of the immunoglobin molecule we set out to answer two important questions. ~irst, is the styrene producing any eonformational change in the protein molecule b y binding to specific sites on the protein? Second, will the styrene treatment prevent binding of heavy atoms to the protein necessary for actual structure analysis? We report the answers to both questions in this paper. I n addition, prompted b y numerous enquiries from other protein crystaUographers, we present the exact experimental details t h a t we have used in the styrene treatment with all the precautions t h a t we think are necessary to achieve optimum results. t Abbreviation used: DOB immunoglobulin, protein purified from the serum of the patient, Dobbs (see Materials and Methods). 22
479
480
R. S A R M A A N D G. Z A L O G A
2. E x p e r i m e n t a l
Methods
(a) Pr6paraZ~n o$ cryata/a All experiments wore ~arrled out using DOB immunoglobulin crystals. These crystals were grown from protein purified from the serum of t h e p a t i e n t (Dobbs) b y s m m o n i u m sulfate fractionation, followed b y colllmn c h r o m a t o g r a p h y on DEAE-eellulose a n d isoelectric focussing on an a~npholyne column (pH 6 to 10). The protein is n o r m a l l y stored in 0.01 ~ - s o d i u m cacodylate (pH 6.0) a t 4~ until needed for crystallization. Crystals are grown a t 4~ in 10-~1 drops sealed in siliconed glass slides, the drop consisting of 3 ~1 protein (30 to 40 mg/ml) a n d 7 ~10-1 ~ - s o d i u m borate buffer (pH 8-4). Styrene-treated crystals were prepared b y cocrystallization. F i v e ml of 0.1 M-sodium b o r a t e buffer (pH 8.4) was overlayed in a test t u b e with a p p r o x i m a t e l y 0.5 ml of 99~/o styrene. (The styrene used in these experiments was purchased from Aldrich Chemical Company. The inhibitor used to prevent spontaneous polymerization was reported b y t h e manufacturer to be t e r t i a r y b u t y l alcohol (I0 to 15 p.p.m.). The remaining i m p u r i t y was n o t identified. No experiments were conducted to purify the styrene.) The m i x t u r e was shaken vigorously a n d allowed to settle for a p p r o x i m a t e l y 1 h a t r o o m temperature. Undissolved styrene separates into a top phase which is t h e n removed with a P a s t e u r pipette. The b o t t o m phase of the mixture is now s a t u r a t e d with styrene a n d is transferred to another test tube. Seven ~1 of this buffered styrene is used with 3 ~1 of the protein solution to prepare the 10 ~1 drop used for crystallization. Great care m u s t be exercised in transferring the buffered styrene for the final crystallization drop because even after several hours of standing, the b o t t o m phase of the mixture has a thin layer of concentrated styrene o n t h e surface which tends to adhere to t h e syringe needle used to transfer this buffer to the slide. The concentration of styrene in the final drop appears to be critical (Zaloga & Sarma, 1974) a n d in the absence of a r a p i d convenient procedure for estimating this concentration, we have found the above procedure quite reproducible. (b) Data collection The monomers of styrene t r a p p e d in the crystal lattice are n o r m a l l y polymerlzed b y the X - r a y s during d a t a collection. However, we have obtained slm~]ar results b y pre-exposing the styrene-treated crystal to X - r a y s with the crystal surrounded b y a small a m o u n t (1 to 2 ~1) of the m o t h e r liquor in the capillary tube. After a p p r o x i m a t e l y I5 rain of such an exposure (40 kV, 25 mA-CuK~ s t a n d a r d X - r a y source), the capillary t u b e is reopened, all t h e excess m o t h e r liquor removed, a n d the capillary resealed for t h e d a t a collection. H e a v y - a t o m derivatives are p r e p a r e d using the buffered styrene described above along with suitable h e a v y - a t o m solutions. W e have been able to prepare the derivatives b o t h b y soaking pregrown crystals in a h e a v y - a t o m solution or b y crystallizing t h e protein in the presence of the h e a v y - a t o m solution. W e have used freshly p r e p a r e d buffered styrene solution every time we set up a new b a t c h of crystals. All diffraction d a t a reported here have been measured on a n E n r a f :l~onius CAD4 a u t o m a t i c X - r a y diffractometer. The X - r a y exposure is measured b y accumulating the actual shutter open time from t h e interface of the diffractometer to an accuracy of 0.01 h. Omega scan of 1.5 ~ is used throughout the d a t a collection. F r o m a t o t a l of 96 separate measurements for each scan, the first a n d the last 16 measurements are separately accumul a t e d to estimate t h e b a c k g r o u n d for each reflection a n d the remaining measurements are accumulated to give the i n t e g r a t e d i n t e n s i t y value for each reflection. The crystal deterioration is monitored b y measuring t h e intensities of a set of 3 reflections periodically during d a t a collection. (c) F o u l e r maps The difference electron density m a p presented in this p a p e r has been calculated using reflections with d > 6-0 A with ([/~[STr - - ]P[NAT), m e x p i ~ A T as coefficients, where ]F]sAT a n d [F[sTr correspond to the structure amplitudes from an u n t r e a t e d a n d t r e a t e d crystal, m is the figure of merit a n d ~NATiS the " b e s t " phase angle for the u n t r e a t e d crystal determined b y S a r m a e t a / . (1971b) using three derivatives, pCMB, Hg(CN)s a n d B a k e r dimereurial.
S T U D I E S ON S T Y R E N E - T R E A T E D
IgG C R Y S T A L S
481
The difference Patterson map is calculated using native and derivative data, both measured from styrene-treated crystals. I n both instances described above the two sets of data were scaled together b y an iterative least-squares procedure similar to that described b y Arnone et ~ . (1971).
3. Results Figure 1 shows the variation in intensity with time for three intensity control reflections from untreated (control) and styrene-treated crystals. The findings are similar to those previously published (Zaloga & Sarma, 1974), but have been extended to nearly 80 hours of exposure time. Figure l(c) shows a reflection (--16 0 4) which appears more sensitive to radiation damage than the other two reflections. Even in such a severe case the protection provided by styrene is approximately seven-fold. It must be emphasized that the degree of radiation protection provided by styrene is not always the same for all crystals. We have interpreted this as being due to small variations in the concentration of styrene in the protein drop. The extreme effects of a much larger variation of the concentration of styrene described in our prelimlnary report are entirely reproducible. Styrene-treated crystals are isomorphous with the untreated crystals and Figure 2 shows 14 sections of a difference electron density map calculated between the styrenetreated and untreated crystals. The first contour in this map is drawn at a density of 0.03 e/A 8 which corresponds to the background level of the map calculated as described by Henderson & Moffatt (1971). Subsequent contours are drawn at intervals H0
o I
.
.
.
o.
1-00@(---o--x. . . . . _~ 0.90 -
.
•
~
.
x~
(0) o .
.
.
x
~x~x
.
o
9
o
x
\
-
" " o - --.....
0.80
0.70
I
I
:,
I
I
I
I (b)
X ----x--X-----x--x
o
I'00 ~X-'--:'T'JX-
~="~-"
o "o--'~
X . . . .
~
(el I'00~"~,.
o
0'90 E
~-o
o
\
.o
O,Oo X- r o y
exposure
( h)
FIG. 1. Variation of AlIo with exposure time ~ for 3 reference reflections (a) (19 5 0); (b), (0 8 0); and (o), (--16 0 4). The filled circles ( 0 ) correspond to data from untreated native crystals, the open oiroles (O) from styrene-treated (2 n~-styrone) native orystals and the orosses for data from styrene-trea~ed protein orystals with pCM.B.
482
R. S A R M A A N D G. Z A L O G A :f
-0-35
-0-55
-0-25
o
0 o o
o~
Y o o o
o
0
~ ~ o
o
9
00 o
o~
0o
+0'50
FIG. 2. A composite of 14 sections (Z = 0.0 to 0.52 in intervals of 0.04) of the 3-dimensional difference electron density map between styrene-treated and untreated crystals of DOB imraunoglobulin. The first contour is at 0.03 elA3 and subsequent contours arc dr~wn at intervals of O.OOO e l k 8.
of 0"005 e / h a. There are no major peaks anywhere in the difference map suggesting that the binding of styrene to the protein is very non-specific and does not produce any major conformational changes detectable at this resolution of 6 .~. In addition there are no regions in the solvent space with large ordered concentrations of styrene or polystyrene. The two positions marked b y a cross are symmetry-related positions and these correspond to the negative features observed in the electron density map of the untreated native crystal (Sarma et el., 19715) at the positions of the mercury binding sites. The small positive values at the above position in the difference electron density map suggests that the negative feature at the heavy-atom binding site in the styrene-treated crystal is not as deep as it is in the untreated crystal. This is probably due to better diffraction data from the treated crystal. Due to the longer Lifetime of the styrene-treated crystal in the X-ray beam, we were able to collect the entire 6 data from one single crystal, whereas it took about six to eight crystals in the untreated case. Figure 3 shows an (h01) projection of the difference Patterson calculated between a mercury derivative and a native crystal, both of which are grown in the presence of styrene. The position of the mercury binding site is identical t~a that observed in the difference Patterson calculated with the untreated crystals suggesting that the presence of styrene in the crystal growing medium does not in any way prevent binding of heavy atoms necessary for structure analysis. Figure 1 also indicates t h a t the protection against radiation provided b y the styrene is observed even with a crystal of the derivative.
STUDIES
ON S T Y R E N E - T R E A T E D
IgO CRYSTALS
488 0.5
O
o
1.0 Fie. 8. An (h01) projection down the b axis of the difference Patterson map between a pCMB derivative of the immunoglobulin and the native crystal. Both crystals were grown in the presence of the same amount of monomers of styrene.
4. D i s c u s s i o n
The data currently available is insu•cient to explain a precise mechanism for the radiation protection or the extension of the diffraction pattern observed in the styrene-treated crystals. However, we speculate that styrene monomers are trapped in the intermolecular volume of the crystal lattice and are polymerized b y the Xradiation either forming a cushion-like mass of polystyrene or forming a cage-like structure surrounding the protein molecule. In fact we have noticed t h a t the crystals appear to become hard after irradiation. The absence of any feature in the difference Fourier maps suggests that the binding of styrene is non-specific, therefore the polymerization appears to be random and probably prevents large vibrational or rotational motions of different parts of the protein, effectively lowering its temperature factor. Radiation damage is prevented b y the phenyl groups of styrene with its multiplicity of energy levels and electronic resonances (Charlesby, 1960). The phenyl group acts as an energy trap and scavenges free radicals and electrons formed in the crystal due to X-ray exposure. They m a y also stabilize damaged and potentially reactive sites on the protein b y donating electrons or hydrogens (Charlesby, 1960) and themselves maintaining an ionized form of low reactivity. Styrene monomers m a y also function b y displacing water in the intermolecular space, thus removing a potent source of H § and O H - radicals. We feel that styrene and other vinyl polymers m a y have widespread use in preventing radiation damage and extending the diffraction pattern from other protein crystals. The treatment m a y be particularly useful with the high-energy synchrotron radiation sources now being developed. We also recognize a possible application of polystyrene for use in taking electron micrographs of specimens t h a t would otherwise be damaged during the experiment. This work was supported by grant number AI10762 from the National Institute of Allergy and Infectious Diseases and grant number NP94A from the American Cancer Society.
484
R. SARMA
AND
G. Z A L O G A
REFERENCES Arnone, A., Bier, C. J., Cotton, F. A., Day, V. W., Hazen, E. E., Richardson, D. C., Richardson, J. S. & Yonath, A. (1971). J. Biol. Chem. 246, 2303-2316. Charlesby, A. (19601. In~A~mic R ~ t i ~ and Polymers, vol. I, p. 492. Pergamon Press, N e w York. Henderson, R. & Moffatt, ,.i..K. (1971). A c ~ Grys~gr. sect.B, 27, 1414-1420. Sarma, R., Silverton, E./ W., Davies, D. R. & Terry, W . D. (1971a). J. Biol. Chem. 246,
3753-3759. S ~ n ~ , R., Davies, D. R., Labaw, L. W., Sflverton, E. W. & Terry, W. D. (1971b). Cold S~.ing Harbor Syrup. Quay. Biol. 36, 413-419. Zaloga, G. & Sarma, R. (1974). Nc~ur6 (London), 251, 551-552.
Structure Studies on Styrene-treated lmmunoglobulin Crystals R. S ~
~ D G. ZALOGX
Department of Biochemistry 8tare University of New York at 8tony Broo~ Etony Broot~, N.Y. 11794, U.8.A.
(Re,dyed I A ~ l 1975) DOBt immunoglobnlln crystals ordinarily arc highly radiation sensitive and give a limited X-ray diffraction pattern to about 6 A resolution, thereby preventing structure analysis at high resolution. Crystals grown in the presence of monomers of styrene last very much longer in the X-ray beam and give a diffTaction pattern extending to about 4 ,~ resolution. This paper describes the experimental conditions we have used for obtaining optimum results. A difference electron density map calculated between the treated and untreated crystals shows no significant features indicating that styrene treatment does not cause any major conformational change in the protein. I t is also possible to prepare heavy-atem derivatives with styrene-treated crystals as seen from a difference Patterson map in the case of a mercury derivative. We speculate that monomers of styrene trapped in the crystal lattice are polymerized by the X-rays and reduce the molecular motion and scavenge the free radicals formed in the crystal lattice.
1. I n t r o d u c t i o n
The major problem preventing a high resolution structure analysis of the D 0 B t immunoglobulln molecule has been its poor diffraction pattern and its extreme sensitivity to X-radiation (Sarma et al., 1971a). I n a recent report (Zaloga & Sarma, 1974) we described some preliminary results of experiments using monomers of styrene in preventing the radiation damage and extending the diffraction pattern from the above crystals. Since then we have extended these studies to understand more fully t h e exact nature of the protection provided b y polystyrene. As a prerequisite to the regular use of styrene in the structure analysis of the immunoglobin molecule we set out to answer two important questions. ~irst, is the styrene producing any eonformational change in the protein molecule b y binding to specific sites on the protein? Second, will the styrene treatment prevent binding of heavy atoms to the protein necessary for actual structure analysis? We report the answers to both questions in this paper. I n addition, prompted b y numerous enquiries from other protein crystaUographers, we present the exact experimental details t h a t we have used in the styrene treatment with all the precautions t h a t we think are necessary to achieve optimum results. t Abbreviation used: DOB immunoglobulin, protein purified from the serum of the patient, Dobbs (see Materials and Methods). 22
479
480
R. S A R M A A N D G. Z A L O G A
2. E x p e r i m e n t a l
Methods
(a) Pr6paraZ~n o$ cryata/a All experiments wore ~arrled out using DOB immunoglobulin crystals. These crystals were grown from protein purified from the serum of t h e p a t i e n t (Dobbs) b y s m m o n i u m sulfate fractionation, followed b y colllmn c h r o m a t o g r a p h y on DEAE-eellulose a n d isoelectric focussing on an a~npholyne column (pH 6 to 10). The protein is n o r m a l l y stored in 0.01 ~ - s o d i u m cacodylate (pH 6.0) a t 4~ until needed for crystallization. Crystals are grown a t 4~ in 10-~1 drops sealed in siliconed glass slides, the drop consisting of 3 ~1 protein (30 to 40 mg/ml) a n d 7 ~10-1 ~ - s o d i u m borate buffer (pH 8-4). Styrene-treated crystals were prepared b y cocrystallization. F i v e ml of 0.1 M-sodium b o r a t e buffer (pH 8.4) was overlayed in a test t u b e with a p p r o x i m a t e l y 0.5 ml of 99~/o styrene. (The styrene used in these experiments was purchased from Aldrich Chemical Company. The inhibitor used to prevent spontaneous polymerization was reported b y t h e manufacturer to be t e r t i a r y b u t y l alcohol (I0 to 15 p.p.m.). The remaining i m p u r i t y was n o t identified. No experiments were conducted to purify the styrene.) The m i x t u r e was shaken vigorously a n d allowed to settle for a p p r o x i m a t e l y 1 h a t r o o m temperature. Undissolved styrene separates into a top phase which is t h e n removed with a P a s t e u r pipette. The b o t t o m phase of the mixture is now s a t u r a t e d with styrene a n d is transferred to another test tube. Seven ~1 of this buffered styrene is used with 3 ~1 of the protein solution to prepare the 10 ~1 drop used for crystallization. Great care m u s t be exercised in transferring the buffered styrene for the final crystallization drop because even after several hours of standing, the b o t t o m phase of the mixture has a thin layer of concentrated styrene o n t h e surface which tends to adhere to t h e syringe needle used to transfer this buffer to the slide. The concentration of styrene in the final drop appears to be critical (Zaloga & Sarma, 1974) a n d in the absence of a r a p i d convenient procedure for estimating this concentration, we have found the above procedure quite reproducible. (b) Data collection The monomers of styrene t r a p p e d in the crystal lattice are n o r m a l l y polymerlzed b y the X - r a y s during d a t a collection. However, we have obtained slm~]ar results b y pre-exposing the styrene-treated crystal to X - r a y s with the crystal surrounded b y a small a m o u n t (1 to 2 ~1) of the m o t h e r liquor in the capillary tube. After a p p r o x i m a t e l y I5 rain of such an exposure (40 kV, 25 mA-CuK~ s t a n d a r d X - r a y source), the capillary t u b e is reopened, all t h e excess m o t h e r liquor removed, a n d the capillary resealed for t h e d a t a collection. H e a v y - a t o m derivatives are p r e p a r e d using the buffered styrene described above along with suitable h e a v y - a t o m solutions. W e have been able to prepare the derivatives b o t h b y soaking pregrown crystals in a h e a v y - a t o m solution or b y crystallizing t h e protein in the presence of the h e a v y - a t o m solution. W e have used freshly p r e p a r e d buffered styrene solution every time we set up a new b a t c h of crystals. All diffraction d a t a reported here have been measured on a n E n r a f :l~onius CAD4 a u t o m a t i c X - r a y diffractometer. The X - r a y exposure is measured b y accumulating the actual shutter open time from t h e interface of the diffractometer to an accuracy of 0.01 h. Omega scan of 1.5 ~ is used throughout the d a t a collection. F r o m a t o t a l of 96 separate measurements for each scan, the first a n d the last 16 measurements are separately accumul a t e d to estimate t h e b a c k g r o u n d for each reflection a n d the remaining measurements are accumulated to give the i n t e g r a t e d i n t e n s i t y value for each reflection. The crystal deterioration is monitored b y measuring t h e intensities of a set of 3 reflections periodically during d a t a collection. (c) F o u l e r maps The difference electron density m a p presented in this p a p e r has been calculated using reflections with d > 6-0 A with ([/~[STr - - ]P[NAT), m e x p i ~ A T as coefficients, where ]F]sAT a n d [F[sTr correspond to the structure amplitudes from an u n t r e a t e d a n d t r e a t e d crystal, m is the figure of merit a n d ~NATiS the " b e s t " phase angle for the u n t r e a t e d crystal determined b y S a r m a e t a / . (1971b) using three derivatives, pCMB, Hg(CN)s a n d B a k e r dimereurial.
S T U D I E S ON S T Y R E N E - T R E A T E D
IgG C R Y S T A L S
481
The difference Patterson map is calculated using native and derivative data, both measured from styrene-treated crystals. I n both instances described above the two sets of data were scaled together b y an iterative least-squares procedure similar to that described b y Arnone et ~ . (1971).
3. Results Figure 1 shows the variation in intensity with time for three intensity control reflections from untreated (control) and styrene-treated crystals. The findings are similar to those previously published (Zaloga & Sarma, 1974), but have been extended to nearly 80 hours of exposure time. Figure l(c) shows a reflection (--16 0 4) which appears more sensitive to radiation damage than the other two reflections. Even in such a severe case the protection provided by styrene is approximately seven-fold. It must be emphasized that the degree of radiation protection provided by styrene is not always the same for all crystals. We have interpreted this as being due to small variations in the concentration of styrene in the protein drop. The extreme effects of a much larger variation of the concentration of styrene described in our prelimlnary report are entirely reproducible. Styrene-treated crystals are isomorphous with the untreated crystals and Figure 2 shows 14 sections of a difference electron density map calculated between the styrenetreated and untreated crystals. The first contour in this map is drawn at a density of 0.03 e/A 8 which corresponds to the background level of the map calculated as described by Henderson & Moffatt (1971). Subsequent contours are drawn at intervals H0
o I
.
.
.
o.
1-00@(---o--x. . . . . _~ 0.90 -
.
•
~
.
x~
(0) o .
.
.
x
~x~x
.
o
9
o
x
\
-
" " o - --.....
0.80
0.70
I
I
:,
I
I
I
I (b)
X ----x--X-----x--x
o
I'00 ~X-'--:'T'JX-
~="~-"
o "o--'~
X . . . .
~
(el I'00~"~,.
o
0'90 E
~-o
o
\
.o
O,Oo X- r o y
exposure
( h)
FIG. 1. Variation of AlIo with exposure time ~ for 3 reference reflections (a) (19 5 0); (b), (0 8 0); and (o), (--16 0 4). The filled circles ( 0 ) correspond to data from untreated native crystals, the open oiroles (O) from styrene-treated (2 n~-styrone) native orystals and the orosses for data from styrene-trea~ed protein orystals with pCM.B.
482
R. S A R M A A N D G. Z A L O G A :f
-0-35
-0-55
-0-25
o
0 o o
o~
Y o o o
o
0
~ ~ o
o
9
00 o
o~
0o
+0'50
FIG. 2. A composite of 14 sections (Z = 0.0 to 0.52 in intervals of 0.04) of the 3-dimensional difference electron density map between styrene-treated and untreated crystals of DOB imraunoglobulin. The first contour is at 0.03 elA3 and subsequent contours arc dr~wn at intervals of O.OOO e l k 8.
of 0"005 e / h a. There are no major peaks anywhere in the difference map suggesting that the binding of styrene to the protein is very non-specific and does not produce any major conformational changes detectable at this resolution of 6 .~. In addition there are no regions in the solvent space with large ordered concentrations of styrene or polystyrene. The two positions marked b y a cross are symmetry-related positions and these correspond to the negative features observed in the electron density map of the untreated native crystal (Sarma et el., 19715) at the positions of the mercury binding sites. The small positive values at the above position in the difference electron density map suggests that the negative feature at the heavy-atom binding site in the styrene-treated crystal is not as deep as it is in the untreated crystal. This is probably due to better diffraction data from the treated crystal. Due to the longer Lifetime of the styrene-treated crystal in the X-ray beam, we were able to collect the entire 6 data from one single crystal, whereas it took about six to eight crystals in the untreated case. Figure 3 shows an (h01) projection of the difference Patterson calculated between a mercury derivative and a native crystal, both of which are grown in the presence of styrene. The position of the mercury binding site is identical t~a that observed in the difference Patterson calculated with the untreated crystals suggesting that the presence of styrene in the crystal growing medium does not in any way prevent binding of heavy atoms necessary for structure analysis. Figure 1 also indicates t h a t the protection against radiation provided b y the styrene is observed even with a crystal of the derivative.
STUDIES
ON S T Y R E N E - T R E A T E D
IgO CRYSTALS
488 0.5
O
o
1.0 Fie. 8. An (h01) projection down the b axis of the difference Patterson map between a pCMB derivative of the immunoglobulin and the native crystal. Both crystals were grown in the presence of the same amount of monomers of styrene.
4. D i s c u s s i o n
The data currently available is insu•cient to explain a precise mechanism for the radiation protection or the extension of the diffraction pattern observed in the styrene-treated crystals. However, we speculate that styrene monomers are trapped in the intermolecular volume of the crystal lattice and are polymerized b y the Xradiation either forming a cushion-like mass of polystyrene or forming a cage-like structure surrounding the protein molecule. In fact we have noticed t h a t the crystals appear to become hard after irradiation. The absence of any feature in the difference Fourier maps suggests that the binding of styrene is non-specific, therefore the polymerization appears to be random and probably prevents large vibrational or rotational motions of different parts of the protein, effectively lowering its temperature factor. Radiation damage is prevented b y the phenyl groups of styrene with its multiplicity of energy levels and electronic resonances (Charlesby, 1960). The phenyl group acts as an energy trap and scavenges free radicals and electrons formed in the crystal due to X-ray exposure. They m a y also stabilize damaged and potentially reactive sites on the protein b y donating electrons or hydrogens (Charlesby, 1960) and themselves maintaining an ionized form of low reactivity. Styrene monomers m a y also function b y displacing water in the intermolecular space, thus removing a potent source of H § and O H - radicals. We feel that styrene and other vinyl polymers m a y have widespread use in preventing radiation damage and extending the diffraction pattern from other protein crystals. The treatment m a y be particularly useful with the high-energy synchrotron radiation sources now being developed. We also recognize a possible application of polystyrene for use in taking electron micrographs of specimens t h a t would otherwise be damaged during the experiment. This work was supported by grant number AI10762 from the National Institute of Allergy and Infectious Diseases and grant number NP94A from the American Cancer Society.
484
R. SARMA
AND
G. Z A L O G A
REFERENCES Arnone, A., Bier, C. J., Cotton, F. A., Day, V. W., Hazen, E. E., Richardson, D. C., Richardson, J. S. & Yonath, A. (1971). J. Biol. Chem. 246, 2303-2316. Charlesby, A. (19601. In~A~mic R ~ t i ~ and Polymers, vol. I, p. 492. Pergamon Press, N e w York. Henderson, R. & Moffatt, ,.i..K. (1971). A c ~ Grys~gr. sect.B, 27, 1414-1420. Sarma, R., Silverton, E./ W., Davies, D. R. & Terry, W . D. (1971a). J. Biol. Chem. 246,
3753-3759. S ~ n ~ , R., Davies, D. R., Labaw, L. W., Sflverton, E. W. & Terry, W. D. (1971b). Cold S~.ing Harbor Syrup. Quay. Biol. 36, 413-419. Zaloga, G. & Sarma, R. (1974). Nc~ur6 (London), 251, 551-552.
