Article pubs.acs.org/Biomac
Engineered Mutations Change the Structure and Stability of a VirusLike Particle Jason D. Fiedler,† Cody Higginson,† Marisa L. Hovlid,† Alexander A. Kislukhin,† Alexandra Castillejos,† Florian Manzenrieder,† Melody G. Campbell,‡ Neil R. Voss,‡,§ Clinton S. Potter,‡ Bridget Carragher,‡ and M.G. Finn*,† †
Department of Chemistry and ‡The National Resource for Automated Molecular Microscopy, Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States S Supporting Information *
ABSTRACT: The single-coat protein (CP) of bacteriophage Qβ self-assembles into T = 3 icosahedral virus-like particles (VLPs), of interest for a wide range of applications. These VLPs are very stable, but identification of the specific molecular determinants of this stability is lacking. To investigate these determinants along with manipulations that confer more capabilities to our VLP material, we manipulated the CP primary structure to test the importance of various putative stabilizing interactions. Optimization of a procedure to incorporate fused CP subunits allowed for good control over the average number of covalent dimers in each VLP. We confirmed that the disulfide linkages are the most important stabilizing elements for the capsid and that acidic conditions significantly enhance the resistance of VLPs to thermal degradation. Interdimer interactions were found to be less important for VLP assembly than intradimer interactions. Finally, a single point mutation in the CP resulted in a population of smaller VLPs in three distinct structural forms.
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helical domains over adjacent β-sheet regions. In this effort, we build on the studies of Stockley and co-workers who found modest variations in thermal stabilities for variants of the MS2 VLP.35 The results of the present work identify certain important determinants for assembly and stability that can be exploited to tailor VLPs to specific applications.
INTRODUCTION Virus-like particles (VLPs) have received increasing attention for their applications in chemistry,1−3 materials science,4−6 and medicine.7−9 They can be highly stable toward extremes of temperature,10,11 pH, and variations in solvent composition12 and, thus, are friendly toward chemical modification, while retaining many advantageous characteristics of biological molecules. VLPs have well-controlled composition and structure,13 are biocompatible,14,15 and can be mutated16−18 and evolved.19,20 Among the most commonly employed VLPs have been the coat proteins (CPs) of MS2, Qβ, and PP7, all RNA phages of the Leviviridae family. Their three-dimensional structures are very similar in spite of substantial diversity in their protein sequences21−23 (see http://viperdb.scripps.edu).24 Expression in E. coli25,26 or yeast27 results in VLPs assembled from 180 copies of these CPs in high yields. The genetic malleability of Leviviridae scaffolds has been demonstrated with an exterior display of peptides and proteins28,29 or point mutations to add chemical reactivity to specific sites.18,30 Many mutations have also been made in MS2 in studies of the mechanisms of natural processes such as RNA binding31,32 and assembly.33,34 Here we report a set of rationally designed genetic mutations of the Qβ capsid protein that have differential effects on assembly and stability of the resulting VLPs. The X-ray crystal structure of the capsid21 is shown in Figure 1; its single small CP (132 amino acids) forms an interdigitated dimer with α© XXXX American Chemical Society
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EXPERIMENTAL SECTION
A. Cloning. Point mutations in the coat protein gene were made with overlap extension PCR. For each mutation (X), PCR reactions were performed with primer CP-X-R1 and primer #1 to amplify the 5′ half of the CP gene and a second PCR using primers CP-X-F1 and primer #2 to amplify the 3′ half of the CP gene (for primer sequences, see Supporting Information, Table S1). PCR products were run on agarose gels to verify size, extracted from the gel and purified. The purified DNA from both reactions were diluted and used as the template for the next PCR reaction with primers #1 and #2. Exceptions were CP-Y132W-R1 and CP-L128W-R1, which were separately amplified with primer #1, and CP-L8W−F1, CP-A1S−F1, CP-K2E-F1, and CP-K2Q-F1, which were amplified with primer #2. The products were purified as above, cut with Nco1/Xho1 restriction endonucleases, and ligated onto the similarly cut pCDF or pET28HpR vector. PCR products ligated into pET28HpR were transcribed by T7 RNA polmerase with the RNA sequence coding for the anti-Rev Received: April 16, 2012 Revised: June 30, 2012
A
dx.doi.org/10.1021/bm300590x | Biomacromolecules XXXX, XXX, XXX−XXX
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incubated with prereduced VLPs overnight at room temperature. Attachment was verified with MALDI mass spectrometry. D. Labeling with OND Electrophile. Various Qβ mutants were reduced and purified as described above. A solution of Qβ (approximately 1 mg/mL = 70 μM in protein; 100 μL) in deoxygenated 0.1 M pH 7.0 phosphate buffer was treated with dihomopropargyl 1-dansylamino-methyl-7-oxanorbornadiene-2,3-dicarboxylate,38 1 (3 mM, 3 μL), and the fluorescence increase was monitored on Varioskan Flash plate reader (λex 332 nm, λem 550 nm). Fluorescence emission intensities were corrected for differences in particle concentration and normalized to 100% for the most extensive reaction (with rC74S) before plotting in Figure 6. E. UV−Vis Measurements of Aggregation. VLPs (0.1 mg/mL = 7 μM protein = 0.4 μM particle) in PBS (pH 7.1) or MES (pH 5.5 and 6.5) buffers were interrogated with a Varian Cary 1 Bio UV− visible spectrometer in 1 cm quartz cuvettes. Oil was layered on top of the solution to minimize evaporation, and the samples were heated from 25 to 90 °C at a rate of 1 °C/min. The solution absorbance at 310 and 350 nm was measured every 0.5 °C. These wavelengths were chosen because the nonheated samples were transparent at these values, meaning that absorption caused by aggregation upon heating could be readily detected. Cloudy precipitates were clearly visible by eye at the end of each experiment. Apparent Tm values were calculated by plotting the optical density (OD) against temperature (T) and fitting the curve to eq 1, where A1 is the initial OD, A2 is the final OD, and dx is the time constant. Triplicate runs were independently analyzed and the reported data are the average values ± standard deviations.
Figure 1. Qβ capsid protein used in this study (PDB 1qbe)21 in ribbon diagram representation showing quasi-equivalent subunits in different colors with the C74−C80 disulfides in yellow.
aptamer upstream of the ribosome binding site and start codon and the natural Qβ hp downstream of the stop codon.36 The fused dimer was made by silently mutating the endogenous Nde1 (CATAG) cut site in the CP gene sequence using overlap PCR with primers #13 and #14. This mutant CP was then ligated into pCDF as above. Whole-plasmid PCR with primers #16 and #17, followed by blunt end ligation, was used to remove the stop codon and insert a Nde1 restriction site at the 3′ of the CP gene to make pCDFCP. Another PCR reaction using WT or any mutant CP gene as the template and primers #15 and #2 was then performed. Purification, digestion with Nde1/Xho1, and ligation into this similarly cut pCDFCP vector created pCDF-CP2 or pCDF-CP-(mutation X). This vector encodes tandem copies of the CP gene or the CP gene and a mutant CP gene with a five amino acid spacer (AYGGM). Ligation products were transformed into DH5α cells (BioPioneer) and plated on selective media (streptomycin for pCDF and kanamycin for pET28HpR). Colonies were picked and cultured overnight in 3 mL of SOB (Amresco) selective media followed by plasmid DNA purification. B. Expression. DNA sequence-verified plasmids were used for transformation into BL21(DE3) (Novagen) or T7 Express (NEB) cells for expression. For coexpressions, compatible plasmids (pET28 and pCDF, Novagen) were maintained under double selection. VLPs were expressed and purified, as previously described.36 All VLPs were assayed for purity and uniformity by size exclusion chromatography (SEC), dynamic light scattering (DLS), and transmission electron microscopy (TEM). The protein content of each sample was analyzed with a Bioanalyzer 2100 Protein 80 microfluidics chip (Agilent). The average number of CP and CP2 gene products per particle was determined from the ratio of the integration of the Bioanalyzer electrophoretic peaks corresponding to the proteins, correcting for their relative masses and multiplying by 180. Total protein concentration was measured using the Coomasie Plus Protein Reagent (Pierce) with bovine serum albumin as standard. C. Cysteine Reactions. WT, C74S, and C74S/C80S mutated particles (70 μM in total protein) were reduced with dithiothreitol (DTT, 3 mM) or tris(2-carboxyethyl)phosphine (TCEP, 10 mM) for 1 h in deoxygenated 0.1 M phosphate buffer (pH 7.0). The reducing agent was removed by exchanging with buffer using 100000 MWCO centrifugal filters (Millipore). The concentration of accessible protein thiol groups was assayed by Ellman’s reagent against an Nacetylcysteine standard curve.37 N-Ethylmalemide (10× excess) was
OD = A 2 +
(A1 − A 2 ) 1 + exp((T − Tm)/dx)
(1)
F. Other Spectroscopy. Mutant VLPs were assayed at roughly equivalent concentrations with a Dynapro dynamic light scattering plate reader (Wyatt Technologies, Santa Barbara, CA) in a 384-well plate with 10% laser power and 30% attenuation with light mineral oil layered on top of the solution to minimize evaporation. The instrument was set to the indicated temperature and the radius was recorded at regular intervals up to 80 min. The radius data was plotted versus time (WT Qβ VLPs have a radius of 14.5 nm). Circular dichroism was performed as described in ref 39. G. Electron Microscopy. Samples were prepared for CryoEM though preservation in vitreous ice via rapid-freeze plunging (FEI Vitrobot) onto plasma cleaned C-flat grids. Electron micrographs were acquired using a Tecnai F20 Twin transmission electron microscope operating at 120 kV using a dose of ∼12 e−/Å2 and a nominal underfocus ranging from 1.5 to 3 μm. For wild-type Qβ, 195 images were automatically collected at a nominal magnification of 80000×, corresponding to a pixel size of 0.0975 nm at the specimen level. For mini-Qβ (L35W), 1177 images were collected at 50000×, representing a pixel size of 0.164 nm. All images were recorded with a Tietz F415 4k × 4k pixel CCD camera (15 μm pixel) utilizing the Leginon data collection software.40 Experimental data were processed by the Appion software package,41 which interfaces with the Leginon database infrastructure. The contrast transfer function (CTF) for each micrograph was estimated using the ACE package.42 For Qβ, two templates created from 500 manually selected and centered particles were used to automatically pick 13,408 particles from the micrographs using a template-based particle picker.43 The particles were extracted at a box size of 400 × 400 pixels. For mini-Qβ, eight templates created using images of the sample preserved in negative stain (data not shown) were used to pick 29892 particles. The particles were extracted at a box size of 256 × 256 pixels. CTF correction was achieved by phase correction of individual particles. After pruning the stack to eliminate obvious junk and contamination, the final Qβ stack contained 11671 particles, which were then aligned and separated into 32 classes using Xmipp clustering 2D alignment.44 The final miniQβ stack contained 29,445 particles, which were aligned and separated into 64, 32, 16, and 8 classes. B
dx.doi.org/10.1021/bm300590x | Biomacromolecules XXXX, XXX, XXX−XXX
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RESULTS
The short form of the Qβ coat protein (amino acids 1−132), lacking the natural A1 extension, was used to make standard VLPs designated here as wild type (WT). Monomers of all the Leviviridae RNA phages form tightly associated noncovalent dimers.34,45 Genetically fusing the two monomers together has been done previously with PP746 and MS247 to facilitate incorporation of larger domains into a loop region or the terminus.34,48 We fused two Qβ CP monomers with a five amino acid spacer (AYGGM) to form a construct designated CP2. E. coli expression of CP2 gave VLPs that were structurally indistinguishable from wild-type particles by SEC, DLS, and TEM (data not shown). A novel capsid was created by coexpression of the CP2 gene with the wild-type CP gene on separate plasmids. The two resulting proteins self-assembled to form “hybrid” particles containing both forms of the CP29 in ratios matching the relative expression levels in the E. coli cell. This is consistent with the expectation that the noncovalent and fused dimers behave similarly in the capsid assembly process. The ratio of CP to CP2 was varied by changing the expression efficiency of the gene sequence of one of these proteins. This was done by appending the sequence coding for the Qβ RNA packaging hairpin (hp, known as TR for MS2) to the 3′ end and an aptamer sequence (coding for Rev-binding element, designated “R”) to the 5′ end of the transcribed mRNA, as was previously performed to package proteins.36,49 When the CP gene sequence was flanked by these functional RNA motifs (CPHpR), its protein product was expressed less abundantly than the coexpressed CP2 protein product, and this difference was reflected in the composition of the resulting assembled particles. The reverse construction, involving coexpression of CP and CP2HpR genes, had the expected effect, diminishing the amount of CP2 expressed and thereby incorporated relative to CP (Figure 2). The stability of many of the capsids against thermal decomposition was assessed by monitoring the optical density of the solution by UV absorption spectroscopy as a function of temperature. Disruption of the capsid structure is accompanied by aggregation and precipitation of the coat protein. As shown in Figure 3, an apparent melting temperature (Tm) was defined as the temperature at which 50% of the aggregation-related absorbance (310 nm) was reached. The direct correlation of the aggregation phenomenon with capsid protein unfolding was established by circular dichroism (CD) spectroscopy39 of WT, CP2, and hybrid particles, showing loss of secondary structure at transition temperatures very similar to those found by UV absorption. Disassembly and aggregation have also been shown by electrophoretic methods to be concurrent processes for VLPs derived from MS2 coat protein.11 Aggregation, precipitation, and loss of secondary structure was irreversible under these conditions: cooling did not allow for refolding of the protein and re-establishment of the capsid structure. Aggregation of the VLPs could also be monitored by DLS. In this assay, the temperature was held constant and the radius of the species in solution was monitored over time (Figure 4). The rapid increase in size is assumed to indicate the initiation of aggregation, which ultimately leads to protein precipitation. Although the DLS and UV-absorption aggregation methods correlated well, the latter was more convenient and robust and so was used for subsequent experiments.
Figure 2. Construction and composition of hybrid VLPs containing the fused dimeric coat protein. “% CP2 expressed” is the amount of the CP2 protein relative to the CP protein present during expression as determined by electrophoretic analysis of cell culture 4 h after induction. Tm is the decomposition temperature (°C) as defined in the text, determined by circular dichroism. All values are averages from independently expressed triplicates; error values are standard deviations of mean. Note that these errors report on the reproducibility of average values of CP2 subunits in the capsids, but do not reflect the width of the distribution of these values. In other words, samples having a value of 50 ± 1 CP2 units per capsid (56 ± 1%) could include a narrow or a broad distribution about that average value when comparing different particles in the same sample. Tm value error was 0.3 °C.
A variety of point mutants were made to test the importance of interactions identified in examination of the X-ray crystal structure of Qβ.21 In part, these studies were intended to give us a fuller picture of the relationship between the static image derived from crystallography and the behavior of the VLP as a dynamic entity in solution. Our guiding hypothesis is that an important mechanism of VLP decomposition is the exposure of hydrophobic patches, usually buried in interfaces between subunits, which both destabilizes the capsid and allows subunits to aggregate in an irreversible manner.34 Thus, anything that assists in the exposure of buried hydrophobic residues, such as disruption of favorable intersubunit interactions (disulfide linkages, salt bridges, hydrogen bonds, etc.) can contribute to this process by making it easier to separate or unfold subunits.50 Mutants containing single amino acid substitutions at ten different positions (L8, L19, L35, C80, R86, F94, F96, E104, E111, and L128) did not provide intact VLPs in spite of robust protein expression in E. coli (Table 1). Several of these mutants were also tested in a less stringent manner by making the mutation in only one of the two subunits in the CP2 gene, but these structures also failed to provide any purified VLPs. The designation of “assembly-defective” denotes only that robust expression of each modified coat protein was observed (by electrophoretic analysis of cell lysates) without isolation of any particles. It is possible that these systems were defective not in particle assembly (quaternary structure), but rather in the achievement of the proper secondary or tertiary structure of C
dx.doi.org/10.1021/bm300590x | Biomacromolecules XXXX, XXX, XXX−XXX
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Figure 3. Representative analyses of thermal stability of Qβ VLPs. (a) UV−vis absorption of WT VLPs before and after heating-induced aggregation. (b) Change in optical density at 310 nm vs temperature.
capsid subunits. No experiments were performed to test these competing hypotheses. Bacteriophages in the Leviviridae family package their genomic RNA by virtue of a strong interaction of the capsid protein with the cognate packaging hairpin (hp) stem-loop sequence.51 In our hands VLPs also encapsidate other cellular RNA in both the absence and the presence of this packaging sequence.52 To probe the role of cognate and noncognate RNA in assembly and stability, we expressed some of the mutants in the presence and absence of the natural Qβ hp (Tables 1 and 3). It was anticipated that hp could add stabilizing interactions
Figure 4. Aggregation of mutant VLPs assayed by dynamic light scattering at a constant temperature of 60 °C.
Table 3. Assembly-Competent Mutants of the Qβ VLP Expressed with the hp and Measurements of Their Expression Yield and Thermal Stability
Table 1. Assembly-Defective Mutants of the Qβ VLP In Standard Particle L8W L35A C80H F94W E111Q K13E/K16E
L19W L35V R86W F96H L128W
C74H
F94H
C74H-hp F94L-hp
L35G Y62E F94H F96W C74S/V108I
L35H C74H F94L E104Q K2E
In CP2 Particle F96H With Hairpin RNA E111Q-hp F96H-hp C74S/V108I-hp F96H/V108I-hp
E111Q F94H-hp
entry
mutation(s)
yielda (mg/L)
Tmb (°C)
1 2 3 4 5 6 7 8
C74S-hp C74S C80S-hp Q65H-hp D91N-hp Q65H D91N-hp Q65H C74S-hp D91N C74S-hp Q65H D91N C74S-hp
20−80 >80 >80 80
