Analysis and Control of Protein Crystallization Using Short Peptide

May 8, 2015 - Analysis and Control of Protein Crystallization Using Short Peptide Tags That Change Solubility without Affecting Structure, Thermal Sta...
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Analysis and Control of Protein Crystallization Using Short Peptide Tags That Change Solubility without Affecting Structure, Thermal Stability, and Function Mohammad Monirul Islam,†,∥ Shigeyoshi Nakamura,§ Keiichi Noguchi,‡ Masafumi Yohda,† Shun-ichi Kidokoro,§ and Yutaka Kuroda*,† †

Department of Biotechnology and Life Science, and ‡Instrumentation Analysis Center, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan § Department of Bioengineering, Nagaoka University of Technology, Niigata 940-2188, Japan ∥ Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong 4331, Bangladesh S Supporting Information *

ABSTRACT: Short tags attached to recombinant proteins are emerging as an important tool for biochemical research. Here, we report the effects of 10 Solubilization Controlling Peptide (SCP) tags on crystallization behavior of a bovine pancreatic trypsin inhibitor (BPTI) variant. The tags did not affect structure, thermodynamics, and activities of BPTI. Moreover, eight of the tagged variants crystallized under the same condition, and six of them diffracted at high resolution. All variants with long-term solubility (LS) between 1 and 6 mg/ mL produced crystals that diffracted well, while variants with LS < 1 and >6 mg/mL did not crystallize, produced poorly diffracting crystals, or crystallized under a different condition. The only exception was a glutamine tagged variant, which had an LS of 5 mg/mL, but fast aggregation kinetics, and produced mere needles unsuitable for further analysis. Crystal structures indicated that most tags were largely invisible, indicating high flexibility, without having interactions with nearby residues. Therefore, short peptides, introducing a mere 5−7 residue elongation, could provide a useful technology for tuning protein solubility without affecting its other properties and hence for overcoming problems associated with excessively low or high solubility, such as in crystallization.

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The production of crystals is a prerequisite first step for determining high resolution structures of biomolecules, but the biophysical mechanism of crystallization remains to be fully understood, and the success rate of protein crystallization projects is surprisingly low, even for well folded proteins with moderate sizes. Though several factors can affect protein crystallization, solubility is a critical, and relatively uncharacterized one.10−13 A few rationally designed point mutations intended to improve a protein’s crystallization behavior by altering its intrinsic properties, such as crystal contacts, solubility, or local flexibility, have been occasionally reported.14−18 The conclusions were somewhat mixed, especially those concerning the role of protein solubility as their effects were entangled with those of other factors.19 Here, we report a systematic investigation of the influence of short solubility-controlling peptide tags on the structure, thermodynamics, and crystallization behavior of our model protein, BPTI-19A, for 10 amino acid types [acidic (D and E), basic (K and R), polar (S, N, Q, P, and H), and hydrophobic

INTRODUCTION

Manipulation of protein solubility is becoming a central issue in several aspects of biotechnological and pharmaceutical usage of recombinant proteins.1 Empirical attempts to control protein solubility by amino acid substitution have been reported,2−4 but the application range of such attempts remains limited as one cannot yet control protein solubility without affecting other properties. Short peptide tags have recently been proposed for improving protein solubility and purification.5,6 They are significantly smaller than traditional solubilizing protein tags, which constitutes a major advantage.7 Furthermore, they are anticipated to function as unfolded “tails”, independent from the proteins to which they are attached.7 This makes short peptide tags particularly attractive as they confer the potential for manipulating a protein’s solubility without affecting its structural or biochemical properties. For example, short fiveresidue Lys and Arg tags increased the solubility of a bovine pancreatic trypsin inhibitor (BPTI) variant by over 4-fold without altering either its NMR spectrum or activity,8 and other five-residue peptide tags could be used to control protein solubility in a pH-dependent manner.9 © XXXX American Chemical Society

Received: January 5, 2015 Revised: April 21, 2015

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DOI: 10.1021/acs.cgd.5b00010 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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Figure 1. Effects of the SCP tags on protein structures. Top: Superimposed backbone structures of untagged BPTI-19A (black lines) and the tagged variants (gray lines). The overall backbone RMS deviations were 0.2−0.3 Å, indicating the retention of the same natively folded structures in all variants. Bottom: Sequences of the untagged and tagged BPTIs are shown with underlined SCP tag residues. Two Gly residues were added as a spacer (C2G), followed by five amino acid residues (C5X) of the same type. Circular Dichroism (CD) Measurements. The variant’s thermal stabilities were monitored using the CD signal at 220 nm measured using a 1 cm cuvette on a Jasco J-820 spectropolarimeter, as described earlier.23 Protein samples were prepared at 10 μM concentration in 20 mM acetate (pH 4.7) and 20 mM Tris-HCl (pH8.7) in the presence of 0.0, 0.5, 0.75, 1.0, and 1.5 M ammonium sulfate. The temperature was raised by 1 °C/min from 5 to 75 °C. Crystallization. Protein stock solutions with 10−15 mg/mL concentration were prepared in 15 mM Tris-HCl, pH 7.0. A 1 μL drop of the stock was mixed with 1 μL of crystallization solution, and set for hanging drop vapor diffusion at 20 °C as reported earlier for BPTI-19A.24 Crystals of C5D, C5H, C5S, C5P, C5Q and C5E were developed under the same condition as for BPTI-19A and by using initial protein concentrations between 5 and 10 mg/mL in 20−30% PEG4000, 0.2 M lithium sulfate, and 0.1 M Tris-HCl pH8.5. The C5R and C5K did not form crystals large enough for diffraction under this condition nor under any of the 98 Hampton conditions. C3R formed large crystals under the above crystallization conditions, but at an initial protein concentration of 20−30 mg/mL. Similar to C5K, C3K formed only needles, and we eventually crystallized C3K using a heteroseeding strategy where microseeds of C3R were used. In the heteroseeding strategy, microseeds were collected from an initial crystallization drop containing over 20 mg/mL of C3R and seeded onto a drop containing 5−10 mg/mL of C3K with all other conditions remaining the same as in the initial C3R drop. Finally, C5N was crystallized in 4 M sodium formate using the Hampton screening kit at 20 °C using the hanging drop vapor diffusion technique (Figure 3). Structure Determination. The X-ray diffraction data were recorded from single crystals using a synchrotron beamline at the Photon Factory (PF, Tsukuba, Japan). The data were processed with the HKL2000 program package, using DENZO for the integration and SCALEPACK for the merging and statistical analysis of the diffraction intensities.25 The structures of all variants were determined by

(I)]. None of the tags affected the trypsin inhibitory activity, thermodynamic properties, or structure of BPTI-19A, whereas the crystallization properties were clearly influenced by the changes in long-term solubility (LS) and precipitation speed generated by the tags.19 The results show that short solubility controlling peptide tags could offer a promising method for analyzing and controlling the effect of solubility on protein crystallization, by isolating it from interfering factors.

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METHODS

Mutant Design, Expression, and Purification. All BPTI variants were constructed using a pMMHa vector as previously described.21 DNA sequences corresponding to two Gly residues (as a spacer) and the SCP tags were added to the C-terminus of the model BPTI using QuikChange site directed mutagenesis, and constructs were confirmed by DNA sequencing (Figure 1). All poly amino acid tagged variants were overexpressed in an Escherichia coli JM109(DE3)pLysS cell line and purified by reverse phase HPLC. The protein identities were confirmed by ESI-TOF mass spectroscopy, and purified proteins were preserved at −30 °C as lyophilized powder. DSC Measurement and Data Analysis. Samples for differential scanning calorimetry (DSC) were prepared by dissolving lyophilized proteins in 20 mM sodium acetate buffer (pH 4.0, pH4.7 and pH5.5), followed by extensive dialysis for 18 h at 4 °C as previously described.20 DSC measurements were performed using a VP-DSC (Microcal, USA) in the temperature range of 5−80 °C at a scan rate of 1 °C/min (Figure 2; Table 1). The apparent heat capacity curves were analyzed with a two-state model using a nonlinear least-squares fitting method and a linear temperature dependence of the heat capacity for the native and denatured states.21,22 B

DOI: 10.1021/acs.cgd.5b00010 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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and the peptide tags. We designed and constructed 10 BPTI19A variants tagged at their C-terminus with 10 representative types of SCP tags (polar: N, Q, and S; hydrophobic: I; positively charged; R and K; negatively charged: D and E, along with P and H).9,19 Two Gly residues were added as a spacer between BPTI and the tags (Figure 1). The variants were named according to the number and type of the amino acid added. For example, C5D stands for BPTI-19A with five Asp attached to its C-terminus (plus two Gly residues as a spacer between BPTI and the C5D tag). Additionally, we also prepared a reference variant, C2G, which has only two glycine residues and no additional residues (Figure 1). The BPTI variants’ solubility was measured as the maximum protein concentration of the supernatant in the presence of 1.5 M ammonium sulfate at pH4.7 and pH7.7.9,19 The C5K and C5N increased protein solubility over 9-fold at pH 7.7, while the C5H, C5Q, C5S and C5R increased protein solubility 2.9 to 5 fold.9 At lower pH, the negatively charged C5D and C5E merely affected protein solubility (1−2 fold solubilization), while at higher pH they introduced over 5−7 fold solubilization. The very hydrophobic Ile markedly decreased BPTI’s solubility,9 and noteworthy Pro, the most soluble amino acid with a solubility of 1600 g/L,32 barely affected BPTI’s solubility. Effects of the SCP Tag on Thermodynamic Properties. The effects of SCP tags on protein stability were assessed using DSC at pH 4.0, 4.7, and 5.5 (Figure 2a,b). All of the variants exhibited a reversible two-state thermal folding-unfolding process, except C5D that aggregated at pH5.5. The melting temperatures (Tm) of the poly amino acid tagged variants were very similar to that of the C2G variant (±0.5−1 °C), which was 25.00 5.15 ± 0.25 17.66 ± ∼0.0

0.74 ± 0.03 0.80 ± 0.02 0.27 ± 0.01 0.64 ± 0.14 1.36 ± 0.27 3.30 ± 0.50 12.1 ± 0.65 3.15 ± 0.10 3.08 ± 0.50 3.82 ± 0.61 >25.00 1.09 ± 0.07 12.14 ± 0.69 ∼0.0

0.023/1.29 0.029/1.32 0.012/1.42 0.014/1.04 0.063/1.43 0.057/0.94 ∼0.0/0.00 0.237/1.63 0.131/1.63 0.117/1.23 ∼0.0/0.00 0.08/1.64 0.115/0.65 −/N.D.

rod N.D.i rod rod/plate rod cubic cubic rod/plate rod/plate needle needle needle hexagonal no crystal

solved N.D.i solved solved solved solved solved not solved not solved not solved not solved not solved solved not solved

100 100 100 100 100 100 100 100 100 100 100 100 100 100

60.18 58.56 57.87 58.98 58.72 N.D. N.D. 60.04 53.03 57.49 54.84 56.09 58.10 58.78

c

d

e

a

Thermodynamic parameters determined by DSC. N.D. stands for not determined. bProtein solubility defined as the protein concentration in the supernatant of supersaturated protein solution measured in the presence of 1.5 M ammonium sulfate.9,19 To ensure reliability and reproducibility of the solubility parameters, all solubility values were measured at least three times, in different working days and with different sets of reagent. The experimental errors (standard deviations) were determined using more than three independent solubility measurements. cTS stands for transient solubility, and it is determined as the amount of protein remaining in solution after 20 min incubation. dLS stands for long-term solubility, and it is determined as the amount of protein remained in solution after 48 h incubation at pH 7.7, as described previously.19 Solubility parameters determined and/or reconfirmed in this study are marked by asterisks. The unmarked values are adapted from ref 19. eThe fraction of protein precipitated per hour of incubation at pH 7.7 calculated from 48 h incubation values. fEffect of the SCP tags on crystallization of BPTI-19A to which they were attached. gTrypsin inhibitory activity measured by monitoring the hydrolysis of N-benzoyl-D,L-arginine-p-nitroanilide at equimolar concentrations of BPTIs and trypsin.40 hC5I precipitated almost completely under the solubility measurement conditions. iCrystallization and structure determination were not attempted for the C2G variant.

Figure 3. Pictures of the crystals. The variant identities are mentioned on the top of each panel. Crystals of untagged BPTI-19A and its tagged variants, except for C5N, were all developed using the hanging drop technique at 20 °C, 20−30% PEG-4000, 0.2 M lithium sulfate, 0.1 M Tris-HCl, pH8.5. Crystals of C5N were grown under the same conditions but with the addition of 4 M sodium formate. In all cases crystals developed within 24−36 h using a hanging drop setting.

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DOI: 10.1021/acs.cgd.5b00010 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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properties per se, and we could crystallize the untagged BPTI19A and its C5D, C5H, C5S, C5P, C5Q, and C5E variants under the same crystallization conditions. Namely, the crystals were grown at 20 °C using the hanging drop vapor diffusion technique under 20−30% PEG4000, 0.2 M lithium sulfate, and 0.1 M Tris-HCl, pH 8.5 at an initial protein concentration of 5−10 mg/mL. The C5N crystals were developed under the same conditions but in the presence of 4.0 M sodium formate (Figure 3). We thus speculate that excessive solubility might render the protein difficult to crystallize, and the high sodium formate concentration could have promoted the crystallization of C5N by decreasing its high solubility. On the other hand, we did not obtain crystals of reasonable sizes for the highly soluble C5R and C5K variants, which formed small needles, even by raising the initial protein concentrations to 40−50 mg/mL or using 4.0 M sodium formate, which was successful for C5N. We thus produced crystals of the less soluble C3R and C3K variants, which had 3.3 and 6.5 fold solubilization (Table 1) and crystallized more easily than the respective five-residue tagged variants. The C3R formed large crystals when the initial concentration was raised to 20−30 mg/mL, whereas C3K formed needles similar to C5K. Eventually, we developed large C3K crystals using a heteroseeding strategy where microseeds from the C3R variants were used as seeds for the C3K variant. The difficulties in getting the large crystals of the C5R and C5K variants could originate from their extreme solubility, but also from strong local repulsive forces arising from their positively charged tags. The C5I was incompatible for crystallization, as precipitation readily appeared in the crystallization drop, under most conditions even at very low initial protein concentration. Similar observations were also made with the C3I, while the C1I remained almost the same as those observed in the case of reference BPTI-19A. For the purpose of discussion, we compared the above crystallization properties with the variant’s transient solubility (TS) as well as ong term solubility (LS), where TS is the protein solubility measured after a 20 min centrifugation, which is a condition often used for preparing crystallization stock solutions, and LS is the protein’s concentration in the supernatant of a saturated protein solution measured after 48 h of incubation.20 Mutants that did not crystallize or yielded poor crystals were located in a TS/LS area where TS is high and LS low (Figure 4a). Similarly, mutants that produced well behaved crystals were located around a dotted line drawn in the TS/LS diagram. All crystals were produced under a similar condition but for C3K which was crystallized using a heteroseeding strategy and C5N which was crystallized in the presence of sodium formate. The crystallization behaviors correlated well with TS and LS measured at pH7.7 (Figure 4a), but not with TS and LS measured at pH 4.7 (Figure 4b; Supplementary Figure S2, Supporting Information) probably because the crystallization pH was 8.5. Finally, we discuss the crystallization behaviors with regard to the precipitation speed and the ratio of TS to LS. Variants with a slow precipitation of