1 Department of Chemistry, Rensselaer Polytechnic Institute, Troy, NY 12180, 3 Nelson Institute of Environmental Medicine, NYU Medical Center, 57 Old Forge Road, Tuxedo Park, NY 10987, 4 Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213 and 5 Wadsworth Center, New York State Department of Health, Albany,NY 12201, USA
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Abstract |
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Keywords: cysteine/crystallization/disulfide/fiber/thread
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Introduction |
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The self-assembly of biomolecules may provide another approach to nanomaterials. Exploitation of the self-assembly inherent in DNA base pairing has generated DNA cubes, junctions and molecular tweezers (Seeman, 1998). Our approach makes use of a different form of self-assembly, protein crystallization. Generation of covalent cross-links between adjacent protein molecules in a crystal lattice should produce fibers upon dissociation of the crystal, if the reacting amino acid residues are appropriately situated with respect to symmetry operators in the crystal space group. A major advantage of this approach is the ability to monitor through the use of X-ray crystallography what happens during the production of the materials. Maltose binding protein (MBP) is a convenient model protein with which to investigate such constructs because it crystallizes in a few days in a crystal form that is amenable to X-ray diffraction data collection (Spurlino et al., 1991
). The materials produced from such a protein should be ordered on the nanometer scale, a size range larger than zeolites but smaller than collections of colloidal particles.
The cross-linking of protein crystals is not a new idea. Quiocho et al. first observed that protein crystals can be cross-linked, as a result of which they are stabilized toward mechanical disruption and will not dissolve (Quiocho et al., 1964). The cross-linking process has been used occasionally to stabilize protein crystals for purposes related to basic research, but its application has otherwise been limited until relatively recently. Cross-linked protein crystals were found to function as catalysts, sometimes in mixed organic solvents and water. Advantages of such cross-linked enzymes over other forms of the enzyme are that stability is enhanced and the ease of separation of the enzyme from reactants and products is improved (St. Clair and Navia, 1992
; Khalaf et al., 1996
).
The crystal cross-linking methods employed to date have largely been random. Proteins are thereby connected through a three-dimensional web of covalent cross-links within which any given protein may react several times, once or not at all. In a clear departure, Yang et al. first attempted to cross-link proteins in an organized manner by introducing cysteine residues at crystal contacts (Yang et al., 2000). However, even under the strongly oxidizing environment of pure oxygen gas, the longest polymers of T4 lysozyme that they could detect had a length of 25 monomers and most of the material produced from the crystals was found to contain three monomers or less. In the present work, cross-link formation in the air extends in one dimension throughout the crystal with close to complete cross-link formation. Protein polymers are long enough that they may be accurately described as fibers. Association of these fibers produces threads.
We denote the protein assemblies described in this work as crystine fibers and threads, since they are derived from protein crystals in which the proteins have been cross-linked with cystine residues. An initial application for crystine fibers might be to introduce enzymes into cloth or filtration materials. However, our present research is focused on the properties of crystine fibers of electron transport proteins and their potential application in microelectronics.
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Materials and methods |
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The plasmid from a commercial protein fusion and purification system, pMALc2 (New England Biolabs, Beverly, MA), was modified to produce the vector for expression of modified MBP used in these experiments, as summarized in Table I. The first two modifications were required to produce a wild-type protein. An insertion at residue 301 was required to modify proteinprotein contacts at crystal interfaces. Residue pairs were substituted with cysteine for covalent cross-linking experiments.
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Modification of the 3' end of the gene
Restriction digests of the pMALc2 plasmid were carried out using SacI and XbaI. Oligomers (Table I) for two complementary strands were designed with appropriate complementary sticky ends to terminate the protein with the correct RITKStop sequence, followed by an AscI restriction site to help identify clones containing the desired sequence. These oligomers were then ligated into the SacIXbaI linearized plasmid using T4 DNA ligase (New England Biolabs). Bluewhite screening (Yanisch-Perron et al., 1985
) and restriction digestion with AscI were used to isolate clones containing the desired plasmid. The entire gene sequence was confirmed by sequencing of both strands.
Mutagenesis
Site-directed mutagenesis, using the QuikChange (Stratagene, La Jolla, CA) system, was carried out to introduce the mutations T2I and A301GS sequentially into the maltose binding protein gene in pGIP3(10), resulting in pUIP1 (Table I). Additional mutations were added sequentially to pUIP1 (Table I
). The mutant DNA was transformed into XL1 Blue Supercompetent cells (Stratagene). Colonies growing on TBYE plus 2x ampicillin plates were selected and DNA was prepared from these colonies for sequencing. For expression, the plasmid was transformed into strain ER2508 (New England Biolabs), an RR1-based strain in which the malE gene (coding for MBP) has been deleted in order to prevent contamination of modified protein with native MBP.
Expression
Cells containing pUIP7 were plated out from a frozen glycerol stock onto TBYE plus 2x ampicillin plates and allowed to grow overnight. Colonies from this plate were used to inoculate three 25 ml overnight cultures in superbroth. These starter cultures were used to inoculate three 1 l superbroth cultures in 2.8 l flasks. Bacteria cultures were grown at 37°C with orbital shaking at 200 rpm. When the absorbance at 600 nm reached 0.25, 4 ml of 0.25 M IPTG were added to each flask to induce overexpression of the MBP. Cultures were allowed to grow for an additional 8 h after induction and then harvested by centrifuging at 5000 rpm for 10 min. The supernatant was discarded and cell pellets were frozen at 80°C.
Protein purification
Each pellet was resuspended in 60 ml of lysis buffer (20 mM TrisCl, 50 mM NaCl, 10 mM EDTA, pH 7.5) to which fresh solid dithiothreitol (DTT) was added to a final concentration of 10 mM. The cell suspension was sonicated eight times for 30 s at 60% power (Sonics CV33, Newtown, CT). The solution was centrifuged for 20 min at 12 000 rpm (Kendro Sorvall SS-34, Newtown, CT) and the pellet was discarded. Since MBP is unusually soluble at high salt concentrations, 0.65 g of magnesium acetate and 16 g ammonium sulfate were added for every 60 ml of lysis buffer used to resuspend the cells. The solution was stirred in the cold until the salts dissolved and then centrifuged again at 12 000 rpm for 20 min. The supernatant was dialyzed against two changes of 4l of 10 mM TrisCl, 1 mM DTT, pH 8.0.
The dialyzed solution was loaded on a 15x5 cm Q-Sepharose column (Amersham Pharmacia Biotech, Piscataway, NJ) equilibrated with 10 mM TrisCl, 1 mM DTT, pH 8.0. MBP was eluted with a 950 ml gradient of NaCl from 0 to 0.175 M, followed by a 300 ml linear gradient from 0.175 to 0.5 M NaCl. All gradient solutions contained 10 mM TrisCl, pH 8.0 and 1 mM DTT. The columns were washed with 0.1 M NaOH and stored in 0.05% sodium azide in water after each preparation. Fractions that contained maltose binding protein, as judged by SDSPAGE, were pooled. The pooled fractions were concentrated to less than 10 ml in a stirred-cell ultrafiltration unit using a YM-30 membrane (Millipore, Bedford, MA). The solution was loaded on a 90x2.5 cm Sephadex G-75 column equilibrated with 10 mM TrisCl, pH 7.3. Two broad peaks were eluted from this column. The second, smaller peak contained mostly maltose binding protein. Pooled fractions were brought to 10 mM DTT and loaded on a 250x10 mm SynChropak AX300 HPLC column (Eichrom Technologies, Darien, IL). MBP was eluted with a 240 ml linear gradient from 0 to 200 mM KCl in 10 mM TrisCl, 1 mM DTT, pH 7.3.
Crystallization
The purified protein from the SynChropak column was dialyzed against two changes of at least 500 ml of 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), 1 mM maltose, 0.02% sodium azide, containing either 10 mM 2-mercaptoethanol or 2-mercaptoethylamine as reducing agent and adjusted to pH 6.2 with either the sodium salt of MES or NaOH. The dialyzed protein was concentrated to 2030 mg/ml using the YM-30 ultrafiltration membrane and stirred ultrafiltration cell and used for crystallization experiments. Crystals were grown by the hanging drop method (drop size 25 ml) under conditions similar to those for the wild-type (Spurlino et al., 1991). Crystals could be obtained under a wide range of conditions from 2.5 to 10 mg/ml MBP, from 9 to 15% (w/v) polyethylene glycol (PEG) 6000, 10 mM NaMES (pH 6.2), 1 mM maltose, 0.02% sodium azide and 1 mM of reducing agent in the droplet, suspended over 1 ml of 19 or 22% PEG 6000 at 4°C. Crystals were sometimes stored in stabilizing solution containing 25% (m/v) PEG 6000, 10 mM NaMES, 1 mM maltose, 0.2% sodium azide, pH 6.2. Small crystals obtained from initial experiments were used to seed additional crystallization experiments of the mutant. Crystals for seeding were transferred to and stored in a special stabilizing solution as described above, but containing 30% PEG 6000. A thin wire or sharp needle was used to transfer seeds into the droplet by dipping the tip first into the seed solution and then into the droplet. The best crystals were obtained from drops containing between 2.5 and 7.5 mg/ml of MBP with 1215% PEG 6000 in drops suspended over 19% PEG 6000 in wells, with all other conditions as reported above.
Protein cross-linking in solution and in crystals
Monomeric protein at a concentration of 8.2 mg/ml was dialyzed as above for crystallization experiments, in the absence of any reducing agent, and incubated at 4°C for 4 months. This protein was found to have cross-linked in solution, whereas protein similarly stored in the presence of reducing agents did not.
To provide material for comparison with the solution cross-linked protein, a droplet containing multiple protein crystals was transferred to a microfuge tube and centrifuged gently for 10 s. The supernatant was removed and the crystals were resuspended by swirling in 100 µl of stabilizing solution containing 10 mM DTT. After 3 h at room temperature the crystals were centrifuged again and the supernatant was removed. The crystals were resuspended in 100 µl of stabilizing solution without DTT, centrifuged and resuspended again in the fresh solution. Gel electrophoresis was performed after 7 days of incubation at room temperature.
X-ray crystallography
Crystals were flash frozen in liquid nitrogen after being exchanged into a cryoprotectant solution. All data collection was at 100 K. Cryoprotectant for the original crystals was composed of 20% (m/v) polyethylene glycol (PEG) 400, 20% PEG 6000, 1 mM maltose and 0.2% sodium azide. For experiments with re-reduced and oxidized crystals, ethylene glycol was substituted at the same concentration for the PEG 400 in the cryoprotectant solution, which provided superior protection of crystal integrity.
Data collection for the original non-cross-linked crystals was carried out on the X12-C beamline at the National Synchrotron Light Source at the Brookhaven National Laboratory, using the B1 CCD detector. The programs Denzo and Scalepack were used for data reduction (Otwinowski and Minor, 1997). An R-AXIS IV image plate area detector, mounted on a Rigaku RU-200 rotating anode equipped with focusing mirrors, was used for the other data set. The R-AXIS data set was integrated with the program MOSFLM (Leslie, 1992
) and merged with the program SCALA (Collaborative Computational Project, Number 4, 1994
). Refinement was carried out with the program CNX 2000.12 (Accelrys) (Brunger et al., 1998
), optimizing both atomic position and individual B-values using the maximum likelihood target. The standard geometry library (Engh and Huber, 1991
) was employed, except that the van der Waals radius of the sulfur in cysteine residues was set at 1.0 Å, in order to allow the two sulfur atoms at the intermolecular cross-link to approach one another in absence of an actual bond defined between asymmetric units, which the program would not allow. Manual adjustment of the model was accomplished with the programs O (Jones et al., 1991
) and Quanta (Accelrys). Crystallographic data processing and refinement statistics are shown in Table II
. Although this protein contains an additional N-terminal methionine compared with the original crystal structure (Sharff et al., 1995
), insufficient electron density was observed to determine coordinates for this amino acid. Coordinates and structure factors have been deposited with the Protein Data Bank (Berman et al., 2000
) as 1JVX and 1JVY.
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Preparation of samples for electron microscopy involved washing the crystals in water to remove polyethylene glycol and buffer components and dissolving them in 0.17 M acetic acid by transferring the crystals one at a time between droplets of liquid with a cat whisker. Specimens for transmission electron microscopy (TEM) were prepared by allowing Formvar-coated copper grids to float on drops of the sample. Grids were retrieved from the drop, excess liquid was removed with filter-paper and then they were stained with 0.5% uranyl acetate. Specimens for scanning electron microscopy (SEM) were prepared by mixing with the droplet containing the dissolved crystals two aliquots of 0.10 M NaOH, each containing half the droplet volume. Fibers were drawn from the neutralized solution by touching the surface of the droplet with a cat whisker. The fibers were laid on a glass slide and shadowed with gold.
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Results |
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The form of maltose binding protein with Ala301 replaced by the dipeptide GlySer (A301GS) proved to be an attractive experimental model (Sharff et al., 1995). Cysteine substitutions were investigated at three sites: between the pairs Asp207Pro316, Gln72Lys306 and Glu172Ala186. None of these disulfide pairs was predicted to have ideal geometry as classified by Sowdhamani et al. (Sowdhamani et al., 1989
). The first pair had poor predicted geometry (Class C, two or more torsion angles outside of the preferred range) and the latter two had fair predicted geometry (Class B, one torsion angle outside of the preferred ranges). The first pair was the only one that produced large crystals.
The X-ray diffraction structure determination of the double-cysteine protein D207C/P316C indicated that the cysteines were in the mixed disulfide form (Figure 1). Treating the crystals for 3 h with 10 mM DTT in crystal stabilization solution, placing the crystals in the same solution without DTT and then allowing the cysteines to air oxidize in the absence of reducing agent for
3 days led to the formation of disulfide cross-links between the adjacent molecules in the crystal lattice (Figure 1
). Although the X-ray crystallographic results indicated essentially complete cross-linking after 3 days of oxidation, the solubility properties (described below) continued to change for ~7 days. These results suggest that all but a few cross-links formed within 3 days and that cross-linking continued at a low level for an additional 4 days.
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Unit cell dimensions from the uncross-linked mutant diverged more from the reference crystal structure than did those dimensions from the cross-linked mutant. These results are consistent with Figure 1c, which shows the relationship between two molecules in the lattice near the contact between residues 207 and 316. Formation of the cross-link actually brought the relative positions of the proteins into orientations more like that in the reference protein crystals than in the non-cross-linked crystals.
Definition of a disulfide bond between asymmetric units is not possible in the refinement program and the positions of the sulfur atoms were not restrained in a conventional manner for a disulfide bond. This technical problem results in the less accurate placement of these atoms than would otherwise be expected, especially at the moderate resolution of this X-ray crystal structure (2.5 Å). Technical problems notwithstanding, the torsion angles formed by the disulfide bond can be estimated. The torsion angles that are most important are the 1 angles (describing the orientation of the bond between the
- and ß-carbons) and the
SS angle (describing the orientation of the bond between the two sulfur atoms). The
1 angles on the 207 and 316 sides of the disulfide bond are approximately 166 and 59°, respectively. These torsion angles fall within commonly observed ranges. The
SS angle is 110°, which falls within the usual range 60° < |
SS| < 120°. A combination of small protein conformational changes and packing rearrangement produced a relatively unstrained disulfide bond (Sowdhamani et al., 1989
).
Figure 2shows one view of the structure of the fibers formed by disulfide linkage of proteins in the crystal. The fiber axis, which is horizontal in this figure, corresponds to the crystallographic b axis. The length of the b axis decreased by 1 Å upon disulfide formation.
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The cross-linked crystals form an apparent solution in mildly acidic conditions, such as 0.1 M acetic acid. Transmission electron micrographs of material found in these solutions show bundles of fibers running approximately parallel to each other (Figure 4). Some order can be seen along the fibers, and also perpendicular to the fiber axis (Figure 5
). Direct measurement of repeat distances from four fiber bundle images produced spacings of 3.4 ± 0.3 nm along the fibers and 4.4 ± 0.2 nm between fibers. The alignment of the connected molecules in the crystal is along the crystallographic b-axis (Figure 2
). Two molecules span the unit cell along the b-axis, which has a length of 6.8 nm. The spacing observed by eye along the fibers probably comes from the distance between protein monomers, that is, half of the unit cell dimension of 6.8 nm. Because the bundles have edges where many fibers end in a flush manner, the bundles are probably due to interactions found in the crystal that were not disrupted in the acidic solution. However, the 4.4 nm spacing does not correspond well to any distance involving the crystallographic a- or c-axes. It is reasonable to assume that the spacing between the fibers may have decreased because of dehydration (Bell, 1999
) during sample preparation, to a greater extent than the spacing along the fiber, where stronger interactions, including a covalent bond, maintain order.
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SEM images are shown in Figure 8of one end of one of the threads that was produced under acid dissociation/neutralization conditions in which the reference protein did not produce threads. The smaller fibers that make up these threads can be clearly seen in the third panel. However, the resolution of the SEM images is insufficient to determine whether individual protein fibers are present. SEM images of the body of several threads show either smooth or grooved surfaces (Figure 9
). The smooth thread was produced when copious precipitate was present, whereas the rough threads were produced in the absence of notable precipitate.
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Discussion |
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Only one of the three MBP variants designed to form disulfide cross-links could be successfully crystallized. Interestingly, the best variant for crystallization was that in which the disulfide was predicted to have the most strained conformation.
The choice of sites for the introduction of cysteine residues will be determined by both the crystallization and the cross-linking steps. The best choice may be sites that are situated so as to avoid excessive strain upon disulfide formation, but not so ideal as to interact during crystallization. The flexibility of the parts of the protein where the cysteine substitutions are incorporated will also have some impact on the results. Falke and co-workers were able to form intramolecular disulfide bonds from cysteine residues much farther apart than would ordinarily be bridged by such a cross-link because of protein conformational fluctuations (Falke and Koshland, 1987; Careaga et al., 1995
; Butler and Falke, 1996
). In one case, disulfide formation occurred at a measurable rate between two residues in the same domain that were 15.2 Å beyond the maximum distance that can form an unstrained disulfide. Long distances between symmetry-related cysteine residues might be advantageous for crystallization but, if cross-linking is accompanied by large changes in protein position, the length of the fibers formed may be limited. Before clear guidelines for cysteine replacement can be formulated, more examples need to be constructed and analyzed.
One-dimensional cross-linking of a protein crystal yielded material that was surprisingly difficult to dissolve, as also observed by Yang et al. (Yang et al., 2000). This property of crystine fibers seems to be an interesting example of cooperativity among many weak interactions (Creighton, 1983
), stabilizing the association of macromolecules through interactions otherwise too weak to produce stable complexes. Mild acidic treatment was effective to dissolve cross-linked forms of both T4 lysozyme and MBP crystals.
The dissociated crystals of MBP formed bundles of fibers and individual fibers. It would seem that some of the bundles were remnants of an association found in crystals, since the ends of individual fibers coincided in some bundles. Whether the proteins were still in their native state in 0.17 M acetic acid (pH 3) is uncertain. Since spacing along the fibers in the fiber bundles is reminiscent of the spacing along the b-axis within the protein crystal, some properties of the intact protein remain in the acid-dissociated material. Other, gentler means of fiber dissociation are needed, together with the development of techniques to monitor conformation during dissociation.
Equally intriguing, but more problematic, is the state of material obtained as threads. Under the usual experimental conditions, threads could be made from cross-linked protein but not from crystals of the reference protein. In the absence of efficient mixing, thread-like objects could also be made from maltose binding protein that is not cross-linked and, therefore, must be denatured. A working hypothesis is that two types of threads can be produced, some denatured and some not, which can be differentiated by the smoothness of the surface observed by SEM (Figure 9). Better methods of thread formation should follow from better methods of fiber dissociation and from experimentation directed towards the question of the physical state of protein in the threads.
The advantage of this approach to biomolecular material production is that it may be used with many proteins that crystallize. Proteins with important properties or enzyme activities could be produced in fibrous form. Such fibers might be used to impart new properties and functions to woven, non-woven and composite materials. Also, nanoscale electronic interconnection through cables of specially designed protein fibers may some day evolve from this technique.
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Notes |
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6 To whom correspondence should be addressed. Present address: Accelrys, Inc., 200 Wheeler Road, Burlington, MA 01803-5501, USA. E-mail: bell{at}mailstation.com
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Acknowledgments |
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References |
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Received September 20, 2001; revised July 29, 2002; accepted August 13, 2002.