Langmuir 2001, 17, 5731-5735
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Point Mutagenesis and Cocrystallization of Wild-Type and Mutant Proteins: A Study of Solid-Phase Coexistence in Two-Dimensional Protein Arrays Sammy J. Farah, Szu-Wen Wang, Wei-Hau Chang, Channing R. Robertson, and Alice P. Gast* Department of Chemical Engineering, Stanford University, Stanford, California, 94305
Langmuir 2001.17:5731-5735. Downloaded from pubs.acs.org by WESTERN SYDNEY UNIV on 01/13/19. For personal use only.
Received February 10, 2001. In Final Form: July 2, 2001
We are studying the molecular organization of protein arrays using two-dimensional streptavidin crystals bound to biotinylated lipid monolayers at the air-water interface. We constructed a mutant form of the streptavidin protein that successfully alters the molecular organization of the streptavidin crystals. Cocrystallization of streptavidin carrying this single targeted point mutation with wild-type streptavidin yields two-dimensional crystals displaying a chiral morphology with molecular coexistence, indicating a solid-phase transition. The phase coexistence and resulting morphologies are reminiscent of two-dimensional crystal behavior of wild-type streptavidin near its isoelectric point, and this analogy is discussed. These results demonstrate the potential to manipulate protein array formation through point mutagenesis and cocrystallization.
Point mutations in proteins alter their intermolecular interactions with macroscopic consequences.1 Directed mutagenesis has been shown to modify protein crystal lattices and habits.2,3 In particular, mutational effects on two-dimensional protein crystals are of interest to understand their influence on protein function and to study fundamental aspects of ordering in reduced dimensionality. Many important systems rely on the function of twodimensional protein arrangements such as the bacteriophage injection apparatus,4 lung surfactant SP-B protein,5 and S-layers on the exterior archaeobacteria and eubacteria cells.6 In this study, using streptavidin as a model protein, we control the structure of two-dimensional protein arrays via a single targeted point mutation. Cocrystallization of this mutant streptavidin and wildtype streptavidin yields two-dimensional crystals displaying unique morphologies with molecular phase coexistencestwo crystal forms existing within a single continuous crystalline domain. We report on the macroscopic and molecular changes observed during cocrystallization, which indicate a solid-phase transition in the protein array caused by the introduction of the point mutation. Two-dimensional crystals of streptavidin on biotinylated lipid monolayers offer an excellent model system for investigating general interfacial and phase change phenomena.7-9 In our laboratory, we have used two* To whom correspondence may be addressed: phone, 650-7253145; fax, 650-725-7294; e-mail,
[email protected]. (1) Ingram, V. M. Nature 1956, 178, 792. (2) Lawson, D. M.; Artymiuk, P. J.; Yewdall, S. J.; Smith, J. M. A.; Livingstone, J. C.; Treffry, A.; Luzzago, A.; Levi, S.; Arosio, P.; Cesareni, G.; Thomas, C. D.; Shaw, W. V.; Harrison, P. M. Nature 1991, 349, 541. (3) Braig, K.; Otwinowski, A.; Hegde, R.; Boisvert, D. C.; Joachimiak, A.; Horwich, A. L.; Sigler, P. B. Nature 1994, 371, 578. (4) Crowther, R. A.; Lenk, E. V.; Kikuchi, Y.; King, J. J. Mol. Biol. 1977, 116, 489. (5) Longo, M. L.; Bisango, A. M.; Zasadzinski, J. A. N.; Bruni, R.; Waring, A. J. Science 1993, 261, 453. (6) Sleytr, U. B.; Messner, P.; Pum, D.; Sara, M. Mol. Microbiol. 1993, 10, 911. (7) Darst, S. A.; Ahlers, M.; Meller, P. H.; Kubalek, E. W.; Blankenburg, R.; Ribi, H. O.; Ringsdorf, H.; Kornberg, R. D. Biophys. J. 1991, 59, 387. (8) Ku, A. C.; Darst, S. A.; Robertson, C. R.; Gast, A. P.; Kornberg, R. D. J. Phys. Chem. 1993, 97, 3013. (9) Calvert, T. L.; Leckband, D. Langmuir 1997, 13, 6737.
Figure 1. Molecular models of P1 and P2 lattice configurations. (a) P1 lattice configuration of wild-type streptavidin at pH 4 with unit cell parameters a ) 58 Å, b ) 50 Å, and γ ) 113°. The contact regions containing the Asp36 residues are shown in red. (b) P2 lattice configuration of wild-type streptavidin at pH 6 and D36K streptavidin at pH 4 with unit cell parameters a ) 116 Å, b ) 58 Å, and γ ) 107°. The contact regions are shown in red.
10.1021/la010227n CCC: $20.00 © 2001 American Chemical Society Published on Web 08/15/2001
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Figure 2. Wild-type and mutant streptavidin crystal morphologies, micrographs, and diffraction patterns: (a) crystal morphologies of pure wild-type and pure D36K streptavidin (images are approximately 400 × 300 µm); (b) micrographs (45 000×) of pure wild-type streptavidin displaying P1 lattice spacing and pure mutant streptavidin displaying P2 lattice spacing; (c) corresponding P1 and P2 diffraction patterns.
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dimensional crystals of streptavidin to study intermolecular protein interactions, protein behavior at interfaces, and phase transitions. In our previous efforts,10-12 we altered the solution pH to manipulate protein-protein interactions during crystallization of streptavidin. Our study of streptavidin crystals grown in environments ranging from pH 4 to pH 7 revealed extensive macroscopic and microscopic changes in crystal structure. To elucidate the specific points of intermolecular interactions causing pH-driven changes in crystal lattice arrangement, we identified an aspartate residue at position 36 (Asp36) implicated in one of the intermolecular contact regions. At pH 4, approximately half the Asp36 residues are charged, allowing for an average of one potential hydrogen bond per aspartate-aspartate contact between adjacent protein molecules. This favorable interaction leads to the tightly packed P1 lattice structure (Figure 1a) prevalent at pH 4. At pH 6, most aspartates are negatively charged leading to electrostatic repulsion, presumably disrupting the P1 lattice packing seen at the lower pH and producing a less compact P2 crystal lattice (Figure 1b) lacking Asp36 contacts.10,13 In subsequent studies to our original pH change experiments, we applied site-directed mutagenesis to control lattice structure through specific interactions. We made a series of mutant forms of the streptavidin protein substituting the residue at position 36 to alter contacts at this point and effect changes in molecular arrangement similar to those induced by the change in pH.14 As previously reported,15 wild type streptavidin crystals grown at pH 4 consist of P1 lattices (Figure 1a) in thin needlelike morphologies (Figure 2a). When Asp36 is changed to a lysine (D36K mutant), repulsive interactions
Figure 3. Crystal morphologies, micrographs, and diffraction patterns for cocrystals formed with greater than 50% wild type streptavidin: (a) crystal morphologies of 90%, 75%, and 67% wild-type streptavidin (images are approximately 400 × 300 µm); (b) micrographs (45 000×) displaying coexistence of P1 or P1 paracrystalline (P1p) and P2 lattice spacing, P1 and P1p are the predominant crystal forms; (c) corresponding P1 and P1p diffraction patterns.
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Figure 4. Crystal morphology, micrograph, and diffraction pattern for cocrystals formed with equal amounts of wild type and D36K streptavidin: (a) crystal morphology of 50% wildtype streptavidin (image is approximately 400 × 300 µm); (b) micrograph (45 000×) displaying coexistence of approximately equal amounts of P1p and P2 lattice spacing; (c) Corresponding P1p and P2 diffraction patterns.
electrostatically and sterically disfavor the formation of the compact P1 crystals at pH 4, producing X-shaped crystal domains (Figure 2a) with the same P2 lattice found with the wild type at pH 6 (Figure 1b).14,16 This result is significant, showing that a single point mutation can produce profound changes in the macroscopic and molecular organization of protein arrays. The results of this work laid the groundwork for further investigations into the phase behavior of two-dimensional protein arrays. The work presented in this study is motivated by the intriguing observations of solid-solid phase coexistence and phase transitions in two dimensions.17,18 In our study of two-dimensional streptavidin crystals, we found a variety of coexisting phases sometimes producing distinc(10) Wang, S. W.; Poglitsch, C. L.; Yatcilla, M. T.; Robertson, C. R.; Gast, A. P. Langmuir 1997, 13, 5794. (11) Yatcilla, M. T.; Robertson, C. R.; Gast, A. P. Langmuir 1998, 14, 497. (12) Wang, S. W.; Robertson, C. R.; Gast, A. P. Langmuir 1999, 15, 1541. (13) Freitag, S.; Trong, I. L.; Klumb, L.; Stayton, P.; Stenkamp, R. Protein Sc. 1997, 6, 1157. (14) Wang, S. W.; Robertson, C. R.; Gast, A. P. J. Phys. Chem. B 1999, 103, 7751. (15) Hemming, S. A.; Bochkarev, A.; Darst, S. A.; Kornberg, R. D.; Ala, P.; Yang, D. S. C.; Edwards, A. M. J. Mol. Biol. 1995, 246, 308. (16) Wang, S. W.; Robertson, C. R.; Gast, A. P.; Koppenol, S.; Edwards, T.; Vogel, V.; Stayton, P. Langmuir 2000, 16, 5199. (17) Brisson, A.; Bergsma-Schutter, W.; Oling, F.; Lambert, O.; Reviakine, I. J. Cryst. Growth 1999, 196, 456. (18) Ribi, H. O.; Ludwig, D. S.; Merger, K. L.; Schoolnik, G. K.; Kornberg, R. D. Science 1988, 239, 1272.
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tive morphologies including chiral-shaped domains.10 The crystallization of our D36K mutant into an array different than the wild-type protein provided a unique opportunity to study the solid-solid phase transition in this twodimensional system. We cocrystallized seven ratios of wild type and D36K mutant streptavidin, ranging from 9:1 to 1:9 wild type to mutant. Prior to performing these experiments, it was not obvious what type of phase transition would be observed, and whether mixtures of wild-type and mutant proteins would simply aggregate, become miscible, cocrystallize into domains with coexisting crystal types, or phase separate into domains with distinct crystal types. As with previous experiments, we grew crystals at pH 4 on biotinylated lipid monolayers at the air-water interface.14 We observed crystal morphology using epifluorescence microscopy and analyzed lattice structure with electron microscopy. Macroscopically, fluorescence microscopy revealed that mixing wild type and mutant streptavidin produced striking “inverse S shaped”, or “chiral”, morphologies. On the basis of a previous experimental observation, which is discussed below, the emergence of crystals with chiral morphologies provided the initial evidence that mixing protein types produced coexisting crystal phases. Introduction of even small amounts of mutant streptavidin (10% D36K) had a noticeable effect on crystal morphology (Figure 3a). The needlelike domains previously grown with 100% streptavidin (Figure 2a), now displayed slightly thinned ends. At 25% mutant streptavidin, chirality was introduced into the crystal morphology (Figure 3a). As the mutant ratio was increased to 33% (Figure 3a) and 50% (Figure 4a), the crystal shapes became smaller, thinner, and more chiral. As the mutant ratio was increased beyond 50%, the crystal morphologies continued to decrease in size and width, while exhibiting decreasing chirality. This trend is evident in the 67% (Figure 5a) and 75% (Figure 5a) mutant crystals. At 90% mutant streptavidin (Figure 5a), the crystal morphologies were very small and thin and had lost all evidence of chirality. Pure mutant streptavidin behaves quite differently forming large X-shaped crystals (Figure 2a). The observed morphological changes raise several important points. First, introduction of mutant streptavidin causes chirality in crystal morphology. This chirality appears to be maximal at 50% wild type (Figure 4a), as evidenced by crystals displaying the most pronounced and dramatic curvature. Second, there are differences in the kinetics of crystal growth. The mutant streptavidin crystals grow at a slower rate (typically over a 24 h period) than the wild-type streptavidin (typically over 4 h). The repulsive interactions in the mutant streptavidin impede crystallization and produce the lower density P2 phase. Accordingly, as the ratio of mutant streptavidin is increased, the mixed domains appear more slowly. And third, there is a profound change in morphology between the thin needles formed with the addition of only 10% wild type to mutant streptavidin (Figure 5a) and the large X-shapes formed with pure mutant streptavidin (Figure 2a). Evidently, the presence of even small amounts (10%) of wild-type streptavidin causes the crystals to form as thin needles. It is possible that this abrupt change in morphology is related to the kinetic differences discussed above, or perhaps the presence of the wild-type protein in the predominantly mutant protein crystals simply alters the growth habit or preferred growth direction of the crystals as they form. To investigate the lattice packing, we cross-linked all crystals with glutaraldehyde, transferred them to carboncoated electron microscopy grids and followed by negative
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Figure 5. Crystal morphologies, micrographs, and diffraction patterns for cocrystals formed with less than 50% wild type streptavidin: (a) crystal morphologies of 33%, 25%, and 10% wild-type streptavidin (images are approximately 400 × 300 µm); (b) Micrographs (45 000×) displaying coexistence of P1p and P2 lattice spacing, P2 is the predominant crystal form; (c) corresponding P2 diffraction patterns.
staining with uranyl acetate for analysis by electron microscopy. We analyzed the transmission electron microscopy (TEM) (Phillips CM12) micrographs (Figures 2b, 3b, 4b, and 5b) by optical diffractometry, digitization protocols and CRISP image processing software (Calidris, Sollentuna, Sweden). The protein arrays showed that all mixed crystals contained coexisting P1 and P2 phases within single continuous crystal domains. The fraction of crystalline regions with P1 or P2 symmetry approximated the fraction of wild type or mutant streptavidin, respectively. P1 and P2 diffraction patterns are shown for the 50% wild-type crystals (Figure 4c). These crystals demonstrated the greatest degree of coexistence and had large regions of protein arrays representing both lattice spacings. The P1 diffraction pattern was more distinct in crystals formed with a majority of wild-type streptavidin (Figure 3c). Although micrographs of these crystals showed coexistence with regions of both P1 and P2 lattice spacing, the P2 regions were too small to produce distinct diffraction spots. Additionally, the P1 symmetry observed in some of the intermediate crystalssshown only for the 67% and 50% wild-type streptavidin crystals but present in all crystals with less than 67% wild-type streptavidinsformed with slightly poorer long-range order than the P1 pattern observed in the pure wild-type crystal (Figure 2c). This pattern was termed P1 paracrystalline (P1p) to indicate a solid in an intermediate state between amorphous and crystalline but containing some degree of crystallographic order with the same lattice parameters as the P1 unit
cell.14,19 The paracrystalline character of this crystal may result from disruption of long-range order by line defects and P2 lattice spacing introduced by the mutant streptavidin. As the fraction of mutant streptavidin increased, the P2 diffraction patterns became more distinct (Figure 5c) and retained their lattice parameters and long-range order. Regions of P1p lattices in these crystals were not sufficiently large to produce distinct diffraction patterns. Phase coexistence in this system was not an obvious outcome. Prior to these experiments, we could not predict whether phase coexistence behavior would be observed. Other possible outcomes included cocrystallization leading to a more “alloy”-like behavior with a single lattice spacing other than P1 or P2 or phase separation leading to individual domains comprised entirely of P1 or P2 lattices. The phase coexistence reflecting the overall mixture composition indicates that this system is undergoing a solid-solid phase transition as the ratio of wild-type and mutant streptavidin is adjusted. The observed trend in the morphologies of these coexisting crystals also illustrates this phenomenon. As mentioned above, phase coexistence and the observation that coexistence leads to chiral domain formation have been documented previously.10 Chiral-shaped crystals seen with wild-type streptavidin crystallized over a subphase at pH 5.5 were also formed by coexistence of P1 and P2 lattice spacingsthe predominant crystal forms of the wild type at pH 4 and pH 6, respectively. Crystals arising from cocrystallization of wild-type and mutant (19) Hindeleh, A. M.; Hosemann, R. J. Mater. Sci. 1991, 26, 5127.
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streptavidin are analogous in shape and structure to these wild-type crystals formed at the intermediate pH. This fact leads to an interesting conclusion. At pH 5.5, coexistence is caused by electrostatic interactions occurring near the isoelectric point of the protein.10 Apparently, coexisting lattice domains within the same crystal are permitted by the amphoteric nature of the protein near its isoelectric point. In this report, combining streptavidin mutants carrying repulsive charged lysine residues with wild-type streptavidin induces a mixture of coexisting P1 and P2 phases in a single crystalline domain with distinct chiral morphologies analogous to those produced by variations in pH. Modification of the crystal arrangements in a targeted and controlled manner is an important step in the study of crystalline behavior, illustrating the
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application of genetic engineering and manipulation of coexisting solid phases as effective methods for directing the formation of two-dimensional protein arrays. Acknowledgment. We gratefully acknowledge Patrick Stayton and Todd Edwards for generously providing the wild-type streptavidin genes and expression protocols. We thank Wray Huestis and Chaitan Khosla and their group members for allowing us to use their laboratory facilities for expression of the mutant proteins. We also are grateful to Roger Kornberg for use of his TEM facilities. This work was funded by Merck & Co., Inc., and NSF Grant BES-9729950. LA010227N
