Nanoparticle Fabrication on Bacterial Microcompartment Surface for

Bacterial microcompartments (MCPs) are polyhedral organelles containing an enzyme cluster wrapped inside a protein shell and carry out specific enzyme...
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Nanoparticle Fabrication on Bacterial Microcompartment Surface for the Development of Hybrid Enzyme-Inorganic Catalyst Naimat Kalim Bari, Gaurav Kumar, Aashish Bhatt, Jagadish Hazra, Ankush Garg, Md. Ehesan Ali, and Sharmistha Sinha ACS Catal., Just Accepted Manuscript • Publication Date (Web): 24 Jul 2018 Downloaded from http://pubs.acs.org on July 25, 2018

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ACS Catalysis

Nanoparticle Fabrication on Bacterial Microcompartment Surface for the Development of Hybrid Enzyme-Inorganic Catalyst Naimat Kalim Bari1, Gaurav Kumar1, Aashish Bhatt1, Jagadish Prasad Hazra2, Ankush Garg1, Md. Ehesan Ali1, and Sharmistha Sinha*1 1

Institute of Nano Science and Technology (INST), Phase-10, Sector-64, Mohali, Punjab, India, 160062. Department of Chemical Sciences, Indian Institute of Science Education and Research (IISER), Mohali, Knowledge City, Sector 81, Mohali, Punjab, India, 140306.

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ABSTRACT: Bacterial microcompartments (MCPs) are polyhedral organelles containing an enzyme cluster wrapped inside a protein shell and carry out specific enzyme reactions in bacteria. These organelles have been explored meticulously using genetic, structural and biochemical tools, however, their application in material science has not been explored much. In this study, we have used the external shell surface of MCP as a scaffold for the fabrication of gold nanoparticles displayed in 3D. This resulted in the formation of a protein scaffolded gold nanoparticle shell enclosing an active enzyme cluster. The surface scaffolded gold nanoparticles demonstrated standard catalysis while the internal enzyme cluster of the MCPs demonstrated no loss in activity due to this fabrication. Under ambient conditions for in vitro inorganic reaction, the shell proteins sturdily maintain the barrier between the luminal enzyme and the surface inorganic catalysts and preserve the functionality of the core enzyme cluster. The MCP-AuNPs hybrid catalysts inspire a pristine class of resources that can be used in a chemical reaction condition without perturbing core biological environment for enzymatic activity. This system provides insight for fabricating uniform size nanoparticles in 3D for the development of orthogonal hybrid catalytic material.

KEYWORDS: Bacterial microcompartments, Hybrid Catalysts, Proteins, Enzymes, Nanoparticle scaffolding, Introduction Bacterial microcompartments (MCPs) are an exclusive example of sophisticated all protein prokaryotic molecular reactors that conditionally optimize specific metabolic processes with the unique spatial organization of structural proteins and enzymes in vivo.1-8 The common paradigms of MCPs in nature are the carboxysomes that play a central role in the carbon fixation and the metabolosomes that help a bacterium to metabolize certain substrates having volatile and toxic intermediates. The prevailing understanding of the structure of this class of prokaryotic organelles is that extended layers of BMC domain proteins self-assemble to make up a polyhedral structure which can be a regular icosahedron as in carboxysomes or irregular polyhedral as in 1,2-Pdu MCP. These structures enclose a set of related enzymes for a particular biocatalysis.6-7 Figure 1a provides a schematic representation of MCP structure and function. The basic building block of any shell protein is the unique BMC domain fold, and six such BMC domain folds assemble to form a cyclic flat disc-shaped hexagonal protein (Figure 1b). Structural studies of individual shell protein show that the hexameric units, because of their complementary shape, interact to form molecular mats that make the outer envelope of the MCPs. Specialized vertex and edge proteins join the extended mats to form the polyhedral structures.9-10 The above detailed structural and functional picture of MCP is realized by several genetic, biochemical and structural studies done in the last decade.9, 11-17 Studies have also

demonstrated the genetic manipulation of the MCPs for biotechnological applications,15, 18-19 however, these organelles have not been explored in the field of material science. Proteins and peptides are used at several instances as scaffolds for nanoparticle (NP) fabrication.20-29 The advantage in having proteins as scaffolds is that the size and shape can be genetically controlled, precisely to the accuracy of few angstroms.21, 30-32 It is reported in the literature that the interaction, reduction and fabrication of the gold precursors or nanoparticles on the proteins is mediated by certain amino acids such as aspartic acid, glutamic acid, histidine, and tyrosine.29, 33-36 Our aim in this work is to use the natural properties of proteins to capture and reduce auric chloride to gold nanoparticles in the context of the MCPs while keeping the property of the MCPs intact. Engineered viral capsids have been used in protein nanotechnology to a great extent37-38, and have been applied widely for various applications ranging from biological to material science while natural systems such as these prokaryotic organelles have still not been explored in detail. The added advantage of the MCP system in evaluating this concept of hybrid catalyst over any other macromolecular protein assembly is its special structure: an internal enzyme core with a sturdy external protein envelope.39 This nature-ready system inspires the fabrication of a catalytic duo with a biocatalytic core within an inorganic catalytic shell.

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Figure 1: Model of MCP showing the metabolism of substrate by localizing a set of related or sequential enzymes within a semipermeable outer protein shell (a); Crystal structure of monomer unit of Pdu A shell protein (PDB ID-3NGK) and six unit of it assemble to form a hexamer (b); In situ formation of AuNPs on MCP scaffold as observed from the characteristic peak of AuNPs with UV spectroscopy; Kinetics of AuNPs formation on the Pdu MCP surface (Inset) (c); X-Ray diffraction for in situ MCP:AuNPs (d); TEM micrograph for the negatively stained MCP(1), AuNPs on the scaffold of MCP shell proteins (2,3,4) (e); size distribution of the AuNPs on the MCP scaffold (red bars), distribution of the distance between the adjacent NPs on the MCP scaffold (blue bars) (f). Here, we have used the shell surface of propane-1,2-diol utilization MCPs (Pdu-MCPs) for scaffolding and in situ reduction of auric chloride (AuCl3) to AuNPs. These Pdu-MCPs in Salmonella encapsulate within their core the diol dehydratase that catalyzes the vitamin B12 dependent degradation of 1,2 propanediol to propionaldehyde (Figure S1) which is taken up by the downstream enzymes and converted to 1propanol and this 1-propanol enters the central metabolic pathway of the bacteria. 40-41 Our results show that the gold nanoparticles are fabricated in 3D over the MCP shell that are confirmed by the characteristic absorbance peak for gold nanoparticles at 540 nm and the X-ray diffraction pattern as shown in Figure 1c and Figure 1d respectively. The Pdu-MCPs are prepared according to a reported protocol42 and their integrity post purification is checked using standard methods (Figure S2). The SDS-PAGE for the PduMCP show the bands for the component shell proteins and the individual enzymes suggesting that the purified MCPs postpurification are intact while the TEM micrographs revealed polyhedral structures. The average size of the MCPs determined by DLS and TEM is 110 ± 30 nm, similar to what has been reported earlier in this class of MCPs.42 The specific activity for the Pdu-MCPs determined by the MBTH assay41, 43 is 23.6 ± 2.2 µmol min-1mg-1. The Pdu-MCPs are easy to purify with high yields and demonstrate remarkable stability under heat and pH-induced insults as shown in Figure S3 and S4 and hence can sturdily act as a barrier between the bio-catalytic core and the fabricated AuNPs in 3D. Under the experimental conditions reported here, the Pdu MCPs remain stable structurally and functionally (Figure S4).

The interaction and in situ reduction of the AuCl3 occur at the MCP surface. We have used tryptophan fluorescence and UV-vis absorption spectroscopy to probe the interaction of the MCPs with the AuCl3 (Figure S5). The extent of the interaction between the gold precursor and the MCP is studied by Isothermal titration calorimetry (ITC) and Biolayer interferometry (BLI) (Figure S5 to S8). The gold precursor binds to the shell surface with a dissociation constant in the range of 10-6. The reduction of surface-bound, gold is assisted by the amino acids29, 35 on the MCP shell protein surface. The signature absorbance peak of AuNPs at 540 nm is observed upon the interaction of auric chloride with MCP surface (Figure 1c), and the intensity of the peak saturates within 30 min (Figure 1c, inset). Titrating the MCPs and AuCl3 in the concentration range 0.1-0.5 mg/ml and 2.5-250 µM respectively is done to determine the optimum ratio for the MCP-AuNP conjugate fabrication. We observed that increasing the concentration of MCP and AuCl3 more than 0.1 mg and 250 µM respectively leads to MCP-AuNPs aggregate formation (Figure S9). Thus, for further experiments, low concentration of MCP as a scaffold (0.1 mg/ml) and AuCl3 as a precursor (5-50 µM) is used. The X-ray diffraction of the AuNPs fabricated on the MCPs in the 3D show peaks at 2θ values of 38.0, 44.3, 64.5 and 77.7 which can be indexed to (1 1 1), (2 0 0), (2 2 0), (3 1 1) reflections of face-centered cubic structure of metallic gold (Figure 1d). In electron micrographs, we observed that the AuNPs are displayed all over the Pdu-MCP surface (Figure 1e) in 3D. Of the several electron micrographs analyzed representative ones

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are shown in Figure 1e and Figure S10. Each electron micrograph of the MCP:AuNP conjugate is analyzed for the average

any specific arrangement of the NPs similar to the one observed for the MCPs, rather NP aggregates are observed.

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Figure 2: Catalytic activity of MCP:AuNPs conjugate showing the conversion p-nitrophenol to p-amino phenol (a) increased catalytic activity of 10 uM HAuCl4 fabricated nanoparticles in comaprision to 5 uM HauCl4 (b); Enzyme catalysis for MCPAuNPs and PduCDE_AuNPs (c) Decreased catalytic activity for MCP-AuNPs in comparison to PduCDE-AuNPs (d). The data are representative of three independent experiments. size (d) and distance between two adjacent AuNPs (D). The Moreover, in the case of BSA scaffolded NPs, no size conaverage size (d) of each of the nanoparticles (NPs) is finement or uniform particle-to-particle distance is observed. 2.5±0.5nm nm with average inter-particle distance (D) being Hence, the spatial distribution of the AuNPs on MCP and their 5.75 ± 0.5nm (Figure 1f). Interestingly, in crystal structures of size confinement is because of the scaffolding of the NPs by most of the shell proteins it has been observed that the centers MCP shell surface. of two adjacent hexamers are placed 5.0-6.5 nm apart9, 17 as Gold nanoparticles are known to show heterogeneous given in Table S1. The arrangement of AuNPs on the hexagocatalytic activity.44-48 Spatial disposition of the AuNPs in 3D nal shell proteins on the MCP shell give rise to their spatial on the have demonstrated effect on the heterogeneous catalytic interaction leading to plasmonic development as observed by activity.49 We next tested if the developed MCP-AuNP conjuUV in Figure S11. Although we see that the spacing and the gates are capable of performing heterogeneous inorganic caparticle size is similar to those seen for the individual proteins talysis for the fabricated nanoparticles and bio-catalysis for the in the crystal structure, the regular array of nanoparticles is not encapsulated enzymes in parallel. For inorganic catalysis, we observed on the MCP surface. This might be due to two reaused the model reaction of reduction of p-nitrophenol to psons. First deformation of the MCPs may occur on the TEM aminophenol44 in the presence of NaBH4 (Figure 2a). The catagrid followed by subsequent drying. Otherwise, it may happen lytic activity is performed using MCP-AuNP conjugates prethat the behavior of the shell are different when expressed pared by different amount of AuCl3, and keeping MCP conindividually compared to in the MCPs. There are certain recentration constant at 0.1 mg/ml. We observe that the heteroports that show that overexpressed PduA and PduB show difgeneous catalytic activity increases with increasing the AuCl3 ferent nanostructures.36 However, the absence of the regular concentration from 5 to 30 µM. Upon mere doubling of AuCl3 array does not affect the formation of the hybrid catalytic duo. from 5 to 10 µM (Figure 2b) we observed a five-six fold enNext, we tested if the scaffolding of the AuNPs is a result of hancement in the catalytic activity of the fabricated gold nathe regular shell scaffold or a result of drying of NPs on the noparticles. This corresponds to a large surface area of a typiproteins. To address this issue, we did similar experiments in cal icosahedral MCP (3.04 × 104 nm2) being provided for NPs the presence of a control protein bovine serum albumin fabrication and hence we observe good catalytic activity (alt(BSA). In the case of BSA, we did not observe (Figure S12) hough the Pdu-MCPs do not have the regular icosahedral

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ACS Catalysis structure, we made an approximation based on the size we see in the TEM images). The fabricated AuNPs on the scaffold of the

and thermal shift assay. The MCP-Au conjugate doesn’t show any decrease in the unfolding Tm when compared with native MCP (Figure S16a and S16b).

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Figure 3: Formation of AuNPs on MCP surface observed by TIRF Microscopy at different time point(a); Excitation and emission spectra of the fluorescent gold nanoparticles on the MCP surface (b); Formation of AuNPs on MCP studied with steady state fluorescence and showing the transition from fluorescent AuNPs to non- fluorescent in the presence of excess precursor (c) in the presence of external reducing agent HEPES (d). The data are representative of three independent experiments. MCP shell protein due to the spatial arrangement in 3D results in plasmonics development as mentioned earlier leading to enhanced catalytic activity.49 Interestingly, we observed that the specific activity of the enzyme core is compromised upon increasing the precursor concentration beyond 10 µM (Figure 2c). The enzyme catalysis we have demonstrated here is that of the diol dehydratase (PduCDE) enzyme encapsulated within the MCPs. The specific activity values indicate concentrations of propionaldehyde produced in one minute by per milligram of protein. PduCDE is the main enzyme of the Pdu MCP system and id involved in the vitamin B12 dependent conversion of 1,2-propanediol to propionaldehyde. The compromise in the enzyme catalysis above 10µM of AuCl3 can be due to two reasons: either the MCP components are getting deformed upon Au-NPs fabrication or AuNPs on the surface are blocking the transport of substrates and co-factors inside the lumen of the MCP. To address the first issue, we checked the conformation of the MCPs and the MCP-AuNPs. CD and FTIR spectroscopy show that the secondary structural elements of the entire Pdu MCP remain preserved upon the formation of NPs on the surfaces ( Figures S13, S14). In the FTIR spectra of the MCP-AuNPs, we observed the amide-I vibrations are smoother than that of bare MCPs suggesting the interaction of the MCPs with the AuNPs. This observation also indicates that the overall conformation and the macromolecular assembly of the MCP remain unaltered upon the formation of NPs on the surfaces. Also, the conformation of the major individual shell proteins (PduA, PduB and PduBB’) with and without AuNPs remain unaltered in the presence and absence of the AuCl3 (Figure 15). The thermal stability of the conjugate material is carried out by fluorescence spectroscopy

These observations are also supported by the light scattering experiments where we observed a slight contraction in the size of MCP:AuNPs (90±10 nm) compared to the size of the MCP (110±30 nm) alone (Figure S17). The decrease in size is observed only when higher concentrations (50-100 µM) of gold precursor are used. At lower concentrations (5-10 µM) no significant size variation is observed (Figure S18). The AuNPs formed in the absence of the MCPs show two size distributions 30±4 nm and 160±25 nm. On the other hand, in the presence of the MCPs, we observe only one size distribution at 90±10 nm. The unimodal size distribution in the presence of MCPs and the precise size of the AuNPs obtained in the electron micrograph suggests that the MCP shell precisely confines the size of the NPs to less than 3nm on the MCP surface. Our studies so far indicate that in the presence of MCP, almost the entire corpus of MCP surface is templated with AuNPs. The conformational stability and the secondary structure of the MCP or shell proteins, in the presence and absence of the AuNPs are same suggesting that AuNPs do not interfere with the conformation of the MCP or shell proteins despite a decrease in the overall size of MCP. We next assessed the specific activity of the bare enzyme PduCDE in the presence and absence of gold nanoparticle fabrication. The experiment is done at different AuCl3 concentrations in the range 0-30µM. We observed that for the bare PduCDE when the enzyme-AuNPs conjugates are made using 10µM of the AuCl3 there is no quenching of the diol dehydratase activity. Beyond this concentration, there is a gradual decrease in the enzyme activity. The decrease in enzyme activity of the MCP-AuNPs is, however, more pronounced compared to the PduCDE-AuNPs (Figure 2d). This observation is perplexing considering the fact that the shell on which the

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ACS Catalysis nanoparticles are scaffolded is supposed to protect the internal enzymes from any chemical or physiological insults. On the contrary, we observe a greater decrease in the activity in the case of MCP-AuNPs compared to the PduCDE-AuNPs. This observation along with the conformational studies supports our second speculation that the gold nanoparticles block the passage of substrates and co-factors from the surface of the MCPs to the central lumen. It is well acknowledged in the literature that the pores on the shell proteins act as conduits for the substrate and cofactor transport.16, 50 As the AuNPs are formed on the surface of the MCPs and considering their size (2.5-3nm) it is likely that they block the conducting pores of the shell proteins thereby retarding the MCP activity. This report is perhaps the first of its kind which directly demonstrates that ectopic blocking of the pore may lead to impaired microcompartment function with an active enzyme cluster inside. Previously, genetic engineering studies have shown that specific blocking of the PduA pore results in the compromise in propane-1,2-diol uptake.16, 51 A recent study through molecular dynamics simulations by Park and co-workers have also proposed the role of the central pore of PduA in selective metabolite transport across the Pdu-MCP.50, 52 Finally, we used fluorescence spectroscopy and imaging to understand the mechanism involved in the fabrication of AuNPs in 3D on the MCP scaffold. As discussed earlier the amino acids on the MCP surface help in the interaction of gold precursor on the protein surface followed by reduction. Our MCP-AuNPs hybrid exhibits a sharp stable fluorescence that peaks at 660 nm at a wide range of excitation as monitored in solution by steady-state fluorescence and total internal reflection fluorescence microscopy (TIRF-M). The TIRF(M) scheme is shown in Figure S19. The fluorescence particles (Figure 3a) evolve after 5-6 minutes of initiation of the reaction and the intensity saturates after 15 min as shown in Figure S20). Control experiments using in the absence of MCP with AuCl3 and HEPES show no appreciable fluorescence in TIRFM. This suggests that fluorescent clusters are formed only in the presence of MCPs (Figure S21a). TIRF imaging for MCP alone in the absence of AuCl3 did not produce detectable as shown in S21b. The fluorescence of the gold nanoclusters disappears upon the addition of excess amount of the gold precursor (Figure 3b) or external reducing agent (Figure 3c) suggesting the formation of bigger non-fluorescent AuNPs. Typically, AuNP of size ≥ 3 nm are reported to be nonfluorescent and size < 3 nm are reported to be fluorescent.53 The formation of non-fluorescent particles in the absence of MCPs suggests that the MCPs scaffold the gold nanoparticles and confine their size below 3nm. This precise size distribution can be obtained by a meticulous balance of the reducing scaffold (MCP) and the precursor (AuCl3).

assisted by the intrinsic nature of the amino acids to nucleate the precursor molecules followed by subsequent reduction. The wrapping of AuNPs on the MCP surface blocks the conduits of substrate and co-factors, thus resulting in decreased enzyme catalytic activity. This unique system demonstrates the use of such a fabrication methodology in the development of a generation of inorganic-organic hybrids. Combining the concepts of ease of encapsulation of ex vivo heterologous enzyme inside the MCPs, their genetic manipulation for getting stable bio-catalytic system11, 54-56, and the shell based fabrication protocol detailed here, would pave the way for a generation of hybrid inorganic-organic materials.

Conclusion and Future Directions In this study, we have successfully fabricated in 3D AuNPs on the surface of MCP to develop a hybrid inorganic-organic catalytic conjugates. The fabrication is carried out with an optimal concentration of auric chloride and under ambient conditions without disrupting the macromolecular assembly of Pdu-MCP. The sturdy nature of shell proteins of Pdu-MCP provides stability to the encapsulated enzymes under wide pH and temperature range. The shell surface of Pdu-MCP acts sturdily as a scaffold for fabrication of gold nanoparticles in 3D and at the same time act as a protective guard for the enzymes encapsulated inside. The fabrication of nanoparticles is

REFERENCES

ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Author

*Sharmistha

Sinha, Institute of Nano Science and Technology, Habitat Centre, Phase-10, Sector-64, Mohali, Punjab, India.

*[email protected] ORCID Sharmistha Sinha: 0000-0003-2459-650X Md. Ehesan Ali: 0000-0001-6607-5484 Naimat Kalim Bari: 0000-0002-2882-5296 Notes The authors declare no competing financial interest.

ABBREVIATIONS MCPs Bacterial microcompartments; Pdu-MCPs Propane-1,2-diol utilization MCPs; AuNPs Au nanoparticles; MD Molecular dynamics; TEM Transmission electron microscope; CD Circular Dichroism; DLS dynamic light scattering.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental details for MCP and PduCDE purification along with protocols for TEM, CD, XRD, DLS etc. are mentioned in the supporting information. Data related to stability of native MCP and gold nanoparticle fabricated MCP are also there in SI.

ACKNOWLEDGMENT The work is supported by SERB, India grant EMR/2015/000746 to SS and SR/NM/NB-1082/2017 to SS and EA. The authors thank Dr. Sabyasachi Rakshit (IISER, Mohali) for helpful discussions.

1. Tanaka, S.; Kerfeld, C. A.; Sawaya, M. R.; Cai, F.; Heinhorst, S.; Cannon, G. C.; Yeates, T. O. Atomic-level models of the bacterial carboxysome shell. Science 2008, 319, 1083-1086. 2. Tanaka, S.; Sawaya, M. R.; Yeates, T. O. Structure and mechanisms of a protein-based organelle in Escherichia coli. Science 2010, 327, 81-84. 3. Sutter, M.; Greber, B.; Aussignargues, C.; Kerfeld, C. A. Assembly principles and structure of a 6.5-MDa bacterial microcompartment shell. Science 2017, 356, 1293-1297. 4. Cheng, S.; Liu, Y.; Crowley, C. S.; Yeates, T. O.; Bobik, T. A. Bacterial microcompartments: their properties and paradoxes. BioEssays 2008, 30, 1084-1095.

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Table of Content

1,2-PD VitB12 PduCDE

-1

Propionaldehyde 30 0.6

-1

Specific Enzyme Activity ∝mol mg ml

25

Downstream pathways

0.5

20

Absorbance a.u

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

15 10

MCP:AuNPs PduCDE:AuNPs

5

5

10

15

20

AuCl3 concentration (∝ ∝M)

Bio-Catalyst

0.3 0.2 0.1

0 0

0.4

30

0.0 250

300

350

400

450

500

Wavelength nm

Inorganic Catalyst

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