J. Phys. Chem. 1993,97, 3013-3016
3013
Molecular Analysis of Two-Dimensional Protein Crystallization Andrew C. Ku,~Seth A. Dam&*Channing R. RoberQon,t Alice P. Cast,? and Roger D. Kornberg'** Departments of Chemical Engineering and Cell Biology, Stanford University, Stanford, California 94305 Received: October 19, 1992; In Final Form: November 24, 1992
Two-dimensional crystals of streptavidin formed on lipid layers at the air-water interface were cross-linked to preserve their structure during transfer to a solid support. The morphology of the crystals viewed at the air-water interface by light microscopy could then be related to the molecular structure revealed in the electron microscope. The preferred growth direction of the crystals proved to be along one of two intersecting rows of intermolecular contacts. A secondary direction arises during a transition to dendritic growth.
Introduction
Experimental Section
The discovery of lateral diffusion in lipid layers by McConnell and colleagues1inspired the development of a general method of forming two-dimensional(2-D)protein In theoriginal version of the method, lipids were linked to ligands for binding specifically to a protein of interest. The orientation and concentration of the protein at the surface of the lipid layer led to 2-D crystal formation. In subsequent studies, proteins adsorbed to lipid layers through electrostatic rather than specific binding were shown to crystallize as weL4 One reason for growing 2-D protein crystals is to permit structure determination by electron microscopy and image proce~sing.~Advantages of structure determination by this approach include the rapidity and the small amount of material required. A general method of growing 2-D crystals is also important for fundamental studies of the crystallization process. Aspects of an order-disorder phase transition can be studied in a system of reduced dimensionality at the molecular level. Streptavidin,a tetrameric protein with four sites of high affinity binding to biotin? has been exploited for studies of the 2-D crystallizationprocess. Fluorescently labeled streptavidinforms 2-D crystals on layers of biotinated lipids large enough to be seen in the light microscope,' where they exhibitstrikingmorphologies. The crystals are generally rectangular, indicating differencesin growth rate in the two primary growth directions, and usually acquire a characteristic notched or H shape, indicatingsome sort of growth instability. Introduction of the related protein avidin as a noncrystallizable contaminant promotes the formation of dendritic crystals by enhancing the growth instability under conditionsof transport limitations.8 It is well-knownthat dendritic growth dependsstrongly on the energetics and hence the molecular structure of the solid-liquid interface, so an understanding of dendritic growth would benefit greatly from a detailed view of the interface. We have therefore initiated studiesof the dendritic growth process by electron microscopy. Electron diffraction from 2-D streptavidin crystals and computed diffraction from electron micrographs of the crystals are indicative of a square lattice with space group symmetry C222 ordered to 2.7-A resol~tion.~,~ Fourier synthesis reveals two molecules in the unit cell, with a pair of biotin-binding sites on each molecule facing the lipid layer and two sites exposed to solution. Here we report on the relationship between the molecular lattice and the macroscopic morphology of dendritic crystals. The results provide a basis for understanding shape formation of 2-D protein crystals analogous to studies of domain shapes in immiscible lipid phases.I0
Streptavidin was fluoresceinatedand crystallizedon lipid layers8 spread over 0.05 M sodium phosphate, pH 7.0, 0.5 M NaCl. With a total concentration of streptavidin and avidin of about 5 pg/mL, abundant crystal growth occurred during 30 min at room temperature. Glutaraldehyde (50%) was injected into the subphaseto a concentrationof 0.5% and cross-linkingwas allowed to proceed for 2-7 h. "Finder" electron microscopegrids (Fullam) coated with nitrocellulose were then placed on the monolayers with the nitrocellulose side down, and fluorescence micrographs were taken to document the locationsof crystals. The grids were then withdrawn, washed with a drop of distilled water, and stained for 5-10 s with 1% (w/v) uranyl acetate. After air-drying, the grid was coated, plastic and protein side up, with a layer of carbon several hundred angstromsthick. Electron microscopyand image processing were performed as described.'
t
Department of Chemical Engineering. Department of Cell Biology.
0022-3654/93/2097-3013$04.OO/0
RHsultS
Two-dimensional crystals of streptavidin were grown on monolayers of phosphatidylcholine containing 2.3 mol % biotinated lipid at a surface pressure of 25-30 dyn/cm at the airwater interface. The streptavidin was labeled with fluorescein isothiocyanate for viewing in the fluorescence light microscope, and the degree of dendrite formation was controlled by the inclusion of 0-90 mol % avidin. The 2-D crystals were initially H-shaped and became X-shaped as they grew larger (Figure 1A,B). Upon still further growth, dendrites appeared, extending at regular intervals from the arms of the X-shaped crystals(Figure 1C). The arms of the X's intersected at a characteristic angle that applied to the intersectionwith dendrites as well. While this angle varied somewhat, it was typically about 33O (Figure 2). Under conditions in which tertiary dendrite growth occurred (not shown), similar spacings and angles were observed. To determine the relationship between dendrite growth directions and the streptavidin molecular lattice, we wished to transfer 2-D crystals intact from the air-water interface to specimen grids for viewing in the electron microscope. Such transfer has been accomplished in the past by touching a carboncoated grid to the backside of the lipid layer and withdrawing the grid with adsorbed lipid and protein crystalline material. The difficulty with the procedure is that the crystals were invariably broken apart by stressesinvolved in the transfer procedure? Small crystalline areas up to 5-10 pm in extent were observed, but no crystals hundreds of micrometers across such as those seen in the light microscope were obtained on grids, and of most concern for our purposes here, no vestige of the originaldendritic morphology of the crystals could be discerned on the grids. When the transfer process was monitored by light microscopy, even the initial step of touching a grid to the lipid layer proved to perturb the crystal morphology (Figure 3A). It occurred to us that cross-linking
0 1993 American Chemical Society
3014 The Journal of Physical Chemistry, Vol. 97, No. 12, 1993
Ku et al.
Figure 1. Fluorescence micrographs of 2-Dstreptavidin crystals. Avidin was present at 70 mol 3'6 of the total protein. Images were recorded with a linear polarizer in the excitation path. (A) Crystals at an early stage of growth. The smallest crystals exhibit an H-shaped structure, while larger ones are in various stages of transition to an X shape. (B) Enlargement of a small crystal from A with an H shape. (C) Crystal at a late stage of growth with an X shape and many dendrites.
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Fi-2. Acute angles formed between armsof X-shaped 2-Dstreptavidin crystals grown at varying streptavidin-avidin ratios. The total protein concentration in the subphase was held constant.
with a reagent such as glutaraldehydemight enhance the rigidity of the protein crystals and help preserve their morphology upon transfer. Indeed, when glutaraldehyde was introduced beneath the surfaceof a lipid monolayer bearing 2-D streptavidin crystals, the mechanical properties of the system werealtered,and transfer to specimen grids was greatly facilitated. The film was noticeably less affected by vibration, and when grids were applied, they tended to remain stationary (rather than drifting about, as occurred before cross-linking). Crystals were unaffected by the application of a grid to the lipid layer (Figure 3b), and after further processing for electron microscopy,the crystal morphology was well preserved. To relate the crystal morphology to the molecular lattice, %der" grids were placed on lipid layers bearing 2-D crystals, edges of H-shaped crystals parallel and perpendicular to the preferred crystal growth direction were noted in the light
microscope, and these same crystal edges were located in the electron microscope(Figure 4). Opticaldiffraction from electron micrographs identified the molecular lattice vectors a and b and thus the orientation of the molecular array (shown as a contour plot of protein density in Figure 4). This determination was repeated several times with consistent results. The preferred growth direction corresponded with the crystallographic (1,l) direction and was parallel to rows formed by a series of identical contacts between streptavidin molecules. Optical diffraction patterns further showed that the unit cell parameters were unaffected by glutaraldehyde treatment, and image processing revealed no structuralperturbation at the resolution of our analysis.
The orientationof the preferred growth direction of H-shaped 2-D streptavidin crystals can be understood in terms of the intermolecular contacts involved. Streptavidin molecules make two types of contacts in a 2-D crystal, those between monomers with biotin-binding sites facing the lipid layer and presumably interactingwith biotinated lipid, and contactsbetween monomers with biotin-binding sites facing toward solution, so free of biotin (Figure 5). Rows of monomers making one type of contact, in the crystallographic(1,l) direction, are at right angles to rows of the other type, in the (1,-1) direction. The preferred growth direction is parallel to one of these rows of contacts (the (1,l) direction), while the other series of contacts (in the (1, -1) direction) supports slower growth. Intermolecularcontacts in the preferred growth direction are between streptavidinmonomers free of biotin, whereasthecontacts in the directionof slower growth are between monomers associated with biotin (Figure 5). The contacts do not involve regions of the protein directly involved in or affected by biotin binding, in particular surface loops containing residues 45-50 and 63-69, shown in previous X-ray crystallographicstudies to be flexible in the absence of biotin and immobilized upon biotin binding." The formation of 2-D crystalsdiffers in this respect from the 3-D crystallizationprocess, which does depend on the immobilization of the flexible surface loops." The reason for the different rates
Two-Dimensional Protein Crystallization
The Journal of Physical Chemistry, Vol. 97, No. 12, 1993 3015
Figure3. Fluorescence micrographs following deposition of electron microscopegridson lipid monolayers bearing uncross-linked (A, left) or cross-linked (B,right) 2-Dstreptavidin crystals.
0 biotin-boundbinding site
Figure 5. Summary of relationships between 2-D crystal morphology and molecular lattice. The projected structure of a crystalline area is shown as in Figure 4. The locationsof unfilled (0)and filled ( 0 )biotinbinding sites are indicated. Lattice vectors, preferred crystal growth direction, and dendrite growth direction are indicated.
Figure4. Relationship between morphology of 2-Dstreptavidincrystals seen in the light microscope and molecular lattice revealed by electron microscopy. A fluorescence micrograph of a grid on a lipid monolayer bearing crystals forms the background. Two electron micrographs of regions (small boxes) containing edges of crystals parallel (top left) and perpendicular (bottom right) to the preferred growth direction are superimposed. The projected structure of a crystalline area from image processing at 14-A resolution,’ with a unit cell boxed in the center, is shown superimposed on the fluorescence micrograph as well. Solid contours correspond to density due to protein and dashed contours to negative stain. Lattice vectors (a and b) are indicated on the electron micrographs and projected structure.
of 2-D crystal growth in the two directionsmay lie in quaternary structure changes upon biotin binding or in the different environments of the two types of monomer (thosebound to biotin lie further from the lipid layer than those without). Theorientation of dendrite growth is between the twodirections defined by rows of intermolecular contacts in the streptavidin lattice. Dendrite formation evidently involves components of growth along both intermolecular directions. While it is unclear at present what determinesthe magnitudeof the two components, there is evidence that a direction of growth, once established, is then maintained by the transport-induced concentration gradient.’* The molecular analysis of 2-D crystal growth reported here was facilitated by cross-linking the proteins in the crystal before
3016 The Journal of Physical Chemistry, Vol. 97, No. 12, 1993
transfer from Langmuir trough to specimen grid. The improvement in preservation of long-range order was dramatic, with no lossofshort-rangeorder detectableat the r d u t i o n of theanalysis, limited to about 12 A by the use of a negative stain for electron microscopy. Any effect on ordering at higher resolution is currently under investigationby electrondiffraction of unstained specimens. Cross-linking may prove helpful in a variety of 2-D crystal systems for preservation during imaging, transfer, and other manipulations.
Ackmwledgment. With respect and affection,we dedicate this article to Harden McConnell. The research was supported by NIH grant A121 144 to R.D.K., NSF grant BCS-9202220 to C.R.R., A.P.G., R.D.K., and S.A.D., an NSERC graduate scholarship (A.C.K.), and the LucilleP.Markey Scholar Award in Biomedical Science (S.A.D.).
Ku et al.
Refe"
lad Notes
(1) Kornberg, R. D.; McConnell,H. M.Proc. Nor/. Acad. Sci. U.S.A. 1971.68,2564-8. (2) Uzgiris, E.E.; Komberg, R. D. Nature 1983,301. 125-9. (3) Kornberg, R. D.; Darst, S. A. Curr. Opinion Struct. Biof. 1991,I , 642-6. (4) Darst, S.A.; Ribi, H. 0.;Pierce,D. W.; Kornberg, R. D. J. Mol. Biol. 1988,203,269-73. ( 5 ) Amos, L. A.; Henderson, R.; Unwin, P. N. T. h o g . Biophys. Mol. Biol. 1982,39, 183. (6) Green, N. M. Ado. Protein Chem. 1975,29,85-133. (7) Darst,S.A.;Ahlers,M.;Meller,P.H.;Kubelek,E. W.;Blankenburg, E. W.; Ribi, H. 0.;Ringsdorf, H.; Kornberg, R. D. Biophys. J. 1991, 59, 387-96. (8) Ku, A. C.;Darst, S. A.; Kornberg, R. D.; Robertson, C.R.; Gast, A. P. Liangmuir 1992,8,2357-2360. (9) Kubalek, E. W.; Komberg, R. D.; Dant, S. A. Uftramicmcopy1991, 35,295-304. (10) knvegnue, D. J.; McConnell, H. M. J . Phys. Chem. 1992, 967. 68204. (11) Weber, P.C.;Ohlendorf, D. H.; Wendoloski, J. J.; Saltmme, F. R. Science 1989,243, 85-88. (12) Huang, S.-C.; Glicksman, M. E. Acta Metall. 1981,29,717-34.
