Molecular Basis for Asymmetrical Growth in Two-Dimensional

At neutral pH, streptavidin forms crystals with C222 symmetry and an X-shaped morphology that arises from asymmetric growth rates along perpendicular ...
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Langmuir 2002, 18, 7447-7451

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Molecular Basis for Asymmetrical Growth in Two-Dimensional Streptavidin Crystals Todd C. Edwards,†,§ Noah Malmstadt,†,§ Sandy Koppenol,† Masahiko Hara,‡ Viola Vogel,† and Patrick S. Stayton*,† Department of Bioengineering, University of Washington, Seattle, Washington 98195-7962, and Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan Received December 26, 2001. In Final Form: May 6, 2002 The two-dimensional crystallization of the protein streptavidin under biotinylated lipids has provided a convenient model system for studying the molecular mechanisms underlying protein crystallization. At neutral pH, streptavidin forms crystals with C222 symmetry and an X-shaped morphology that arises from asymmetric growth rates along perpendicular axes. The presence or absence of biotin association in the subunits has been connected to the growth asymmetry, but the molecular mechanism coupling biotin binding and growth kinetics has remained uncertain. Here we show evidence that a cooperative interaction between the Asn 23 hydrogen bond to biotin and a protein-protein hydrogen-bonding interaction at Tyr 22-Thr 20 across the crystal contact interface is acting as the structural/energetic connection between biotin binding and the asymmetric crystal growth kinetics. Brewster’s angle microscopy and atomic force microscopy studies of a Thr20Ala site-directed mutant revealed that it crystallizes as symmetric squares, while maintaining the molecular C222 arrangement of wild-type protein crystals. These results suggest that the T20-Y22 hydrogen-bond interaction across the protein-protein contact interface is the crystallization contact that is altered by biotin binding to give growth asymmetry in wild-type crystals.

Introduction A better understanding of the molecular interactions underlying protein crystallization may provide insight leading to improved techniques for growing better crystals. The tetrameric protein streptavidin has served as a wellstudied 2D crystallization model, and we have utilized site-directed mutagenesis to probe the roles of specific side chains in controlling crystal morphology.1 Streptavidin crystallizes in X-shaped crystals with C222 symmetry at neutral pH beneath monolayers of a biotinylated lipid (Figure 3a). Two of the streptavidin subunits are oriented at the interface and are bound to the biotinylated lipids, while the other two subunits are oriented away from the monolayer and remain unbound.2,3 The four protein-protein contacts in the C222 unit cell geometry are essentially identical due to the internal 222 (D2) symmetry of the streptavidin tetramer. The asymmetry in crystal growth axes arises due to the faster growth kinetics along the axis defined by the contacts between biotin-bound subunits as compared to the axis along the contacts between unbound subunits.4 It has been suggested on this basis that biotin binding could induce structural differences at the protein-protein contacts between bound subunits compared to those * To whom correspondence should be addressed. Patrick S. Stayton, Box 351721, Department of Bioengineering, University of Washington, Seattle, WA 98195. Phone: (206) 685-8148. Fax: (206) 685-8256. E-mail: [email protected]. † University of Washington. ‡ The Institute of Physical and Chemical Research (RIKEN). § Joint first authors. (1) Edwards, T. C.; Koppenon, S.; Frey, W.; Schief, W. R.; Vogel, V.; Stenkamp, R. E.; Stayton, P. S. Langmuir 1998, 14, 4683-4687. (2) Ahlers, M.; Blankenburg, R.; Grainger, D. W.; Meller, P.; Ringsdorf, H.; Salesse, C. Thin Solid Films 1989, 180, 93-99. (3) Blankenburg, R.; Meller, P.; Ringsdorf, H.; Salesse, C. Biochemistry 1989, 28, 8214-8221. (4) Wang, S.; Robertson, C. R.; Gast, A. P. Langmuir 1999, 15, 15411548.

between unbound subunits, giving rise to the asymmetrical growth seen in 2D streptavidin crystals with C222 symmetry.5-7 This growth asymmetry is removed when streptavidin is crystallized in the absence of biotin beneath copper-chelating lipids.6 In this case, streptavidin crystallizes with square-shaped crystals that retain the C222 symmetry. The changes in protein-protein interactions (or dynamics) induced by biotin binding must be extremely subtle, since they are not observable in a 1.8 Å resolution X-ray crystallographic streptavidin structure with biotin molecules bound in the same subunits as in the case of streptavidin bound to a biotinylated monolayer (pdb entry 1swd).8,9 Our computer model of the C222 contacts revealed several potentially interesting interactions. The first is an apparently unfavorable electrostatic interaction between two adjacent lysine 132 (K132) residues across the protein-protein contact interface. Earlier, we showed that this interaction is likely counterion mediated, which gives rise to the requirement for a high ionic strength buffer for crystallization.1 Additionally, there is a hydrogen bond between threonine 20 and tyrosine 22 (T20-Y22) that appears twice in each of the four protein-protein contacts per tetramer due to the dyad symmetry of streptavidin and the C222 crystal symmetry (Figure 1a). The direct proximity of these T20-Y22 interactions to the key Asn 23 side-chain hydrogen bond to biotin (Figure 1b) suggests that this protein-protein interaction may provide the coupling between biotin association and crystal (5) Ku, A. C.; Darst, S. A.; Robertson, C. R.; Gast, A. P.; Kornberg, R. D. J. Phys. Chem. 1993, 97, 3013-3016. (6) Frey, W.; Schief, W. R. J.; Pack, D. W.; Chen, C. T.; Chilkoti, A.; Stayton, P.; Vogel, V.; Arnold, F. H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4937-4941. (7) Vogel, V.; Schief, W. R.; Frey, W. Supramol. Sci. 1996, 4, 163171. (8) Frey, W.; Brink, J.; Schief, W. R.; Chiu, W.; Vogel, V. Biophys. J. 1998, 74, 2574-2579. (9) Freitag, S.; Le-Trong, I.; Klumb, L.; Stayton, P. S.; Stenkamp, R. E. Protein Sci. 1997, 6, 1157-1166.

10.1021/la0118457 CCC: $22.00 © 2002 American Chemical Society Published on Web 08/27/2002

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Figure 1. Molecular models created with Molscript (ref 31) of (A) the two threonine 20-tyrosine 22 hydrogen bonds in the streptavidin C222 2D crystal contact and (B) the hydrogen bond formed between biotin and asparigine 23. Threonine 20 and tyrosine 22 may be affected through the backbone upon biotin binding.

growth kinetics. In this series of experiments, we investigated the effect on 2D crystallization of two mutations designed to disrupt the T20-Y22 hydrogen bonds at the C222 contact. Materials and Methods Molecular Modeling. Construction of the computer model of the C222 crystal has been previously described.1 Computer modeling was done on an O2 workstation (Silicon Graphics Computer Systems, Mountain View, CA) with the programs PSSHOW (Swanson, E. PSSHOW, 4D version; Silicon Graphics: Seattle, WA, 1990) and XtalView.10 Atomic coordinates for a single streptavidin subunit were obtained from the Protein Data Bank as the file 1sld.pdb.11 Those coordinates were chosen because the two-dimensional C222 plane is represented in the 3D crystal of space group I122 and thus has similar protein-protein contacts. The coordinates of the monomer were transformed to produce the entire tetramer, which was then copied and translated according to the published unit cell2,3,12 to model the proteinprotein contact. (10) McRee, D. E. J. Mol. Graphics 1992, 10, 44-46. (11) Katz, B. A. Biochemistry 1995, 34, 15421-15429.

Edwards et al. Site-Directed Mutagenesis and Protein Chemistry. The mutant coding sequences were generated by cassette mutagenesis of the previously constructed 13-136 streptavidin truncation mutant gene in the pET 21a(+) plasmid. Synthetic sense and antisense oligonucleotides (Integrated DNA Technologies, Inc., Coralville, IA) were annealed to form the mutant cassettes. The T20A and Y22F mutant cassettes were subcloned between the Nde I and BamH I restriction sites in the truncation mutant. Plasmids containing the mutant genes were sequenced and then transformed into BL21(DE3) Escherichia coli cells for expression. Cells were grown in 2xYT medium until OD600 ) 0.6-1.0, at which point protein expression was induced with 200 µM isopropyl-β-D-thiogalactopryranoside (IPTG). The cells were harvested after 3 h. Isolated inclusion bodies were then denatured in solubilization buffer (50 mM Tris, 6 M Guanidine HCl, pH 7.5) and refolded by slowly dripping into a stirred refolding solution (50 mM Tris, 100 mM NaCl, 5 mM EDTA, 0.1 mM PMSF, pH 7.5) at 4 °C. Refolded protein was concentrated by ultrafiltration and purified via iminobiotin affinity chromatography (Pierce, Rockford, IL). The purified streptavidin mutants were compared to 13-136 truncated wild-type by electrospray ionization mass spectrometry, which yielded a molecular mass of 13 042 Da as a control. Additional characterization was performed for 13-136/ T20A to determine the off-rate of bound biotin with a competition assay that has been used on previous streptavidin mutants.13 Crystallization Studies. 2D crystallization experiments were performed in a 6 mm deep Teflon Langmuir trough with subphase buffers consisting of 50 mM NaH2PO4 and 500 mM NaCl, at pH 7.0 and 4.2. The trough was equipped with computer-controlled mobile barriers to control the surface pressure. Surface pressure was measured using a Wilhelmy plate balance. The total area of the trough was 8 cm by 25 cm. The barrier distance ranged from approximately 3 to 20 cm. A biotinylated lipid mixture consisting of N-((6-(biotinoyl)amino) hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (B-x-DPPE) (Molecular Probes, Eugene, OR) and dimyristoylphosphotidylcholine (DMPC) (Avanti Polar Lipids, Birmingham, AL) in a 1:10 molar ratio was spread from chloroform to a measured surface pressure of 28-29 mN/m. After time was allowed for chloroform evaporation, protein was injected into the subphase to yield a final concentration of 13 nM. Prior to injection, all protein samples used for crystallization were diluted with buffer. Crystals typically appeared 5-10 min after protein injection. Brewster’s Angle Microscopy. Monolayer binding and crystal formation were monitored with a home-built Brewster’s angle microscope (BAM) based on a previously described design.14 The laser and polarizer are linearly arranged along a single axis, eliminating the need for mirrors or prisms. A 1 mm diameter beam of light from a 10 mW He-Ne laser was p-polarized using a dichroic sheet polarizer (extinction ratio ) 10-4) and then reflected from the air/water interface at an angle of incidence of 53.12° with respect to the surface normal, that is, the Brewster’s angle for the air/water interface. A portion of the reflected light was focused directly on the sensing unit of a CCD camera (CCD72 with an active area of 6.6 × 8.8 mm, Dage-MTI, Michigan City, IN). A custom configuration for the focusing optics provided a combination of long working distance (ca. 50 mm) to avoid interference with the barriers of the Langmuir trough, a relatively high magnification (ca. 26×) to visualize crystals typically 20200 microns in size, and a largely uniform and speckle-free image illumination to allow reliable, quantitative gray scale analysis. With this apparatus, the lateral resolution is approximately 5 microns. The images from the CCD camera were captured at 18 Hz with a SG-9 Scion video capture card (Scion Corp., Frederic, MD) in a StarMax 3000/180 PowerPC personal computer running NIH-image 1.60. Reflected intensity in gray scale units was continuously measured from unprocessed images before and after crystals appeared. The gray scales of the noncrystalline and crystalline phases were measured separately, as previously (12) 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, 387-396. (13) Chilkoti, A.; Stayton, P. S. J. Am. Chem. Soc. 1995, 117, 1062210628. (14) Cohen-Stuart, M. A.; Wegh, R. A.; Kroon, J. M.; Sudho¨lter, E. J. R. Langmuir 1996, 12, 2863-2865.

Asymmetrical Growth in Streptavidin Crystals described.15 Using a previously developed model to quantitatively interpret BAM gray scales of streptavidin binding and crystallization beneath a biotinylated lipid monolayer,15 changes in gray scale were converted first into changes in refractive index of the protein layer and then into changes in the protein surface density. Thus changes in protein surface density with time could be calculated for both noncrystalline and crystalline phases.15 Image background subtraction was not performed. Cross-Linking and Transmission Electron Microscopy (TEM). Crystals were transferred to TEM grids both with and without prior cross-linking. For cross-linked samples, crystals were chemically cross-linked with either gluteraldehyde (Sigma, St. Louis, MO) or 1,5-difluoro-2,4-dinitrobenzene (DFDNB, Pierce), an amine-reactive cross-linking reagent.16 The crosslinking solution was injected directly into the subphase, pure gluteraldehyde to a final concentration of 5 µL per mL of buffer and DFDNB as a methanol solution to a final concentration of 50 µM. Cross-linking reactions were allowed to proceed from 2 to 16 h before transfer was attempted. The Langmuir-Schaefer17 method was used to transfer crystals to 400 mesh copper finder grids coated with a lacey carbon film (Ted Pella, Inc., Redding, CA) for TEM. Such lacey carbon supports have been shown to be the least damaging transfer substrate for 2D protein crystals in preparation for electron microscopy.18,19 The grids were rinsed in chloroform immediately before placing them on the lipid monolayer. The samples were rinsed with water, stained with 2% uranyl acetate for 1 min, and allowed to air-dry before storage. The stained crystals were imaged with a JEOL 1200 transmission electron microscope. Atomic Force Microscopy (AFM). Crystals were transferred to freshly cleaved highly ordered pyrolytic graphite (HOPG, SPI Supplies, West Chester, PA) by the LangmuirSchaefer method. Slabs of HOPG were affixed by waterproof epoxy to steel AFM pucks. Immediately prior to transfer, the surface of the HOPG was cleaved by affixing to it a piece of electrical tape and pulling off the outer layer. Transfer was accomplished by bringing the freshly cleaved surface of the HOPG into contact with the surface of the lipid monolayer, holding it in place for approximately 30 s, and withdrawing it. Samples were then air-dried. All AFM was performed with a NanoScopeIII MultiMode scanning probe microscope (Digital Instruments, Santa Barbara CA). This microscope was equipped with an E-type scanner and a crystal silicon cantilever and tip (125 µm length, spring constant ∼20 N/m), which was oscillated near its resonant frequency in air (tapping mode). Samples were scanned at 1-3 Hz, and images were captured at 256 × 256 pixel resolution. Images were captured at various scales, from 8 × 8 µm to 100 × 100 nm, and at a variety of scan angles. Height, amplitude, and phase data were recorded. The image processing steps of flattening, plane fitting, and 2D fast Fourier transform were performed in ImageSXM (Steve Barrett, http://138.253.47.16/). Images were masked with an annular Gaussian decay envelop prior to Fourier transform. Further noise and distortion reduction was performed via correlation averaging20,21 using inhouse software (http://www.students.washington.edu/noahmalm/ image_process.html).

Results and Discussion Mutants were based on a truncated streptavidin gene consisting of residues 13-136 of the originally identified streptavidin amino acid sequence. This gene has been designed to express a recombinant version of the “core” streptavidin natively produced by post-translational pro(15) Frey, W.; Schief, W. R.; Vogel, V. Langmuir 1996, 12, 13121320. (16) Kornblatt, J. A.; Lake, D. F. Can. J. Biochem. 1980, 58, 219224. (17) Langmuir, I.; Schaefer, V. J. J. Am. Chem. Soc. 1938, 59, 28032810. (18) Kubalek, E. W.; Kornberg, J. A.; Darst, S. A. Ultramicroscopy 1991, 35, 295-304. (19) Brisson, A.; Bergsma-Schutter, W.; Oling, F.; Lambert, O.; Reviakine, I. J. Cryst. Growth 1999, 196, 456-470. (20) Saxton, W. O.; Baumeister, W. J. Microsc. 1982, 127, 127-138. (21) Frank, J. Optik 1982, 63, 67-89.

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Figure 2. Biotinylated monolayer binding curves for 13-136 and 13-136/Y22F proteins. The observed gray scale is directly related to the concentration of protein bound to the interface. Both proteins demonstrate first-order binding kinetics to the monolayer, and they bind at similar saturation levels. At the 25 min time point shown on this curve, the 13-136 protein begins to crystallize; the 13-136/Y22F protein never forms crystals. The similarities in these binding curves indicate that the failure of the 13-136/Y22F to form crystals is due to a change in proteinprotein interactions on the monolayer rather than a deficiency in monolayer binding.

teolysis in Streptomyces avidinii.22 The 13-136 truncation was selected for its ability to crystallize in all known 2D streptavidin crystal packing arrangements.23 Conservative mutations were constructed at the T20 and Y22 positions. The 13-136/Y22F streptavidin failed to crystallize. Figure 2 shows the change in BAM gray scale with time for 13136/Y22F adsorbing to the biotinylated lipid monolayer. This binding curve indicates that interaction between the mutant protein and the monolayer (first-order binding kinetics) is similar to that between the 13-136 protein and the monolayer. The failure of 13-136/Y22F to crystallize is therefore due to an alteration in the interactions between monolayer-bound protein molecules caused by the mutation. The 13-136/T20A mutant formed square crystals at pH 7 (Figure 3b). The gray scale of the square crystals relative to that of the lipid monolayer was the same as control C222 crystals, a result that left the nature of the T20A crystal packing arrangement ambiguous, since C222 and P2 crystals have similar relative gray scales.23 To resolve this ambiguity, efforts were made to transfer these crystals onto TEM grids for structural analysis. Transmission electron microscopy, however, revealed no crystalline regions in the transferred samples, even following chemical cross-linking. The loss of the hydrogen bonds at the crystal contact thus resulted in crystals that are exceptionally fragile. HOPG provided a molecularly smooth hydrophobic substrate onto which crystals could be transferred with a minimum of structural disruption.24 Height and phase data from tapping mode AFM of crystals transferred to HOPG and air-dried revealed a crystal lattice. Tapping mode AFM phase images show contrasts in the surface stiffness of the sample on a molecular scale,25 while height images give the elevation of the sample at each point on the scan plane. The phase data (Figure 4b) show the 13-136/T20A crystal lattice at higher resolution (22) Pa¨hler, A.; Hendrickson, W. A.; Kolks, M. A. G.; Argaran˜a, C. E.; Cantor, C. R. J. Biol. Chem. 1987, 262, 13933-13937. (23) Wang, S. W.; Robertson, C.; Gast, A.; Koppenol, S.; Edwards, T.; Vogel, V.; Stayton, P. Langmuir 2000, 16, 5199-5204. (24) Scheuring, S.; Muller, D. J.; Ringler, P.; Heymann, J. B.; Engel, A. J. Microsc. 1998, 193, 28-35. (25) Magonov, N. N.; Elings, V.; Whangbo, M.-H. Surf. Sci. 1997, 375, L385-L391.

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Figure 3. Brewster’s angle microscopy images of 2D streptavidin crystals grown under a biotinylated lipid mixture of B-xDPPE and DMPC in a 1:10 molar ratio on a buffer containing 50 mM NaH2PO4 and 500 mM NaCl, pH 7.0. (A) 13-136 streptavidin displaying typical X-shaped morphology with asymmetric growth axes. (B) 13-136/T20A streptavidin. The square morphology suggests that the molecular interactions that give rise to the asymmetric growth have been removed by the mutation.

and with less noise than the height data (Figure 4a). Five phase data images were Fourier transformed, and the reciprocal space vector data from these transforms were averaged; an example of the transformed data is shown in Figure 5. This analysis yielded a ) 9.9 ( 0.2 nm, b ) 8.8 ( 1.4 nm, and γ ) 88.5 ( 1.6°. The data are at rather low resolution, with reflections only up to 1,1 visible (at 6.8 nm). The quality of the AFM data is not sufficient to reconstruct in detail the crystal packing arrangement a priori. A detailed reconstruction is not necessary, however, since the packing arrangements available to 2D streptavidin crystals have been well established.4 The power spectrum presented herein is experimentally determined to a resolution sufficient to be consistent with only one known streptavidin 2D crystal packing arrangement: the C222 packing (see Table 1). The literature contains several examples1,26,27 of power spectra at similarly low resolution used to determine the packing arrangement of 2D streptavidin crystals by comparison to power spectra of known packings. We conclude therefore that the T20A (26) Wang, S. W.; Robertson, C. R.; Gast, A. P. J. Phys. Chem. B 1999, 103, 7751-7761. (27) Wang, S.; Poglitsch, C. L.; Yatcilla, M. T.; Robertson, C. R.; Gast, A. P. Langmuir 1997, 13, 5794-5798.

Figure 4. AFM images based on data for a 13-136/T20A crystal transferred to highly ordered pyrolytic graphite. These data encompass a 450 × 450 nm square area and were taken at a scan rate of 1 Hz. (A) An image based on the height data, showing heights from 0 (dark) to 4.5 nm (bright). Though some periodicity is apparent, there is no clear lattice structure. Inset: A swath of this image processed by correlation averaging, in which the periodic structure is slightly easier to see. (B) An image based on the phase data, showing a phase angle shift range from 0 (dark) to 10° (bright). Changes in phase angle shift correspond to changes in surface stiffness of the sample. The crystal lattice is readily apparent as a pattern of light and dark corresponding to the difference in surface stiffness between protein molecules and the transferred lipid monolayer underneath them. Inset: A swath of this image processed by correlation averaging, in which the periodic structure is further clarified.

streptavidin mutant forms 2D crystals in the C222 molecular packing arrangement. The C222 packing arrangement has been studied at high resolution.8,24,28,29 Square streptavidin crystals have been previously observed in two cases. The first was in crystals formed beneath Cu-IDA lipid monolayers, and the second was with the N23A biotin binding pocket mutant formed under biotinylated lipids. The former result has been largely (28) Furuno, T.; Sasabe, H. Biophys. J. 1993, 65, 1714-1717. (29) Avila-Sakar, A. J.; Chiu, W. Biophys. J. 1996, 70, 57-68.

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Figure 5. Two-dimensional fast Fourier transform of the data shown in Figure 4b. Reflections corresponding to the reciprocal lattice vectors a* and b* are indicated. The angle between these reflections is approximately 87°, consistent with a C222 crystal. Averaging the data from several similar images yields a mean lattice angle of 88.5 ( 1.6° and lattice spacings a ) 9.9 ( 0.2 nm and b ) 8.8 ( 1.4 nm. These data correspond to a C222 crystal packing arrangement, as shown in Table 1. Table 1. Lattice Parameters for Known Streptavidin 2D Crystal Packing Arrangements C222, P1, and P2, as Reported in Reference 27, alongside Lattice Data for the 13-136/T20A Streptavidin 2D Crystalsa C222 P1 P2 13-136/T20A

a (nm)

b (nm)

γ (deg)

8.5 5.8 11.6 9.9 ( 0.2

8.4 5.0 5.8 8.8 ( 1.4

90 113 107 88.5 ( 1.6

a The 13-136/T20A data correspond to a C222 crystal packing arrangement; the slight difference in lattice spacing may be an artifact of crystal drying or a result of inexact AFM calibration.

interpreted as arising due to the loss of growth axis asymmetry that occurs concurrently with replacement of biotin binding by the metal-chelating binding mode, although the interfacial binding dynamics are also sub-

stantially faster. The N23A result could be attributed either to the faster interfacial protein binding dynamics, due to the large increase in biotin and biotinylated lipid off-rate, or alternatively to the breaking of coupling between biotin binding and an essential protein-protein crystal contact interaction.30 The effects of altered interfacial binding dynamics complicate both cases. Because the T20A mutant exhibits an off-rate of 13 × 10-6 s-1, which is comparable to that of wild-type, this ambiguity does not exist. The square crystal morphology for T20A is thus a result of the altered protein-protein hydrogenbonding contact, where the growth rate differences for the two axes associated with the bound or unbound streptavidin subunits are removed. The directly adjacent Asn 23 hydrogen bond to biotin provides a straightforward structural connection between biotin binding and the crystal contacts at Tyr 22 and Thr 20. Both the Asn 23 mutant and Thr 20 mutant remove crystal growth asymmetry, consistent with this direct coupling mechanism. The coupling is structurally subtle, as no differences in Thr 20 or Tyr 22 atomic coordinates can be discerned in biotin-bound versus unbound subunits in the high-resolution X-ray crystal structure of streptavidin with two biotin ligands bound.9 Nevertheless, this coupling does not need to be large to alter the proteinprotein activation barrier that is the determinant of this kinetic effect on growth axis rates. The retention of the C222 symmetry dictates that the general protein-protein contact area is similar in the 13-136/T20A mutant crystal and the wild-type protein crystal. Small changes in structure and/or dynamics associated with biotin binding at the Asn 23, Tyr 22, and Thr 20 positions thus account for important barrier changes at the protein addition step that dramatically affect final crystal morphology. Acknowledgment. We are grateful to Drs. Ken Nakajima and Keita Mitsui for assistance with and advice regarding atomic force microscopy. We further gratefully acknowledge the support of NASA (NAG8-1149), the National Science Foundation (through the UWEB Engineering Research Center and for support for N.M. through the Summer Institute in Japan), and the Pharmaceutical Research and Manufacturers Association of America (postdoctoral fellowship to S.K.). LA0118457 (30) Koppenol, S.; Klumb, L. A.; Vogel, V.; Stayton, P. S. Langmuir 1999, 15, 7125-7129. (31) Kraulis, P. J. J. Appl. Crystallogr. 1991, 24, 946-950.