Natural Promoters of Calcium Oxalate Monohydrate Crystallization. Supporting Information

Transcription

1 Natural Promoters of Calcium Oxalate Monohydrate Crystallization Sahar Farmanesh 1, Jihae Chung 1, Ricardo D. Sosa 1, Jun Ha Kwak 2, Pankaj Karande 2, and Jeffrey D. Rimer 1* 1 University of Houston, Department of Chemical and Biomolecular Engineering, Houston, TX Rensselaer Polytechnic Institute, Department of Chemical and Biological Engineering, Troy, NY *Correspondence sent to jrimer@central.uh.edu Supporting Information Table of Contents Page S1. Experimental methods S2 S2. Protein amino acid sequence and secondary structure S3 S3. COM bulk crystallization S4 S4. Correction of COM growth solution ph S5 S5. ISE measurements of COM growth in the presence of amino acids S8 S6. In situ atomic force microscopy S9 S7. Supporting references S10 List of Figures, Tables, and Movies Figure S1: Protein crystal structure and amino acid sequence of lysozyme from chicken egg white Figure S2: Protein crystal structure and amino acid sequence of lactoferrin from bovine milk Figure S3: Circular dichroism spectra of lysozyme and lactoferrin at different temperatures Figure S4. Optical micrographs of COM crystals in the presence and absence of proteins Figure S5. Changes in COM bulk crystal dimensions with increasing lactoferrin concentration Figure S6. Effect of lysine and arginine on COM crystal aspect ratio Figure S7. Analysis of ph correction for COM growth in the presence of basic amino acids Figure S8. COM growth promotion in the absence and presence of HEPES buffer Figure S9. ISE measurements of COM growth in the presence of basic and acidic amino acids Figure S10. Deflection mode AFM image of a growing COM (010) hillock in 5 g/ml lysozyme. Table S1. Measurements of COM growth solution ph with the addition of modifiers Movie S1. In situ AFM measurement of COM (010) surface growth in the absence/presence of lysozyme. S1

2 S1. Experimental methods Materials. The following reagents for calcium oxalate monohydrate crystallization were purchased from Sigma Aldrich (St. Louis, MO) and used without further purification: calcium chloride dihydrate (ACS Reagent, 99+%), sodium oxalate (Na 2 C 2 O 4, >99%), hydrochloric acid (HCl, ACS Reagent, 37%), sodium hydroxide (NaOH, 98%), lactoferrin from bovine milk ( 85%), lysozyme from chicken egg white (lyophilized powder, 90%), human transferrin (bioreagent, 98%), L-aspartic acid (Asp, C 4 H 7 NO 4, 98%), L-glutamic acid (Glu, C 5 H 9 NO 4, 99%), L-lysine (Lys, C 6 H 14 N 2 O 2, 90%), L-arginine (Arg, C 6 H 14 N 4 O 2, 98%), poly-l-lysine hydrobromide (poly-lys, 4-15 kda), poly-dl-aspartic acid sodium salt (2-11 kda), poly-l-glutamic acid sodium salt (3-15 kda), and poly-l-arginine hydrochloride (poly-arg, 5-15 kda). Sodium chloride (99.9% ultrapure bioreagent) and HEPES sodium salt (Ultrapure Bioreagent, C₈H₁₇N₂NaO₄S, 99.6%) were purchased from JT Baker (Center Valley, PA). The following reagents for peptide synthesis were purchased from AGTC Bioproducts (Wilmington, MA) and used without further purification: O-(benzotriazol-1-yl)-N,N,N,N -tetramethyluronium (coupling reagent), piperidine (99.5%), dichloromethane (DCM, 99.9%), N,N-dimethylformamide (DMF, 99.8%), and methyl tert-butyl ether (MTBE, 99.5%). The following reagents were purchased from Sigma Aldrich and used without further purification: acetonitrile (CAN, 99%), acetic acid (99.7%), triisopropylsilane (TIPS, 99%), N-methylmorpholine (99%), 1-methyl-2-pyrrolidinone (99.5%), and 2,2 - (ethylenedioxy)diethanethiol (95%). Trifluoroacetic acid (99.5%) was purchased from Alfa Aesar (Ward Hill, MA). The following amino acids purchased from 21st Century Biochemicals (Marlborough, MA) exhibit 99% purity: Fmoc-L-Ala-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L- Asp(OtBu)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Ile-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Met-OH, Fmoc-L-Phe-OH, Fmoc-L-Pro-OH, Fmoc-L-Ser(OtBu)-OH, Fmoc-L-Thr(OtBu)-OH, Fmoc-L-Trp(Boc)- OH, Fmoc-L-Tyr(OtBu)-OH, and Fmoc-L-Val-OH. Calcium oxalate monohydrate (COM) crystallization. COM crystals were prepared according to a previously reported procedure. 1 In brief, we prepared a solution with molar composition 0.7 mm CaCl 2 : 0.7 mm Na 2 C 2 O 4 : 150 mm NaCl by first dissolving NaCl in deionized water, followed by the addition of CaCl 2 stock solution (10 mm). The sample was placed in an incubator at 60 ºC for one hour prior to the addition of Na 2 C 2 O 4 stock solution (10 mm) dropwise while stirring. The sample was returned to the incubator for three days. Crystals prepared by this method are referred to as the control. The procedure for preparing COM crystals in the presence of growth modifiers was the same as the control, with the addition of an appropriate amount of modifier to the solution prior to the addition of Na 2 C 2 O 4. A clean glass slide was placed at the bottom of the synthesis vial to collect crystals for microscopy. After crystallization was complete, the glass slides were removed from the mother liquor, gently washed with deionized water to remove the residual supernatant, and dried in air prior to analysis. For zeta potential studies, control crystals were transferred from the glass slide to an aqueous solution by scraping the slide to produce a powder of COM seeds. For AFM studies, crystals were transferred to AFM sample disks by gently pressing the glass microscope slide on metal disks (Ted Pella) coated with partially-cured epoxy. Peptide synthesis. Peptides were synthesized using an automated peptide synthesizer (Multipep RS, Intavis Inc., Germany), which can synthesize 384 peptides in four 96-well plates. Fmoc solid-phase peptide synthesis chemistry was used to synthesize the peptides from their C-termini to N-termini on a TentaGel amide resin (Intavis Inc.). The resin has a loading capacity of 0.25 mmol/g and yields peptides with amidated C-termini. Pre-synthesis, the requisite amount of resin was weighed out and transferred to each well of a 96-well plate. The resin was swollen by a 2:1 N,N-dimethylformamide: dichloromethane (DCM) solution. Post-synthesis, the resin was washed with DCM and peptides were cleaved off with a water: triisopropylsilane: trifluoroacetic acid (TFA) cocktail. The peptides were then precipitated in icecold methyl tert-butyl ether (MTBE). The precipitate mixture was centrifuged to perform a second MTBE S2

3 wash for removal of TFA. After another round of centrifugation, the supernatant was decanted and the peptides were air-dried. The dry peptides were dissolved in 1:5 acetonitrile:water, flash frozen in liquid nitrogen, and lyophilized. The lyophilized peptides were stored at -20 C until needed. S2. Protein amino acid sequence and secondary structure The cationic proteins examined in this study are rich in basic amino acids L-arginine (Arg, R), L- lysine (Lys, K), and L-histidine (His, H). Here we show the reported structures and amino acid sequences of lysozyme and lactoferrin. The amino acids with acidic side chains (D and E) and basic side chains (R, K, and H) in the primary amino acid sequences are highlighted in red and green, respectively. Figure S1. Structure and amino acid sequence of lysozyme from chicken egg white. The 3D image was generated from the reported crystal structure 2 using the program Visual Molecular Dynamics. Amino acids with basic and acidic side chains are highlighted in green and red, respectively. Figure S2. Structure and amino acid sequence of lactoferrin from bovine milk. The 3D image was generated from the reported crystal structure 3 using the program Visual Molecular Dynamics. Amino acids with basic and acidic side chains are highlighted in green and red, respectively. S3

4 Circular dichroism (CD) spectra were obtained for lactoferrin and lysozyme in aqueous solution. As shown in Figure S3, these proteins contain combinations of secondary conformations that qualitatively agree with the secondary structures of their crystal structures. The CD spectra of lactoferrin and lysozyme taken at 25 C (i.e., ISE data) and 60 C (i.e., bulk crystallization studies) are nearly identical, which reveals that changes in secondary structure are negligible. CD confirmed that both of these proteins are thermally stable (i.e., no unfolding) within the temperature range of our experiments. Figure S3. Circular dichroism spectra of aqueous solutions containing 150 mm NaCl, 0.7 mm CaCl 2, and 25 g/ml of either (A) lactoferrin or (B) lysozyme. CD spectra were collected at 25 o C (dashed lines) and 60 o C (solid lines), which correspond to the conditions used for ISE measurements and bulk crystallization studies, respectively. S3. COM bulk crystallization The size of COM crystals was not significantly affected by the presence of either lactoferrin or lysozyme. Figure S4 compares optical micrographs of a representative control sample (panel A) with bulk crystals prepared in the presence of 20 g/ml lactoferrin (panel B) and lysozyme (panel C). The measurements of bulk crystal aspect ratio (c/b ratio) and [100] thickness for COM crystals prepared in growth solutions of varying lactoferrin concentrations is shown in Figure S5. Analogous to lysozyme (Figure 4A), there is a small increase in aspect ratio (Figure S5A) that occurs at low concentration of modifier (ca. 1 g/ml). The changes in [100] dimension for lactoferrin (Figure S5B) are statistically similar to the control, which suggests there is little, if any, modification in COM crystal thickness compared to what was observed for lysozyme (Figure 4B). S4

5 Figure S4. Optical micrographs of COM crystals in the presence of (A) no additive (control), (B) 20 µg/ml lactoferrin, and (C) 20 µg/ml lysozyme. The scale bar is identical for all three panels. Figure S5. Changes in COM bulk crystal dimensions with increasing lactoferrin concentration. (A) Aspect ratio of the (100) basal plane measured as the ratio of [001] length to [010] width. (B) Measurements of thickness along the [100] direction. Error bars equal two standard deviations and solid lines are interpolations of the data (symbols). S4. Correction of COM growth solution ph The addition of select modifiers to COM growth solutions resulted in a change in ph relative to the control (6.11 ± 0.05). Measurements of ph at different modifier concentrations are provided in Table S1. The basic amino acids (Lys and Arg) increased the ph, while the acidic amino acids (Asp and Glu) reduced the ph. The addition of polyamino acids (poly-lys and poly-arg) and cationic proteins (lactoferrin and lysozyme) had no appreciable effect on growth solution ph. Calcium oxalate supersaturation can vary with solution ph; therefore, changes in ph can potentially lead to differences in the rate of COM crystal growth. To this end, we performed bulk COM crystallization studies in the presence of amino acid (5-30 µg/ml) without ph correction, The COM crystal habit was assessed from three separate batches using optical microscopy to measure the crystal aspect ratio within the (100) plane. We also prepared COM growth solutions at different ph by adding an appropriate quantity of either NaOH or HCl. Here we show data for Lys (Figure S6A) and Arg (Figure S6B) where the addition of modifier increased the ph from 6 to 10. As such, solutions with similar ph (in S5

6 the absence of the amino acid) were prepared by the addition of NaOH stock solution (Figure S6, open diamonds). Comparison of the aspect ratio reveals very little difference between samples prepared with basic amino acids and those altered by the addition of NaOH (modifier-free solutions). We also examined the effect of poly-arg and poly-lys on COM crystal aspect ratio at a single concentration, 20 µg/ml polymer. COM crystals prepared in the presence of poly-lys exhibited an aspect ratio of 2.7 ± 0.1, which is comparable to the control (2.8 ± 0.1). Similar measurements with poly-arg revealed a slight increase in the aspect ratio (3.2 ± 0.2). Table S1. Initial ph of COM growth solutions in the presence of growth modifiers Growth modifiers a Modifier concentration (µg/ml) b Amino acids Lysine 6.11 ± ± ± ± ± ± 0.1 Arginine 6.11 ± ± ± ± ± ± 0.09 Aspartic acid 6.11 ± ± ± ± ± ± 0.03 Glutamic acid 6.11 ± ± ± ± ± ± 0.02 Polyamino acids Poly-lysine 6.11 ± ± Poly-arginine 6.11 ± ± Poly-glutamate 6.11 ± ± Poly-aspartate 6.11 ± ± Proteins Lactoferrin 6.11 ± ± ± ± ± ± 0.2 Lysozyme 6.11 ± ± ± ± ± ± 0.1 a COM crystallization was performed in aqueous solutions containing 0.5 mm CaC 2 O 2 and 150 mm NaCl b Solution ph was measured at room temperature prior to COM crystallization Figure S6. Effect of L-lysine (A) and L-arginine (B) on COM crystal aspect ratio measured along the [001] and [010] directions of the basal surface. Open diamond symbols correspond to solutions prepared with the addition of NaOH to a control solution (in the absence of modifier). Closed circle symbols are measurements of COM crystals that were prepared in the presence of amino acid (see Table S1 for modifier concentrations). Data points are the average of 3 or more separate batches, and error bars equal two standard deviations. The solid line is a linear regression of data for modifier-free COM growth solutions (R 2 = 0.97). S6

7 All ISE measurements reported in the manuscript were performed at constant ph. The correction of growth solution ph was only required for studies using amino acids as modifiers. We corrected for the ph change in growth solution by adding either HCl (for basic amino acids) or NaOH (for acidic amino acids). In Figure S7 we provide an example of basic amino acids, showing a comparison between solutions with and without ph correction. These studies reveal that growth solutions with increasing Arg and Lys concentration and ph correction have no observable effect on the rate of COM crystallization. Figure S7. Comparison of the relative growth rate (RGR, Eq. 1) of COM crystallization with the addition of amino acids Lys and Arg without ph correction (open symbols) and with ph correction (closed symbols). The ph values of the latter are provided in Table S1. Growth solutions with ph correction were prepared by adding an appropriate amount of NaOH to maintain the solution at ph 6.1. Data are the average of 3 separate measurements and error bars equal two standard deviations. Dashed lines are interpolations to facilitate better visualization of the trends. The addition of lysozyme or lactoferrin to COM growth solutions had a minor effect on the ph (see Table S1). As such, there were no corrections made to adjust solution ph. To ensure that COM growth promotion in the presence of cationic proteins was not affected by the small change in ph, we examined COM growth using ISE with varying concentrations of lysozyme in the presence of 0.1 mm HEPES buffer. The HEPES buffer maintained the growth solution at constant ph (ca. 7.8). As shown in Figure S8, we observed an accelerated rate of COM growth (analogous to the trend in Figure 3). S7

8 Figure S8. Relative growth rate (RGR, Eq. 1) of COM crystallization in the presence of lysozyme (circles) and with lysozyme and HEPES buffer (squares). Data are the average of 3 separate measurements and error bars equal two standard deviations. The solid line is interpolated for improved visualization of the trend. The dashed line is drawn along RGR = 1, which is equal to the control (open diamond symbol). S4. ISE measurements of COM growth in the presence of amino acids Here we present a systematic analysis of COM growth in the presence of basic and acidic amino acids over a broad range of modifier concentration (Figure S9). The results of this study reveal that the basic amino acids Lys and Arg have a negligible effect on the RGR of COM crystallization. There is a monotonic decrease in the RGR of COM with increasing concentration of acidic amino acid (Glu and Asp). The addition of Asp to COM growth solutions results in greater inhibition of COM crystallization compared to Glu; however, within experimental error their RGR values are similar. S8

9 Figure S9. Relative growth rate (RGR, Eq. 1) of COM crystallization at ph 6.1 in the presence of acidic and basic amino acids. Data are the average of 3 separate ISE measurements and error bars equal two standard deviations. Solid lines are interpolations and the dashed line represents growth rates equal to the control (RGR = 1). S6. In situ atomic force microscopy Figure 10. In situ AFM measurements of hillock growth on a COM (010) surface in a supersaturated CaOx solution (S = 3.8). This deflection mode image is a snapshot of hillock growth in the presence of 5 µg/ml lysozyme. S9

10 Movie S1. In situ AFM measurement of COM (010) surface growth in a solution of composition 0.18 mm CaCl 2 and 0.18 mm Na 2 C 2 O 2 at 25 C. Growth solution is continuously supplied to the AFM sample cell using an in-line flow configuration at a combined feed rate of 0.2 ml/min. The movie was taken over the course of 30 min by continuously scanning a 2.4 x 2.4 m 2 area of the crystal surface in contact mode. The initial portion of the movie pertains to growth in the absence of modifier (i.e. control). After 693 seconds, lysozyme is introduced into the growth solution (as indicated in the upper right hand corner of the image). Upon addition of lysozyme, there is a noticeable increase in the rate of step advancement. S7. Supporting references 1. Farmanesh, S., J. Chung, D. Chandra, R.D. Sosa, P. Karande, and J.D. Rimer, High throughput platform for design and screening of peptides as inhibitors of calcium oxalate monohydrate crystallization. Journal of Crystal Growth, : p Evans, G. and G. Bricogne, Triiodide derivatization and combinatorial counter ion replacement: two methods for enhancing phasing signal using laboratory Cu K alpha X ray equipment. Acta Crystallographica Section D Biological Crystallography, : p Karthikeyan, S., S. Yadav, M. Paramasivam, A. Srinivasan, and T.P. Singh, Structure of buffalo lactoferrin at 3.3 angstrom resolution at 277 K. Acta Crystallographica Section D Biological Crystallography, : p S10