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Time lapse microphotography of protein crystal growth using a color VCR
340
Journal of Crystal Growth 90 (1988) 340—343 North-Holland, Amsterdam
TIME LAPSE MICROPHOTOGRAPHY OF PROTEIN CRYSTAL GROWTH USING A COLOR VCR Stanley KOSZELAK and Alexander McPHERSON Department of Biochemistry, University of California, Riverside, California 92521, USA
Received 15 September 1987; manuscript received in final form 7 November 1987
A time lapse video color recorder (VCR) interfaced to a color video camera mounted on a light microscope was used to observe and record the growth of three protein crystals: lysozyme, canavalin, and a fungal protease. The studies produced a number of interesting observations regarding the formation of crystal habit, crystal aggregates, and crystal polymorphs. It further provided a simple and easy means for the measurement of growth rates. The system is described here along with a brief collection of some of our conclusions based on the time lapse video sequences.
I. Introduction
2. Materials and methods
Direct observation and recording of the growth of conventional, small molecule crystals has traditionally been used to approach questions regarding the kinetics and structural changes characterizing crystal development. Such questions include the formation of crystal habit, polymorphic transitions, rates of growth, occurrence of defects, and the appearance of crystal aggregates or twins, Traditionally, direct observation of protein crystal growth, except for a few well-studied cases such as lysozyme [1—3],has not been employed. Presumably this was due to the difficulty in promoting growth within a specified interval of time, the apparent slowness of protein crystal growth, and the lack of appropriate means to observe growing protein crystals over long periods of time. We have attempted, using conventional and readily available methods and instruments, to overcome some of the problems and to obtain compact visual records of growing protein crystals. We believe we have succeeded to some extent, and have, indeed, been able to make some interesting observations bearing on the mechanisms and kinetics of protein crystal growth.
The proteins we employed in our experiments were lysozyme [4], canavalin [5,6] and the serine protease from penicillium cyclopium [71.These were chosen for their availability, the ease and rapidity with which they form crystal nuclei, and the relatively rapid rates of growth of their crystals. The solution conditions used for the crystallization of lysozyme and canavalin are given in a second paper found in these proceedings (McPherson and Shlichta [8]). The fungal protease was crystallized as described by Day et al. [7] from 8% PEG 4000 (polyethylene glycol) at neutral pH. Vapor diffusion in hanging drops from 22 mm glass cover slips over the wells of Linbro tissue culture plates [9] were used in some experiments. In other trials, sandwich drops between glass slides using the plastic crystallization plates from FLOW Labs were employed. Again, the crystallization was promoted by vapor transport between protein droplets and a 0.5 ml reservoir. The experiments were carried out on the stage of a Swift microscope so that the events in the protein droplet could be observed as equilibration proceeded. 10 x and 4 x objective lenses were used for high and low power magnification.
0022-0248/88/$03.50 © Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)
S. Koszelak, A. McPherson et a!.
/
Time lapse microphotography of protein crystal growth using color VCR
341
Fig. 1. The system utilized for recording time lapse video sequences of growing protein crystals. The three elements are a standard microscope (Swift), a color video camera (Sanyo) mounted on the microscope, and a time lapse VCR (GYYR).
The image at the specimen was monitored with a Sanyo color video camera mounted on the microscope by a special adaptor. The image was displayed in real time on a color television placed next to the microscope and, at the same time, was recorded by a GYYR time lapse VCR to which the video camera was interfaced. The time lapse VCR had the capability of compressing real time events from 1 to 240-fold, so that maximally, 240 h could be recorded on a single 2 h magnetic tape. In general, we found that a compression of 72-fold was optimal for our experiments, A polarizer was fitted between the specimen stage and the light source so that color variations could be used to enhance contrast, improve the quality of the images, and to make observation of birefringence possible. The light source was cooled by a small fan to reduce heating at the microscope stage. It was found that image quality at high magnification could be substantially improved by removing the light diffuser so as to obtain a brighter source. The bottoms of the plastic plates were quite adequate in diffusing the light, The entire system is seen in fig. 1. It can be purchased as a complete unit from Resolution Technologies, San Juan Capistrano, California, for about $6000.00 and is ready for experiments immediately. Using a second standard VCR, it is a generally straightforward matter to edit the tapes
from several different experiments into a single sequence for review and storage. It is also possible, although we have not yet carried the work so far, to apply image analysis methods to individual frames and to a series of observations. An important feature of the system shown in fig. 1 is that the VCR provides a visible running clock and calendar and which is superimposed on the corner of every video frame. Thus, when a tape is reviewed, the clock in the lower corner of the screen continuously informs the viewer of the time elapsed and the actual time each frame was recorded. It is, therefore, possible to obtain reasonably accurate measures of growth rates by simply using a ruler held against the growing crystal imaged on the television monitor.
3. Some experiences and observations It is, of course, difficult to describe clearly and succinctly a sequence of visual images over a period of time, and we will make no attempt to do so here. We can, however, note some of the observations that we found particularly interesting. (1) Protein crystal growth rates are substantially greater than we had believed. In numerous cases, we observed for canavalin rates of growth that were greater than 5000 A per minute. For
342
S. Koszelak, A. McPherson et a!.
/
Time lapse microphotographv of protein crystal growth using color VCR
rhombohedral canavalin, this corresponds to roughly 40 unit cell thicknesses per minute. Crystals as large as 1 mm in size were seen to nucleate and reach that size in three hours, (2) Frequently, the rates of crystals, both mdividually and as an ensemble, were seen not to be continuous but the enlargement was observed to occur in spurts and stops. In most other cases, it indeed appeared almost constant. (3) Several microcrystalline showers were promoted to occur by altering temperature. We observed that these showers could be complete and return the solution to an apparently non-supersaturated state in less than 15 mm (fungal protease). In other cases, the showers would persist for as long as several hours. (4) Protein crystals tend to nucleate on particulate matter or solid surfaces in the protein droplet before anywhere else. If no particles are available, they will then preferentially form at the air—liquid interface. (5) In hanging drops [9], all crystals as large as visible nuclei (at 100 >< magnification) fall immediately and collect at the bottom of the drop. This is convenient for observation but hardly an advantage if the objective is to grow large, single crystals. The potential advantages of crystal growth in microgravity were clearly evident in hanging drop experiments, (6) There is much more convective turbulance present in the protein droplets than we suspected. This flow, and the micro currents present, can be readily seen if there is a small amount of protein precipitate present. This acts as dust in the wind and delineates well the motion present. Although part of this turbulance is undoubtedly due to temperature gradients, it seems likely to us that it may also be a consequence of other kinds of convective phenomena. (7) We observed that large crystals tend not to be obstructed by smaller crystals, but simply continue growing and push them out of the way. Using a position marker, we have recorded a large growing canavalin crystal pushing a smaller crystal abutting its face some distance across the droplet. We have also observed small crystals contacting or adhering to the faces of larger ones, dissolve and
incorporate into the larger crystal as the dominant crystal continues to grow. (8) Large masses and aggregates of protein crystals were never observed to form from secondary crystals nucleating and growing from a single initial crystal, nor from each other. Very few, if any cases were observed of crystals initiating and growing from the surface of another. All aggregates and masses of crystals that we observed originated from individual nuclei that came together due to gravity or from the solution currents present in the samples. (9) Crystals tended, in general, to grow so as to preserve the habit and edge ratio evident at the earliest stage of visibility unless they were obstructed by surfaces or other crystals. Except when physical forces due to physical contact altered form, ultimate shape appeared to be determined at or shortly after nucleation. (10) Crystals of very different habit (polymorphs) were observed to nucleate, grow, and coexit simultaneously in the very same sample with no apparent difference in the initial time of nucleation or rate of growth. 4. Intentions Our intentions for the future are to expand the range of proteins that we can observe and experiment upon so that some generalizations may be made. Currently the protein sample size is quite small and we cannot say with any certainty what effects may be common to all protein crystals and which observations are particular to a single protein. A second objective at this point is to introduce some rudimentary image analysis procedures. These will permit us to better measure growth rates, compare edge ratios as a function of time, and in general, institute a greater degree of quantitation into our studies. We have been surprised and pleased at the observations presented us, and would encourage others to extend these investigations to other proteins. In this way, a body of experience and a collection of observations can be compiled from which some new principles may emerge.
S. Koszelak, A. McPherson et a!. / Time lapse microphotography ofprotein crystalgrowth using color VCR
Acknowledgements We gratefully acknowledge the financial support of the National Science Foundation (BBS 8614585) and the California Biotechnology Training Program for this research.
References [1] Z. Kam, H.B. Shore and G. Feher, J. Mol. Biol. 127 (1978) 539. [2] M.L. Pusey and R. Naumann, J. Crystal Growth 76 (1986) 593.
343
[3] R.W. Fiddis, R.A. Longman and PD. Calvert, Trans. Faradat Soc. 75 (1979) 2753. [4] C.C.F. Blake, L.N. Johnson, G.A. Mair, A.C.T. North, D.C. Phillips and V.R. Sarma, Proc. Roy. Soc. (London) B167 (1967) 378. [5] A. McPherson, J. Biol. Chem. 255 (1980) 10472. [6] S.C. Smith, S. Johnson, J. Andrews and A. McPherson, Plant Physiol. 70 (1982) 1199. [7] J. Day, S. Koszelak, D. Casciao and A. McPherson, J. Biol. Chem. 261 (1986) 1957. [8] A. McPherson and P.J. Shlichta, J. Crystal Growth 90 (1988) 47. [9] A. McPherson, in: Methods in Enzymology, Vol. 114, Eds. C.H.W. Hirs, S.N. Timasheff and H.W. Wyckoff (Academic Press, Orlando, FL, 1985) pp. 112—119.
Journal of Crystal Growth 90 (1988) 340—343 North-Holland, Amsterdam
TIME LAPSE MICROPHOTOGRAPHY OF PROTEIN CRYSTAL GROWTH USING A COLOR VCR Stanley KOSZELAK and Alexander McPHERSON Department of Biochemistry, University of California, Riverside, California 92521, USA
Received 15 September 1987; manuscript received in final form 7 November 1987
A time lapse video color recorder (VCR) interfaced to a color video camera mounted on a light microscope was used to observe and record the growth of three protein crystals: lysozyme, canavalin, and a fungal protease. The studies produced a number of interesting observations regarding the formation of crystal habit, crystal aggregates, and crystal polymorphs. It further provided a simple and easy means for the measurement of growth rates. The system is described here along with a brief collection of some of our conclusions based on the time lapse video sequences.
I. Introduction
2. Materials and methods
Direct observation and recording of the growth of conventional, small molecule crystals has traditionally been used to approach questions regarding the kinetics and structural changes characterizing crystal development. Such questions include the formation of crystal habit, polymorphic transitions, rates of growth, occurrence of defects, and the appearance of crystal aggregates or twins, Traditionally, direct observation of protein crystal growth, except for a few well-studied cases such as lysozyme [1—3],has not been employed. Presumably this was due to the difficulty in promoting growth within a specified interval of time, the apparent slowness of protein crystal growth, and the lack of appropriate means to observe growing protein crystals over long periods of time. We have attempted, using conventional and readily available methods and instruments, to overcome some of the problems and to obtain compact visual records of growing protein crystals. We believe we have succeeded to some extent, and have, indeed, been able to make some interesting observations bearing on the mechanisms and kinetics of protein crystal growth.
The proteins we employed in our experiments were lysozyme [4], canavalin [5,6] and the serine protease from penicillium cyclopium [71.These were chosen for their availability, the ease and rapidity with which they form crystal nuclei, and the relatively rapid rates of growth of their crystals. The solution conditions used for the crystallization of lysozyme and canavalin are given in a second paper found in these proceedings (McPherson and Shlichta [8]). The fungal protease was crystallized as described by Day et al. [7] from 8% PEG 4000 (polyethylene glycol) at neutral pH. Vapor diffusion in hanging drops from 22 mm glass cover slips over the wells of Linbro tissue culture plates [9] were used in some experiments. In other trials, sandwich drops between glass slides using the plastic crystallization plates from FLOW Labs were employed. Again, the crystallization was promoted by vapor transport between protein droplets and a 0.5 ml reservoir. The experiments were carried out on the stage of a Swift microscope so that the events in the protein droplet could be observed as equilibration proceeded. 10 x and 4 x objective lenses were used for high and low power magnification.
0022-0248/88/$03.50 © Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)
S. Koszelak, A. McPherson et a!.
/
Time lapse microphotography of protein crystal growth using color VCR
341
Fig. 1. The system utilized for recording time lapse video sequences of growing protein crystals. The three elements are a standard microscope (Swift), a color video camera (Sanyo) mounted on the microscope, and a time lapse VCR (GYYR).
The image at the specimen was monitored with a Sanyo color video camera mounted on the microscope by a special adaptor. The image was displayed in real time on a color television placed next to the microscope and, at the same time, was recorded by a GYYR time lapse VCR to which the video camera was interfaced. The time lapse VCR had the capability of compressing real time events from 1 to 240-fold, so that maximally, 240 h could be recorded on a single 2 h magnetic tape. In general, we found that a compression of 72-fold was optimal for our experiments, A polarizer was fitted between the specimen stage and the light source so that color variations could be used to enhance contrast, improve the quality of the images, and to make observation of birefringence possible. The light source was cooled by a small fan to reduce heating at the microscope stage. It was found that image quality at high magnification could be substantially improved by removing the light diffuser so as to obtain a brighter source. The bottoms of the plastic plates were quite adequate in diffusing the light, The entire system is seen in fig. 1. It can be purchased as a complete unit from Resolution Technologies, San Juan Capistrano, California, for about $6000.00 and is ready for experiments immediately. Using a second standard VCR, it is a generally straightforward matter to edit the tapes
from several different experiments into a single sequence for review and storage. It is also possible, although we have not yet carried the work so far, to apply image analysis methods to individual frames and to a series of observations. An important feature of the system shown in fig. 1 is that the VCR provides a visible running clock and calendar and which is superimposed on the corner of every video frame. Thus, when a tape is reviewed, the clock in the lower corner of the screen continuously informs the viewer of the time elapsed and the actual time each frame was recorded. It is, therefore, possible to obtain reasonably accurate measures of growth rates by simply using a ruler held against the growing crystal imaged on the television monitor.
3. Some experiences and observations It is, of course, difficult to describe clearly and succinctly a sequence of visual images over a period of time, and we will make no attempt to do so here. We can, however, note some of the observations that we found particularly interesting. (1) Protein crystal growth rates are substantially greater than we had believed. In numerous cases, we observed for canavalin rates of growth that were greater than 5000 A per minute. For
342
S. Koszelak, A. McPherson et a!.
/
Time lapse microphotographv of protein crystal growth using color VCR
rhombohedral canavalin, this corresponds to roughly 40 unit cell thicknesses per minute. Crystals as large as 1 mm in size were seen to nucleate and reach that size in three hours, (2) Frequently, the rates of crystals, both mdividually and as an ensemble, were seen not to be continuous but the enlargement was observed to occur in spurts and stops. In most other cases, it indeed appeared almost constant. (3) Several microcrystalline showers were promoted to occur by altering temperature. We observed that these showers could be complete and return the solution to an apparently non-supersaturated state in less than 15 mm (fungal protease). In other cases, the showers would persist for as long as several hours. (4) Protein crystals tend to nucleate on particulate matter or solid surfaces in the protein droplet before anywhere else. If no particles are available, they will then preferentially form at the air—liquid interface. (5) In hanging drops [9], all crystals as large as visible nuclei (at 100 >< magnification) fall immediately and collect at the bottom of the drop. This is convenient for observation but hardly an advantage if the objective is to grow large, single crystals. The potential advantages of crystal growth in microgravity were clearly evident in hanging drop experiments, (6) There is much more convective turbulance present in the protein droplets than we suspected. This flow, and the micro currents present, can be readily seen if there is a small amount of protein precipitate present. This acts as dust in the wind and delineates well the motion present. Although part of this turbulance is undoubtedly due to temperature gradients, it seems likely to us that it may also be a consequence of other kinds of convective phenomena. (7) We observed that large crystals tend not to be obstructed by smaller crystals, but simply continue growing and push them out of the way. Using a position marker, we have recorded a large growing canavalin crystal pushing a smaller crystal abutting its face some distance across the droplet. We have also observed small crystals contacting or adhering to the faces of larger ones, dissolve and
incorporate into the larger crystal as the dominant crystal continues to grow. (8) Large masses and aggregates of protein crystals were never observed to form from secondary crystals nucleating and growing from a single initial crystal, nor from each other. Very few, if any cases were observed of crystals initiating and growing from the surface of another. All aggregates and masses of crystals that we observed originated from individual nuclei that came together due to gravity or from the solution currents present in the samples. (9) Crystals tended, in general, to grow so as to preserve the habit and edge ratio evident at the earliest stage of visibility unless they were obstructed by surfaces or other crystals. Except when physical forces due to physical contact altered form, ultimate shape appeared to be determined at or shortly after nucleation. (10) Crystals of very different habit (polymorphs) were observed to nucleate, grow, and coexit simultaneously in the very same sample with no apparent difference in the initial time of nucleation or rate of growth. 4. Intentions Our intentions for the future are to expand the range of proteins that we can observe and experiment upon so that some generalizations may be made. Currently the protein sample size is quite small and we cannot say with any certainty what effects may be common to all protein crystals and which observations are particular to a single protein. A second objective at this point is to introduce some rudimentary image analysis procedures. These will permit us to better measure growth rates, compare edge ratios as a function of time, and in general, institute a greater degree of quantitation into our studies. We have been surprised and pleased at the observations presented us, and would encourage others to extend these investigations to other proteins. In this way, a body of experience and a collection of observations can be compiled from which some new principles may emerge.
S. Koszelak, A. McPherson et a!. / Time lapse microphotography ofprotein crystalgrowth using color VCR
Acknowledgements We gratefully acknowledge the financial support of the National Science Foundation (BBS 8614585) and the California Biotechnology Training Program for this research.
References [1] Z. Kam, H.B. Shore and G. Feher, J. Mol. Biol. 127 (1978) 539. [2] M.L. Pusey and R. Naumann, J. Crystal Growth 76 (1986) 593.
343
[3] R.W. Fiddis, R.A. Longman and PD. Calvert, Trans. Faradat Soc. 75 (1979) 2753. [4] C.C.F. Blake, L.N. Johnson, G.A. Mair, A.C.T. North, D.C. Phillips and V.R. Sarma, Proc. Roy. Soc. (London) B167 (1967) 378. [5] A. McPherson, J. Biol. Chem. 255 (1980) 10472. [6] S.C. Smith, S. Johnson, J. Andrews and A. McPherson, Plant Physiol. 70 (1982) 1199. [7] J. Day, S. Koszelak, D. Casciao and A. McPherson, J. Biol. Chem. 261 (1986) 1957. [8] A. McPherson and P.J. Shlichta, J. Crystal Growth 90 (1988) 47. [9] A. McPherson, in: Methods in Enzymology, Vol. 114, Eds. C.H.W. Hirs, S.N. Timasheff and H.W. Wyckoff (Academic Press, Orlando, FL, 1985) pp. 112—119.