Self-Assembly of Spider Silk-Fusion Proteins ... - ACS Publications

*corresponding author: e-mail (M.H.) [email protected]. Abstract: The recombinant spider silk protein eADF4(C16) was genetically fused...
0 downloads 0 Views 5MB Size
Subscriber access provided by READING UNIV

Article

Self-Assembly of Spider Silk-Fusion Proteins Comprising Enzymatic and Fluorescence Activity Martin Humenik, Madeleine Mohrand, and Thomas Scheibel Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00759 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 16, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28 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

Bioconjugate Chemistry

Self-Assembly of Spider Silk-Fusion Proteins Comprising Enzymatic and Fluorescence Activity Martin Humenika*, Madeleine Mohranda and Thomas Scheibela-f a

Lehrstuhl Biomaterialen, Faculty of Engineering Science

b

c

Research Center Bio-Macromolecules (BIOmac)

d

e

Bayreuth Center for Colloids and Interfaces (BZKG)

Bayreuth Center for Molecular Biosciences (BZMB)

Bayreuth Center for Material Science (BayMAT)

f

Bavarian Polymer Institute (BPI)

a-f

Universität Bayreuth, Universitätsstraße 30, 95440 Bayreuth, Germany

*corresponding author: e-mail (M.H.) [email protected]

Abstract: The recombinant spider silk protein eADF4(C16) was genetically fused either with esterase 2 (EST2) or green fluorescent protein (GFP). The fusions EST-eADF4(C16) and GFP-eADF4(C16) were spectroscopically investigated and showed native structures of EST and GFP. The structural integrity was confirmed by the enzymatic activity of EST and the fluorescence of GFP. The spider silk moiety retained its intrinsically unstructured conformation in solution and the self-assembly into either nanofibrils or nanoparticles could be controlled by the concentration of phosphate. Particles, however, showed significantly 1 ACS Paragon Plus Environment

Bioconjugate Chemistry 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

lower activity of the EST and GFP domains likely caused by a steric hindrance. However, upon self-assembly of EST-eADF4(C16) and GFP-eADF4(C16) into fibrils the protein activities were retained. In general, the fusion of globular enzymes with the spider silk domain allow the generation of fibrillar biomaterials with catalytic or light emitting properties.

TOC:

Introduction Silk cross-β nanofibrils possess highly ordered H-bonded β-sheets oriented perpendicularly along the fibril axis. Since cross-β structures represent a thermodynamically stable fold, especially intrinsically unfolded proteins will form ß-sheet rich fibrils when exposed to appropriate physical and chemical triggers.1-3 These fibrils can further self-assemble into nanoscaled scaffolds with high aspect ratios, high chemical and thermodynamic stability and mechanical rigidity.4-5 This enable various applications, such as nanoscaffolds for catalysts or light harvesting.6-12 Enzymes reflect building blocks, which in combination with fibrillar proteins allow to achieve functional nanomaterials. There are several interesting examples where enzymes were used in combination with fibrillary proteins to develop functional nanomaterials. For instance, cytochrome-b562 was fused with a self-assembling tandem repeat 2 ACS Paragon Plus Environment

Page 2 of 28

Page 3 of 28 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

Bioconjugate Chemistry

of the bovine PI3-SH3 domain. The fusion formed fibrils and was used to study electron transfer along the fibrils.13 A self-assembling domain of Saccharomyces cerevisiae Sup35(161) was fused with E2GFP, a pH sensitive GFP mutant, and methyl parathion hydrolase (MPH). Fluorescence emission could be detected upon substrate hydrolysis and release of H+ ions upon a MPH catalyzed reaction.14 Various fluorescent proteins have also been combined with low-complexity (LC) sequence domain of fused in sarcoma (FUS) protein, an RNAbinding protein with essential roles in RNA transcription, processing, and transport. The fusions were used to study assembly of LC sequences and an organizing principle for cellular structures in vivo,15 as well as to prepare multiblock supramolecular fibrous structures in vitro.16 A self-assembling yeast Ure2 prion domain was fused with barnase, carbonic anhydrase (CA), glutathione S-transferase (GST), and green fluorescent protein (GFP), showing only slight alteration of their activities upon fibril assembly.17 The system was also used to display more complex enzymes, such as alkaline phosphatase (AP) and horseradish peroxidase (HRP).7 Here, however, a significant drop of AP activity was observed upon fusion and assembly (>10 fold). Generally, it has been shown that enzymes, such as HRP, CA or barnase, which enzyme kinetics are diffusion controlled, reveal a high activity loss if displayed on the fibril surface. In the presented study, the recombinant spider silk protein eADF4(C16), based on 16 repetitions of the consensus sequence of ADF4, a MaSp2 dragline silk protein from Araneus diadematus,18 was fused with the C-terminus of thermophilic Esterase 2 (EST2) or with the His6-tagged enhanced variant of green fluorescent protein (eGFP) (Figure 1). EST2 from Alicyclobacillus acidocaldarius is a monomeric serine hydrolase (34 kDa), which possesses typical α/β-fold and highest catalytic activities at 70 °C. However, sufficient activities at ambient temperatures and different pH values as well as long-term stability19-21 are rendering EST2 an interesting model for biosensor development.22-25 GFP from Aequorea victoria is an 3 ACS Paragon Plus Environment

Bioconjugate Chemistry 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

11-stranded β-barrel threaded by an α-helix. GFP is stable and fluorescent at temperatures up to 65°C. The chromophore, resulting from the spontaneous cyclization and oxidation of the sequence Thr65-Tyr66-Gly67, requires the native protein fold for its formation. Denatured GFPs or small proteolytic fragments carrying the chromophore are, however, essentially nonfluorescent. There are many variants of GFP, some with enhanced and red or blue shifted fluorescence bands, and they are widely used as fusion-tags to study fundamental principles ranging from biochemistry, cell biology, developmental biology, neurobiology, ecology to nanotechnologies.26-29 The recombinant eADF4(C16) is an intrinsically unfolded protein in solution. Low concentration of phosphate ions (Pi) (< 300 mM)30-31 trigger self-assembly of the spider silk protein into cross-β structured nanofibrils, whereas high concentration (>400 mM) induce salting-out yielding spider silk particles.32 Here, genetic fusions ESTeADF4(C16) and GFP-eADF4(C16) (Figure 1) were produced, purified and characterized with the aim to evaluate the compatibility of the spider silk self-assembly with the integrity and functionality of the fused globular domains. Thus, the silk transformation into β-sheet rich structures and the morphology of the self-assembled fusion proteins were studied in the presence of different phosphate concentrations. Vice versa, protein structures, enzymatic and fluorescent activities of the globular moieties in soluble and assembled forms were compared.

4 ACS Paragon Plus Environment

Page 4 of 28

Page 5 of 28 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

Bioconjugate Chemistry

Figure 1. Schematic representation of the eADF4(C16) fusion with EST and GFP. The structure of the globular moieties were reproduced from protein database entries (PDB ID 1EVQ for EST220 and 2Y0G for eGFP33, www.rcsb.org). The protein backbone is represented as a ribbon model with a ball model highlighting residues of the catalytic S155/H282/D252 and the fluorophore forming T65/Y66/G67 triad in the active centers of EST2 and eGFP, respectively. Results and Discussion Purification, Activity and Structure of the Soluble Silk-Enzyme Fusions. Globular EST2 and eGFP as well as intrinsically unstructured eADF4(C16) represent thermostable proteins. Consequently, the purification approach of EST- and GFP-eADF4(C16) took advantage of this thermal stability allowing a simple, column-free strategy including a heat step and ammonium sulfate precipitation.18 The analysis of the heat treatment (Figure S1A and B) revealed 70°C as an optimal temperature, which allowed precipitation of E. coli proteins and, at the same time, solubility of the fusion proteins. After purification, however, re5 ACS Paragon Plus Environment

Bioconjugate Chemistry 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

solubilization required 6 M guanidine thiocyanate (GdmSCN) due to the compact and insoluble β-sheet rich structure of the spider silk moiety after the salting out step.18 Thus, for the functionality of the EST and GFP moieties a refolding step was required, which was achieved by dialysis against urea and a stepwise reduction of the urea concentration. The dialysis resulted in active proteins as demonstrated in the native polyacrylamide gels (Figure S1C and D) using activity staining19, 23 and fluorescent scanning, respectively. The identity of the proteins was confirmed by MALDI-TOF (Figure S2).

6 ACS Paragon Plus Environment

Page 6 of 28

Page 7 of 28 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

Bioconjugate Chemistry

Figure 2. Spectroscopy analysis of soluble and assembled fusion proteins. A) and B) CD spectra of the soluble fusions and corresponding unmodified ancestors, which were also used to calculate theoretical averaged spectra; D) and E) Amid I regions of FT-IR spectra of the soluble proteins. C) CD and F) FT-IR spectra of fibrils assembled in KPi buffer.

7 ACS Paragon Plus Environment

Bioconjugate Chemistry 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

The solution structures of eGFP (β-barrel),34 EST2 (α/β fold)35 and eADF4(C16) (intrinsically unstructured)18 were compared to structures of the corresponding fusion proteins using CD spectroscopy (Figure 2A and B). The theoretical averaged spectra, calculated from the unmodified counterparts, reflect contributions from the native structures36 of the globular EST/GFP domains as well as the unstructured spider silk. Apparently, the globular moieties refolded properly in the fusions without influences of the silk domain. The FT-IR spectroscopy (Figure 2D and E) of the soluble GFP- and EST-fusions with maxima in the Amid I region at 1645 cm-1 and 1652 cm-1, respectively, showed that the random coil and αhelical structures are dominant due to the presence of the intrinsically unstructured eADF4(C16) (maximum at 1648 cm-1). In contrast, eGFP revealed a clearly β-sheet rich spectrum with a maximum at 1627 cm-1, and the α/β fold of the EST led to separated maxima of α-helix (1652 cm-1) and β-sheet (1621 cm-1). The observations were also confirmed by quantitative FSD analysis of the Amid I regions37 (Figure S3, Table S1 and S2). The fluorescence of GFP-eADF4(C16) was compared to that of eGFP using fluorescence spectrometry (Figure 3). The average activity of the fusion protein reached 88.9% in the concentration regime between 0.5-5 µM (Figure 3A), however, at concentrations above 1µM the trend was non-linear due to the innerfilter effect.

8 ACS Paragon Plus Environment

Page 8 of 28

Page 9 of 28 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

Bioconjugate Chemistry

Figure 3. Fluorescent activities of eGFP compared to that of soluble GFP-eADF4(C16) as well as assembled fibrils and particles made thereof. A) Relative activities of soluble GFP-eADF4(C16) and assembled forms thereof compared to that of native eGFP. Significantly higher variations in the activity of the particles were due to quick sedimentation; B) Examples of fluorescence spectra at 3 µM protein; spectra were recorded at an excitation wavelength of 488 nm. Hydrolytic activity of the EST-eADF4(C16) was tested using p-nitrophenyl acetate (pNPA) which is hydrolyzed to p-nitrophenol (pNP), a spectrometric active substance detectable at 405 nm.19, 23 The enzymatic activity of the silk fusion reached 80 % of the wild type EST2 (Table 1). The preservation of the fluorescence as well as the hydrolytic activity in the fusion constructs is consistent with their high structural integrities after refolding (Figure 2). 9 ACS Paragon Plus Environment

Bioconjugate Chemistry 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

Page 10 of 28

Table 1: Specific and relative hydrolytic activities of esterase in the spider silk fusion. The average values of the enzymatic activities and the standard deviations (SD) were calculated from triplicates. Protein

Specific activity 3 -1 x10 (U.µmol )

% of EST2 activity

34.3

80.4

1.9

fibrils

32.1

75.4

0.9

particles

8.9

20.8

0.7

Protein state

SD

soluble EST-eADF4(C16)

soluble, after refolding assembled

controls EST2

dilution from storage buffer

42.6

100.0

0.6

eADF4(C16)

in 10 mM Tris/HCl, pH 8

1.3

3.0

0.1

Salting-Out of Spider Silk Fusions. Addition of 1 M Pi induces salting-out of eADF4(C16) from the solution yielding regularly shaped particles32, 38 (Figure 4A). Treatment of the ESTand GFP-fusions at the same protein and phosphate concentration resulted apparently in smaller particles (Figure 4C and E), which were not colloidally stable and sedimented within 1 h. In contrast, particles of the unmodified silk protein remained in suspension over several days. The particles distribution analysis (Figure S4) revealed mean particles diameter at 160 nm for the fusions and 710 nm for the unmodified eADF4(C16) particles, the later showing a much broader distribution range. The measurement of protein activities showed a significant loss for both, the GFP and the EST domain (