CRYSTAL GROWTH & DESIGN 2007 VOL. 7, NO. 11 2134–2139
Articles Calorimetric Approaches to Characterizing Effects of Additives on Protein Crystallization† Joanne I. Yeh*,‡ and Samuel I. Beale§ Department of Structural Biology, UniVersity of Pittsburgh Medical School, 3501 Fifth AVenue, BST3 1040, Pittsburgh, PennsylVania 15260, and Department of Molecular Biology, Cell Biology and Biochemistry, Brown UniVersity, ProVidence, Rhode Island 02912 ReceiVed July 26, 2007; ReVised Manuscript ReceiVed September 19, 2007
ABSTRACT: Introduction of additives to the crystallization solution can improve the quality of macromolecular crystals, presumably by enhancing crystal contacts, conformational stability, and/or solvent interactions. However, despite their widespread use, the mechanisms by which these additives exert their effects remain largely unknown. We have used differential scanning calorimetry and isothermal titration calorimetry (ITC) to characterize the effects of additives on the crystallization of several proteins. We have found that the positive effects on crystallizability exerted by an additive can be correlated with stabilization of the macromolecule in solution, as evidenced by an increase in the midpoint melting transition temperature. Additionally, the additives’ effect on the thermodynamic variables ∆H and ∆G, as determined by ITC, can be used to distinguish whether the additive functions by binding to the macromolecule or by altering the properties of the crystallization solution. The use of ultrasensitive instrumental methods to evaluate the potential effectiveness of additives can facilitate rapid screening of a large number of additives prior to crystallization and provide a means to rationally limit the crystallization solution search space. Most physical and chemical processes have an associated heat effect, and this can be used as the basis for a number of analytical techniques and determination of relative thermodynamic quantities. We have applied microcalorimetry to characterize crystallization behavior of several proteins. Results from the microcalorimetry indicate that conditions that produce highquality crystals have a positive effect on the protein’s solution stability as reflected by an increased transition temperature. These results show the promising potential of developing a more rational approach to defining crystallization conditions. The crystallization step is one of the primary obstacles to obtaining detailed structural knowledge of a macromolecule by X-ray diffraction. Crystallization of macromolecules has remained a generally empirical process: successful crystallization conditions are usually identified by an extensive screening procedure, typically based on the sparse matrix method.1 Various methodologies are currently employed for the crystallization of biological macromolecules,2–4 mostly centered on additional screening procedures to broaden crystallization phase space. Most initial screening procedures are based on observing the effects of varying pH and buffer components as well as salts † Part of the special issue (Vol 7, issue 11) on the 11th International Conference on the Crystallization of Biological Macromolecules, Québec, Canada, August 16–21, 2006 (preconference August 13–16, 2006). ‡ University of Pittsburgh Medical School. § Brown University. * Corresponding author: E-mail:
[email protected].
and/or organic precipitants, both in the concentrations used and their chemical class. Commercial screens such as those available from Hampton Research, Emerald BioStructures, and others, start at fairly high concentrations of salt or organic precipitant. Although these screens allow one to observe the effects of various chemical components on the crystallization behavior of a macromolecule fairly readily, many conditions can result in non-productive precipitation as screening begins in an area of the phase diagram that is high in both macromolecular and precipitant concentrations, that is, the “labile region”. As the labile region is penetrated further, the probability of spontaneous and uncontrolled nucleation is enhanced, resulting in extensive precipitation.5 However, the creation of crystals that are suitable for X-ray diffraction analysis requires the formation of an ordered lattice. The ability of compounds, for example, salts, to gradually bring a macromolecule out of solution by “salting out” can result in the formation of an ordered crystal. The mechanism by which these compounds promote ordered lattice formation is related to the interaction of these compounds not only with the macromolecules but also with the solvent molecules. Most chemicals used for crystallization can be categorized broadly into five classes: ions, organics, cosmotropes, chaotropes, and detergents, with many compounds spanning more than one category. To illustrate, one of the commonly used salts in crystallization, ammonium sulfate, is ionic in solution but
10.1021/cg7007045 CCC: $37.00 2007 American Chemical Society Published on Web 11/07/2007
Calorimetric Crystallization Additive Evaluation
additionally possesses cosmotropic properties in that it stabilizes proteins and orders water molecules. Consequently, addition of ammonium sulfate lowers the availability of free water to interact with the macromolecule to keep it in solution, and the protein eventually precipitates out of solution. This effect has been utilized not only in crystallization, which is a process of bringing macromolecules out of solution in an orderly manner but also in protein purification, to separate proteins by selective fractionation. Chaotropes such as urea have also been used in crystallization. These have the opposite effects of cosmotropes, that is, they increase the availability of free water and thereby increase the solubility of macromolecules. This class of compounds can destabilize proteins by disrupting intramolecular hydrogen bonding interactions, resulting in unfolding of proteins. This effect has been utilized for solubilizing inclusion bodies and for refolding. Another class of compounds is detergents, which are used for solubilizing and crystallizing membrane proteins2,6–8 but have also been found to be helpful with soluble protein crystallization at concentrations below their critical micellar concentration.2,9 Other compounds, such as glycerol, stabilize proteins by amphiphilic interactions.10 These compounds stabilize hydrophobic patches on the macromolecule and prevent aggregation but are polar enough to help maintain solubility of the macromolecule. At lower concentrations, these classes of compounds have been used to aid in optimizing crystal formation, even though the mechanisms by which they improve the diffraction qualities of crystals remain unknown.9,11–13 Although extensive understanding of the mechanism of macromolecular crystallization is far from complete, empirical methods that have been employed successfully include use of additives. Macromolecules are structurally dynamic, changing conformation in the presence of ligands. More subtly, macromolecular interactions can be affected by the presence of various added compounds. The use of a compound to modify intermolecular interactions, namely, to enhance crystal lattice contacts, defines the compound as an additive. In contrast, the use of compounds to bring the macromolecule into the labile zone for crystal formation in an orderly manner categorizes the compound as a precipitant. Although a compound can be both an additive and a precipitant, empirically, precipitants are used at high concentrations, and additives are usually employed at lower concentrations. Utilization of additives typically occurs after preliminary crystallization conditions have been found. The mechanisms by which additives affect crystallization or the lattice formation have remained largely unknown. Differentiating between direct interaction with the macromolecules versus interactions with the solvent can help to define the mechanism by which a compound exerts its effects on crystallization behavior. DSC can be useful for discriminating between specific interactions of an additive with the macromolecule and solvent-mediated effects. When an additive binds to a macromolecule and stabilizes it, a shift in Tm can result. An increase in Tm indicates binding of the additive to sites on the macromolecule to help maintain the protein’s structural integrity. These can be at intra- or intermolecular interfaces, in the cores or the interior of globular proteins, or they can stabilize helical motifs. Hence, a change in Tm is indicative of direct macromolecular interactions. The binding of a ligand to a macromolecule can be accompanied by an enthalpic change that can be detected through the amount of heat released or absorbed, which can be correlated to the free energy of binding. Positive, negative, and cooperative changes in free energies accompanying binding events can be detected and differentiated by ITC experiments. ITC can also
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provide binding affinity values. Conversely, solvent effects are typically characterized as being largely entropically driven, and the ordering or freeing of water molecules does not have a significant total enthalpic (heat) component. Consequently, the combined use of ITC and DSC measurements can be used to determine the degree to which a compound interacts directly with the macromolecule or if it exerts its influence through solvent-mediated effects. We are studying the effects of additives to improve crystallization behavior of several proteins and have applied DSC and ITC to correlate the thermodynamic effects of the additives with the improvements observed. Particularly striking is the positive correlation observed between an increase in Tm of the protein and the enhanced crystal quality obtained in the presence of the additive. Understanding how various compounds interact with both solvent and macromolecule can lead to a more rational utilization of them for crystallization. Moreover, studying how various compounds affect crystallization behavior of specific macromolecules can result in insights into the general mechanisms of crystal growth. The ability to utilize DSC and ITC to help narrow the number of variables and their ranges has the potential to vastly improve the probability of growing useful macromolecular crystals. With the development of highly sensitive instruments having enhanced signal-to-noise ratio at the microscale level, detailed characterization of the effects of various compounds on conformational stability as correlated to crystallizability becomes possible. Here we report correlations between the effects of additives on three thermodynamic parameters, Tm, ∆H, and ∆G, with the quality of crystals obtained for five proteins glycerol kinase (GK), glycerol-3-phosphate dehydrogenase (GlpD), R-glycerophosphate oxidase (GlpO), hen egg white lysozyme (HEWL), and NADH peroxidase (Npx). Results Optimization of initial additive conditions resulted in significant improvements in crystal sizes and quality of all five proteins. In addition, two of the proteins, GK and GlpO, went through an additional round of additive screening after it was found by ITC and DSC that the additive bound to the protein (i.e., additive was an effector). The second round of screening of crystallization conditions in the presence of the effector, as described in Materials and Methods, led to much improved crystals with respect to their size and quality for yielding highresolution diffraction data. Whether the additives had any effects on the biological activities of the proteins was not determined. Correlation of the observed improvements in crystal morphology with DSC results indicated that there was concomitant stabilization of the protein in solution when additives were present (Figure 1). Additionally, reversibility of folding also indicates protein stabilization under the crystallization conditions, enabling the protein to refold into its native state after heating, as in the case of HEWL (Figure 2). These changes indicate that conditions that increase the Tm of the protein in solution also increase the diffraction quality of the crystals. In all the cases shown here, improvements in resolution of diffraction accompanied the increased size, although mosaicity values also increased in two cases (data not shown). Particularly striking were the DSC results; four of the five proteins, GlpO, GK, and Npx, and HEWL, exhibited increases in Tm ranging from 3 to 9 °C in the presence of some additives, indicating stabilization of the protein by the additive (Figures 1 and 2, Table 1). In contrast, GlpD did not exhibit a shift in Tm in the presence of the additives tested. Not surprisingly, the
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Yeh and Beale
Figure 1. DSC results (left) and crystal morphologies obtained (right) for GlpO (A) and GK (B) in the presence and in the absence of the indicated additives. For GlpO, neither of the additives, fructose 6-phosphate or hexaglycine plus glycerol, is a known effector molecule, that is, they do not appear to bind to the enzyme specifically. However, fructose 6-phosphate appears to stabilize the enzyme as indicated by its effect on increasing the Tm, and both of these additives enhance the crystallization of GlpO. Glycerol is a substrate for GK, and it raises the Tm, indicating enhanced stability of the protein. Poly L-aspartic acid also raises the Tm of GK and exerts a positive effect on the crystallization behavior, but it has not been identified as an effector.
Figure 2. Stabilization of proteins by certain additives is evidenced by an increased Tm. The shift to higher Tm values under the crystallization conditions, compared to the Tm in buffer alone, indicates stabilization, as in the case of Npx (top). For HEWL, the protein was stabilized such that refolding after heating was observed (bottom). In the DSC scans, the “solution” refers to the crystallization solution, as described in the text.
two effector molecules, glycerol and glycerol 3-phosphate, which are known substrates for the enzymes GK and GlpO, respectively, caused substantial increases in the Tm values. The
structure of GK has been solved in the presence and in the absence of glycerol.14 Binding of glycerol results in domain closure of the enzyme. On the basis of the calorimetric results
Calorimetric Crystallization Additive Evaluation
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Table 1. Tm Measurements by DSC for Four of the Five Proteins in the Studya protein
Tm (buffer only)
Tm (crystallization solution)
Tm (crystallization solutions plus additive)
additives (concentrations shown are amounts in the protein drop)
GlpOb
50
GKb
42
Npx
47
Lss
57
55 52 65.7 57.2 57 58 53 73.6 75.2 63
60 51.6 69.1 59.1 63 65 59 77.7 75.5 63
5 mM fructose-6-phosphate 5 mM hexaglycine and 1 mM glycerol 2.5 mM glycerol 2 mM poly L-aspartic acid 7.5 mM 1,12-dodecanedioic acid 1 mM hexaglycine 2 mM -amino-n-caproic acid 5 mM 1,2,3-heptanetriol 3 mM 1,8-diaminooctane 7 mM dextran sulfate
a Tm values are shown in buffer and in the presence and absence of the additives in the optimal crystallization solution for each additive. Tm Values shown are the average of at least three measurements. note that the DSC results for GlpD are more complex and harder to interpret due to the presence of detergents and possibly endogenous lipids; therefore they are not shown here. b The DSC curves for these two proteins are shown in Figure 1.
Figure 3. Additives can affect protein solubility so that the concentration of the protein may need to be readjusted with respect to the original conditions. The initial crystallization condition (0.1 M acetate buffer pH 4.5, 8% PEG 4000) was found for GK at 10–15 mg/mL protein without glycerol. After glycerol was added, the protein became significantly more soluble so that the protein concentration was eventually increased by more than 3-fold (up to 40 mg/mL), and the additive concentration was halved to obtain the best crystals.
presented here and our previous structural results, the closed form is presumably the more stable, less dynamic structure. In addition to changing the solution behavior of proteins, additives can modify solubilities significantly, increasing or decreasing solubilities of a protein for a particular buffer and pH condition (Figure 3). Consequently, additives can further expand the crystallization screening space. The DSC results convincingly showed that changes in Tm can provide an indication of how the native protein behaves under various conditions. Shifts in Tm in the presence of additives can be used to “quantitate” the effects of the additive and help to determine optimal concentrations of additives to use for enhancing crystallization. The combination of DSC and ITC gives a good indication of the effects of a particular additive compound on the protein, where an increase in the Tm and binding enthalpy can indicate direct interaction of a compound with a protein. In our study, an increase in Tm also correlated with a favorable effect on the crystallization behavior of the protein. When the additive appeared to act via by solvent effects, the resulting change in Tm was not as dramatic, and the ITC showed little or no change in the enthalpy of binding (data not shown). These results indicate that enhanced crystallization behavior of the protein under various conditions may be predicted by use of calorimetric methods. Enhancement of lattice formation could be linked to the ability of an additive to decrease the entropic penalty for ordered lattice
formation. Our results from this study suggest that facilitation of crystallization by an additive may be correlated to direct interactions between the additive molecules and the proteins in solution. These solution interactions could already constrain the system (i.e., decrease the number of degrees of freedom in solution), possibly lowering the overall entropic difference between the solution verses solid phases and consequently aiding in crystal lattice formation. Discussion Our study of additive effects on five proteins to characterize the changes observed with respect to crystallization behavior indicates that DSC and ITC can be very useful for systematic screening to determine potential crystallization conditions. This is especially helpful for conserving macromolecules or other reagents and to gain initial insight into potential crystallization conditions. These results signify that this approach can be applied to determine useful initial crystallization parameters and conditions, for prescreening solutions that stabilize the macromolecule in solution prior to crystallization. It should be emphasized, however, that thermodynamic parameters are related to a particular set of experimental conditions and are not absolute values. Use of Tm to differentitate between specific additives and conditions can be useful in ranking those that enhance protein stability, provided that the
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experimental setup and evaluation procedure remain the same. The thermodynamic parameters obtained from microcalorimetry are macroscopic properties and should be interpreted with caution when applied on a molecular level. The absolute effect of the binding of a ligand on protein stability cannot be fully delineated by microcalorimetry. Differentiation between enthalpically versus entropically driven binding processes is complex to deconvolute: when ∆H is greater than or close to zero in ITC measurements, the binding is not necessarily entropically driven. The heat effects registered by ITC are the sum of the heat effects of all conformational changes, desolvation, buffer deprotonation, etc. Consequently, the differential binding of ligands to a protein can be explained in terms of their ∆H and ∆S contributions only when their mechanism of binding is the same. Therefore, the major emphasis based on the results reported here is pragmatic in nature; microcalorimetry is a means of finding solution conditions that are more stabilizing for a protein, and our results indicate a positive correlation between the solution stability and increased probability of obtaining diffracting crystals of the protein. Traditional crystallization approaches use extensive screening of conditions to cover a wide range of pH, salt, and organic concentrations. This typically requires large amounts of material to adequately sample enough conditions in the sparse matrix screen to find potential crystallization conditions. Although our study is on a small number of proteins, the positive correlation between enhancement of thermodynamic parameters, mainly ∆H, and crystal improvement suggests that microscale instrumental approaches can be useful as a means of defining more optimal screening conditions. Our original hypothesis, that conditions that give rise to high-quality crystals increase the macromolecule’s Tm, appears to be substantiated by the proteins in this study. Advancement of microcalorimetric instrumentation now allows high throughput screening using sample amounts of 100 µg (MicroCal). We believe that the use of instrumental approaches such as the ones discussed here, in conjunction with detailed observation of crystallization behavior, can result in more methodical and ultimately more efficient means of determining crystallization conditions and identifying useful additives so that crystallization of macromolecules can be more rationally defined. Materials and Methods Protein Expression and Purification. All proteins were cloned, expressed, and purified in-house except for GlpO (Genzyme) and HEWL (Sigma). These two commercially obtained proteins were additionally purified through ion-affinity chromatography followed by gel-filtration before the crystallization trials. Expression and purification protocols for Npx, GK, and GlpD have been extensively described elsewhere.15–18 Generally, starter cultures were grown overnight at 37 °C and supplemented with appropriate antibiotic(s) for selection. These cultures were diluted 100-fold into fresh media and antibiotics and grown to an OD600 of ∼0.7 before protein expression was induced by addition of IPTG to a final concentration of 1 mM. The cultures were then grown for an additional 4 h. Cells were harvested by centrifugation at 6000g for 10 min at 4 °C. Cells were resuspended in 1:100 culture volume of cold Buffer A (10 mM Tris pH 7.5, 2 mM DTT) and lysed by three passages through a French pressure cell (American Instruments Co., Silver Spring, MD). Cell debris and unbroken cells were removed by centrifugation at 9000g for 45 min at 4 °C. The supernatant was then ultracentrifuged at 100000g for 90 min at 4 °C to separate soluble proteins from membrane proteins. For soluble proteins (Npx, GK), the supernatant from the ultracentrifuge spin was collected and purified through various chromatography steps. For the GlpD, a membrane protein, the pellet containing membrane proteins was resuspended with a homogenizer into a buffer supplemented with 0.2 M KCl at a volume 1:100 of the original culture. The solution was ultracentrifuged for 90
Yeh and Beale Table 2. Classes of Additives additive cadmium chloride dihydrate zinc chloride D-fructose-6-phosphate ethylene glycol glycerol anhydrous 1,6 hexanediol 2-methyl-2,4-pentanediol 1,3-propanediol hexaglycine 6-aminocaproic acid 1,8-diaminooctane 1,12-dodecanedioic acid spermidine poly L-aspartic acid 1,2,3-heptanetriol nondetergent sulfobetaine 195 (NDSB) trimethylamine HCl ammonium sulfate EDTA sodium salt 1,10-phenanthroline NAD ATP disodium salt
class ion ion organic, nonvolatile organic, nonvolatile organic, nonvolatile organic, nonvolatile organic, nonvolatile organic, nonvolatile linker linker linker linker linker linker linker chaotrope chaotrope kosmotrope chelator chelator cofactor cofactor
min at 100000g at 4 °C to remove peripheral membrane proteins from the integral membrane proteins. Detergent solubilization followed by chromatography yielded purified GlpD for crystallization.18 Crystallization Screens. Crystallization screening set-ups were done on all proteins included in this study. We screened 350–400 solutions initially to obtain preliminary crystallization conditions; these screens included Crystal Screen (Hampton Research), Cryo I and II (Emerald BioStructures), and our own in-house screens. The sparse matrix screening using Greiner plates was done with 150 µL of solution per well and sitting drops containing 1 µL protein and 1 µL of solution. The protein concentrations used for crystallization were 10 mg/mL in 50 mM Tris-HCl, pH 8 for GK, 8 mg/mL for GlpD in 50 mM TrisHCl, pH 7.5, 13 mg/mL in 50 mM HEPES pH 7.5 for GlpO, 25 mg/ mL in 50 mM acetate pH 4.5 for HEWL, and 15 mg/mL in 50 mM Tris-HCl pH 7.5 for Npx. Once one or more promising conditions were identified, we optimized the conditions through modifying concentrations of precipitants (organic and/or salts), pH, and temperature of the set-ups. For some proteins, this optimization procedure is sufficient to obtain crystals suitable for data collection; however, for the proteins described in this study, production of highest-quality crystals for diffraction required further optimization, through additive screens. Additive Screens. In-house additive screens were prepared by making stocks of the compounds in Table 2. The working concentrations, that is, the concentrations of the additive in the protein drop, are listed in Table 1. Additives were introduced either to the crystallization drop setup or by premixing them with the macromolecules, prior to the crystallization setup. The latter approach was used for proteins GK and GlpO, when the additive was found by DSC and ITC to be an effector molecule. Examples of effectors are ligands and inhibitors, which stabilize the macromolecule in a defined way by interacting directly with it, rather than with the solvent molecules. It was found that one of the most important parameters for successful additive screening is concentration of the additive. Similar to the critical importance of protein concentration in the initial crystallization screening, working at the proper concentration of an additive can be essential. Determining the working concentration of an additive is empirical and difficult to generalize without detailed characterization of a particular macromolecule. However, we typically started at a relatively high concentration of the additive (detailed below) and then adjusted its concentration higher or lower, depending on the initial observations. Starting at a high concentrations of the additive can allow gross determination of how the additive affects the crystallization, for example, whether the additive increases or decreases solubility of the macromolecule under the specific crystallization conditions. The appearance of crystalline matter different from that observed in the absence of additive (i.e., the control drop) is a positive sign that the presence of the additive might be helpful. The general starting concentrations for different classes of compounds ranged from 1 to 50
Calorimetric Crystallization Additive Evaluation mM, depending on the chemical class of the particular additive (e.g., ions, detergents, organics, etc.). Control drops, without additive, were always used as a reference point with every setup. Using the approach described above, we studied the effects of various additives on the proteins GK, GlpO, HEWL, Npx, and GlpD. These proteins were screened as described above using a 2:1:1 ratio of protein/ crystallization solution/additive in the drops, using crystallization conditions previously identified. In some cases, a nanoscale crystallization approach was used to minimize the amount of protein needed, using drop volumes of 100 nL.19 Crystallization set-ups were stored at ambient temperature and checked every 1–3 days for the first month followed by approximately once per week until about 3 months. After this time, the crystal trays were checked once every 3–4 weeks until they were no longer viable (i.e., dried out). Frequent observation, particularly initially, is important to observe nucleation, crystal growth, and other changes due to the presence of additives. Once crystals were obtained, after completion of growth (indicated by cessation of further increase in crystal size), crystals were flash-cooled and diffraction images taken. Changes in diffraction limits and mosaicity were noted (data not shown). Isothermal Titration Calorimetry. ITC measurements were carried out using a VP-ITC titration calorimeter (MicroCal, Northampton, MA) to obtain enthalpy and heat capacity changes. All protein samples for microcalorimetry were concentrated and then exhaustively dialyzed against buffer. For the ITC experiments, phosphate buffer was used for GK, GlpO, Npx, and GlpD and acetate buffer for HEWL. These buffers were chosen for their low enthaply of ionization,20 that is, to minimize artifactual heats. Exhaustive dialysis was necessary to minimize heats due to buffer mixing. Protein solutions were degassed before addition to the calorimeter cell. The additives were injected in 10 µL increments into the reaction cell (cell volumes 1.30–1.41 mL) until complete saturation was reached. A 250 µL injection syringe and 310–400 rpm stirring rate were used to give a series of 10 µL injections at 3-min intervals. Control experiments, without protein, were done to determine heats of mixing and dilutions were performed under identical conditions. The control data were then used for baseline standardization correction in the subsequent analysis. Data acquisition and subsequent nonlinear regression analysis were done in terms of a simple binding model, using Microcal ORIGIN software. Differential Scanning Calorimetry. DSC measurements were carried out using a VP-DSC differential scanning calorimeter (MicroCal) at a scan rate of 60 °C/h in buffer and in crystallization solution, with and without the additive. The proteins were dialyzed against the crystallization solution to minimize mixing heat effects caused by differences in solution composition. The protein sample and reference solutions (the dialysis solutions) were degassed for approximately 5 min before being loaded into the cell. Samples were scanned from 20 to 110 °C and then rescanned if denaturation, as indicated by precipitation, was absent. DSC scans were corrected by subtraction of data from suitable controls and concentrations were normalized to determine the Tm.
Acknowledgment. We thank T. Bergfors for insightful comments. Funding was from NIH (GM/66466) and the March of Dimes Foundation (5-FY00-564). We are grateful to the late Mary Walsh for encouragement and advice, and for her contribution in obtaining the initial DSC results.
Abbreviations and Symbols DSC GK GlpD GlpO ITC HEWL Npx Tm
differential scanning calorimetry glycerol kinase glycerol-3-phosphate dehydrogenase R-glycerophosphate oxidase isothermal titration calorimetry hen egg white lysozyme NADH peroxidase midpoint melting transition temperature
Crystal Growth & Design, Vol. 7, No. 11, 2007 2139 poly asp F-6-PO4 hexgly
poly L-aspartic acid fructose-6-phosphate hexaglycine
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CG7007045
