A Model Sea Urchin Spicule Matrix Protein ... - ACS Publications

Subscriber access provided by UNIV OF ARIZONA

Article

A model sea urchin spicule matrix protein, rSpSM50, is a hydrogelator that modifies and organizes the mineralization process. Gaurav Jain, Martin Pendola, Yu-Chieh Huang, Denis Gebauer, and John Spencer Evans Biochemistry, Just Accepted Manuscript • Publication Date (Web): 06 May 2017 Downloaded from http://pubs.acs.org on May 7, 2017

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.

Biochemistry 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 44

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

Biochemistry

A model sea urchin spicule matrix protein, rSpSM50, is a hydrogelator that modifies and organizes the mineralization process.

Gaurav Jain,1¶ Martin Pendola,1¶ Yu-Chieh Huang,2 Denis Gebauer,2 and John Spencer Evans1*

1

Laboratory for Chemical Physics, Center for Skeletal and Craniofacial Biology, New York University,

345 E. 24th Street, NY, NY, 10010 USA. 2

Department of Chemistry, Physical Chemistry, Universität Konstanz, Universitätstrasse 10, Konstanz D-

78457, Germany.

Keywords:

Sea urchin / spicules / calcite / amorphous calcium carbonate / protein aggregation / hydrogels / intrinsic disorder / amyloid-like

¶

Both authors contributed equally to this work

*To whom correspondence should be addressed. Email: [email protected]

1

ACS Paragon Plus Environment

Biochemistry

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 2 of 44

ABBREVIATIONS TrxHis6

=

recombinant

isopropylthiogalactoside;

thioredoxin

poly(His)6

affinity

tag;

β-IPTG

=

beta-

IDA = iminodiacetic acid; SA = sinapinic acid; DHA = 2',5'-

dihydroxyacetophenone; MALDI-TOF-MS = matrix-assisted laser desorption/ionization time-offlight mass spectrometry; SM = spicule matrix; SpSM50 = Strongylocentrotus purpuratus spicule matrix protein 50, rSpSM50 = E. coli recombinant Strongylocentrotus purpuratus spicule matrix protein 50; SpSM30 = Strongylocentrotus purpuratus spicule matrix protein 30; rSpSM30B/C = insect cell expressed recombinant Strongylocentrotus purpuratus spicule matrix glycoprotein 30, hybrid B/C isoform; PMC = primary mesenchyme cells; PNC = pre-nucleation cluster; ACC = amorphous calcium carbonate; MgC = magnesium carbonate; ID = intrinsic disorder; CTLL = C-type lectin like binding domain; MAQPG = Met/Asn/Gln/Pro/Gly-rich sequences common to spicule matrix proteins.

2


Page 3 of 44

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

Biochemistry

ABSTRACT In the purple sea urchin Strongylocentrotus purpuratus the formation and mineralization of fracture-resistant skeletal elements such as the embryonic spicule requires the combinatorial participation of numerous spicule matrix proteins such as SpSM50. However due to limited abundance and solubility issues it has been difficult to pursue extensive in vitro biochemical studies of SpSM50 protein and deduce its role in spicule formation and mineralization. To circumvent these problems we expressed a tag-free bacterial model recombinant spicule matrix protein rSpSM50. Bioinformatics and biophysical experiments confirm that rSpSM50 is an intrinsically disordered, aggregation-prone C-type lectin-like (CTLL)-domain containing protein that forms dimensionally and internally heterogeneous protein hydrogels that control the in vitro mineralization process in three ways: 1) kinetically stabilize the aqueous calcium carbonate system against nucleation and thermodynamically destabilize the initially formed ACC in bulk solution; 2) promote and organize faceted single crystal calcite and polycrystalline vaterite nanoparticles; and 3) promote surface texturing of calcite crystals and induce subsurface nanoporosities and channels within both calcite and vaterite crystals. Many of these features are also common to mollusk shell nacre proteins and the sea urchin spicule matrix glycoprotein, SpSM30B/C, and we conclude that rSpSM50 is a spiculogenesis hydrogelator protein that exhibits traits found in other calcium carbonate mineral-modification proteins.

3


Biochemistry

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

Sea urchin embryo skeletal elements (spicules) are important research models for investigating the chemical mechanisms and biological processes that organisms use to control crystal growth and create sophisticated functional nanocomposites 1 under very mild and sustainable conditions.1-5 In the purple sea urchin embryo, Strongylocentrotus purpuratus, the spicule is a single crystal of magnesium-bearing calcite (Ca.95Mg.05CO3 or MgC) that is formed by primary mesenchyme cells (PMCs) via deposition of amorphous calcium carbonate (ACC) within the extracellular spicule matrix (SM) created by protein families designated as SpSM. 6-24 This ACC phase then undergoes an amorphous-to-crystalline transition to form MgC and subsequently the SpSM proteins become occluded within the crystalline mineral phase. 12,22-24 Unfortunately the mechanistic details of these processes remain unknown. Thus there is a strong motivation to gain further information regarding the spicule matrix, the SpSM proteome, and the spatial and functional relationships between SpSM proteins and mineral phase. Genomic and proteomic studies reveal that all expressed SM proteins feature a C-type lectin-like domain (CTLL),2-5,11 which may play an important role in matrix assembly and mineral formation.11 Second, in ten SpSM proteins there also exists Met/Asn/Gln/Pro/Gly-rich (MAQPG) redundant sequence that bears a striking similarity with known elastomeric sequences.2-5,11,14 These common sequence elements may convey important ECM functions, such as matrix protein assembly and elasticity, protein – mineral phase interaction/stabilization, or matrix-mineral organization to this family of proteins. Of the known SM proteins, the best characterized member is SpSM50 (pI = 10.7, 44541 Da, 428 AA, Uniprot: P11994, GenBank: AAA30071.1),2-5,13-21 the most abundant member of the SM proteome whose individual domains have been found to possess several important features related to protein self-assembly and ACC

4


Page 4 of 44

Page 5 of 44

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

Biochemistry

formation.11 The pI of this protein is primarily alkaline due to the presence of 25 Arg groups (Figure 1). SpSM50 plays a central role in spicule matrix assembly and biomineralization as evidenced by SpSM50 knockdown experiments which interfered with spicule growth and mineral formation.10

SpSM50 is localized to both the outer and inner regions of triradiate

spicules13,18,21 and concentrates in the radially growing portions of the spicules (normal to the caxis of calcite).18 It has been proposed that SpSM50 defines the extracellular space in which mineral deposition occurs,18 and if so, then the assembly properties of SpSM50 become tremendously important. Due to the strong aggregation properties of SpSM50 and the small size of spicules, very few in vitro experiments have been conducted with spicule-extracted protein to test its mineralization and association properties. Instead, recombinant variants or protein fragments have been utilized. In one case a recombinant form of SM50 was created for the exclusive purpose of demonstrating kinetic stabilization of ACC in vitro,24 thus supporting the hypothesis that one of the roles assigned for SM50 in spiculogenesis is ACC stabilization and/or transformation to calcite.22-24 Subsequently a study utilized recombinantly-expressed solubilitytagged CTLL domain, a glycine rich region (GRR), and a proline rich region (PRR) 11 found that under various mineralization conditions CTLL is monomeric and influences calcium carbonate mineralization, GRR aggregates into large protein superstructures, and PRR modifies the early calcium carbonate mineralization stages as well as crystal growth. 11 More recently, a 26 AA SM50 repeat polypeptide was studied and found to self-assemble into matrix subunits and promote growth of calcium carbonates within the subunits themselves, as well as promoting fusion of adjacent crystals into a wide-spread network. 25 Unfortunately, these fragment-based

5


Biochemistry

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

studies do not capture the entirety of the SM50 sequence and thus at present our understanding of SpSM50 protein aggregation and mineralization functions are incomplete.

Figure 1. The mature, Signal-P transmembrane-deleted primary sequence of the SpSM50 protein. Predicted intrinsically disordered (DISOPRED, IUP algorithms, solid underline) and amyloid-like cross-beta strand aggregation (AGGRESCAN, ZIBBER DB algorithms, dashed underline) sequences are shown. The putative CTLL domain is denoted in italics (Q1 - G100). Note that the MAQPG region (boldface) runs from F101 to Q392. Anionic (red) and cationic (blue) amino acid residues are highlighted in color.

One way to better clarify the role of SpSM50 within spicule mineralization is to utilize a tag-free model recombinant SpSM50 for in vitro studies, where the absence of a solubility tag can provide a better understanding of SpSM50 mineralization and aggregation functionalities within a defined laboratory environment. Using E. coli for recombinant expression we created and characterized a model recombinant protein, rSpSM50, that could help us address three issues: (1) Does SpSM50 affect the early and later events in nucleation and can these provide insights into the potential role(s) that SpSM50 play within spiculogenesis? (2) How does the mineralization behavior of rSpSM50 compare to published results obtained for isolated SpSM50 domains,11 the previously reported recombinant SpSM50,24 and for the recombinant spicule matrix glycoprotein, rSpSM30B/C?26 (3) what similarities and differences exist between a calcitic 6


Page 6 of 44

Page 7 of 44

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

Biochemistry

spicule matrix protein and other well-documented calcium carbonate biomineralization proteins, such as those associated with the mollusk shell nacre aragonite? 27-35 As documented below, we find that rSpSM50 possesses a high aggregation propensity and intrinsic disorder, two factors that promote porous protein hydrogel formation in solution and on charged surfaces. These hydrogels not only organize and promote calcite and vaterite mineral nanoparticle formation within the gel environment, they also modify the surface and subsurface (intracrystalline) regions of forming calcite and vaterite crystals. Further, with regard to the early events in nucleation, rSpSM50 hydrogels kinetically stabilize pre-nucleation stages, progressively inhibiting nucleation, and induce the formation of ACC that is thermodynamically less stable than the one formed in the absence of the protein. Thus, the results obtained with the model rSpSM50 protein establish three important overarching concepts: (1) similar to the rSpSM30B/C glycoprotein, rSpSM50 is capable of participating in spicule matrix assembly and mineralization processes; 6-24 (2) the rSpSM50 is an ACC destabilizer, which may be relevant for spiculogenesis with regard to ACC to calcite transformation; (3) intrinsically disordered, aggregation-prone biomineralization proteins and their modulatory effects on nucleation, nanoparticle organization, and crystal growth are common to calcium carbonate invertebrate skeletal structures such as mollusk shell 2734

and sea urchin spicules.26

MATERIALS AND METHODS

Bacterial recombinant expression and purification of rSpSM50.

7


Target DNA sequence of

Biochemistry

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

SpSM50 (Accession number P11994, SM50_STRPU UniProtKB) mature expressed form sans 17 AA membrane leader sequence was codon optimized using the BiologicsCorp Maxima codon optimization program and synthesized into the pUC57 vector for bacterial expression. The cloning strategy included the incorporation at the N-terminus of the mature SpSM50 sequence a Trx-His tag + enterokinase cleavage site (Figure S1, Supporting Information). 26,36,37 For the purposes of optimizing enterokinase cleavage, a Met residue was added to the first position of the mature, native SpSM50 sequence, yielding a theoretical MW of mature rSpSM50 = 44690. 36 This combined sequence was subcloned into Expression Vector E6 plasmid for E. coli expression as performed by BiologicsCorp USA (Indianapolis, IN, USA; http://www.biologicscorp.com/) using their proprietary system. E. coli BL21 (DE3) was transformed with this recombinant plasmid and a single colony was inoculated into LB medium containing kanamycin; cultures were incubated at 37 oC until cell density reached an OD 600 nm of 0.6 – 0.8, at which point β-IPTG was introduced for induction.25 A total of 10 liters of bacterial culture were raised and cells were lysed by sonication and rSpSM50 protein was obtained from cell supernatants. The supernatant after centrifugation was loaded onto Ni(II)-IDA resin columns for purification and eluted with gradient concentrations (30 – 300 mM) of imidazole.26 Eluted fractions were then subjected to dialysis against enterokinase buffer (50 mM NaCl, 20 mM Tris pH 7.4)36 followed by digestion with recombinant enterokinase protease which contained a N-terminal His tag for 1 hr at 25 oC.25,26 This digestion mixture was then again purified on Ni-IDA affinity chromatography to remove the solubility/His tag and enzyme and subsequently dialyzed against storage buffer (50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 500 mM L-Arg, pH 8.0).26 Total protein yield was 4 mg per 10 L of culture and established as >

8


Page 8 of 44

Page 9 of 44

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

Biochemistry

90% pure by densitometric analysis of Coomassie Blue-stained SDS-PAGE 4-20% gradient gels (Figure 2). Protein concentrations were determined using UV spectrophotometry at 280 nm and the EXPASY ProtParm tool (http://web.expasy.org/protparam/). N-terminal sequencing of the first 8 residues confirmed the expected sequence + 1 Met residue (Table S1, Supporting Information).

Note that the rSpSM50 protein is largely disordered (Figure 1) and thus no

refolding protocols were utilized for this preparation. However, we are not absolutely certain that the CTLL domain exists in a proper fold. The purified protein was stored at -80 oC until needed. MALDI-TOF-MS analysis of rSpSM50. To precisely determine the actual molecular masses of rSpSM50 this protein was subjected to matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS)26,27,38-44 using a Bruker OmniFlex MALDI MS spectrometer in the negative ion mode.

Two matrices, Sinapinic acid (SA) and 2',5'-

dihydroxyacetophenone (DHA) were used (10 mg/mL, ThermoFisher Scientific).38-44

For SA,

the protein sample was premixed with an equal volume of sinapinic acid (SA) and a 1.5 µL aliquot of the protein + matrix sample was placed on the steel grid target, allowed to air dry and the sample was covered with 1.5 µl of matrix and the drying process was repeated again. For DHA, 0.6 µL of the matrix mixture was first spotted onto the target, followed by 1 µL of the protein sample, and then air dried. Dried samples were analyzed using a Bruker UltrafleXtreme MALDI-TOF/TOF in linear mode with a detection range of 10-95 kDa, 1.00 m/Z resolution, 500 shots/sec, pulsed laser power of 200 mJ at 337 nm.26 For calibration and comparison, the SIGMA Aldrich ProteoMass Peptide and Protein MALDI-MS Calibration kit (SIGMA-Aldrich, USA) was run using identical matrices and conditions. The mMass 5.50 software program 45 was employed for m/Z determinations.

9


Biochemistry

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 44

AFM and light microscope imaging of rSpSM50 protein phases. The aggregation characteristics of 300 nM rSpSM50 assemblies on mica substrates were performed using tapping mode AFM. 2628,33,34

The apo form [i.e., Ca(II) free] was imaged in 10 mM HEPES buffer (pH 8.0) and the

Ca(II) form was imaged in 10 mM HEPES buffer containing 10 mM CaCl 2 (pH 8.0). AFM experiments were conducted in the range of 27-31 °C using a MFP-3D-BIO instrument operating in tapping mode in a buffer solution. Olympus BL-AC40TS-C2 rectangular-shaped, gold-reflex coated silicon-nitride tips with a spring constant of approximately 0.09 N/m with a resonance frequency of 110kHz were used to achieve the best image quality were used for imaging. All samples were aliquoted onto a freshly stripped surface of mica (0.9 mm thick, Ted Pella, Inc.) and incubated for a period of 20 min at ambient temperature prior to measurement. Gwyddion software was implemented for image processing, noise filtering, and analysis, including the calculation of Rq, i.e., the surface roughness of the imaging surface.26-28,33,34 For detection of mesoscale protein hydrogel particles, 5 µL of a 10 µM solution of rSpSM50 in 10 mM HEPES, pH 8.0 and 10 mM HEPES, 10 mM CaCl2 (pH 8.0) was placed on a clean glass slide and imaged using bright field microscopy (100x lens, Nikon DS-U3 Light Microscope). Note that higher protein concentrations were required to generate sufficiently large hydrogels for visualization purposes. Flow cytometry experiments of rSpSM50 protein aggregates. The aggregation of rSpSM50 (1.5 µM final concentration) was studied in both of the AFM buffers. Samples were constituted and allowed to sit for 5 min prior to analysis. Testing was performed using a multi-parameter cell analyzer BD LSRFortessa (BD Biosciences, USA). The solution was tested in a continuous flow rate of 25 µL/min to complete 150 µL of sample tested, using four laser excitation lines: 405nm,

10


Page 11 of 44

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

Biochemistry

488nm, 561nm, and 640nm to register two light-scattering parameters (FSC-A and SSC-A) 46-48 and the number of events for each sample. Data was collected using the BD FACS DiVa (BD Biosciences, USA) software designed for the instrument. Files were later processed using FlowJo software (TreeStar, OR, USA). In vitro mineralization assays. Mineralization assays were adapted from published protocols 26-34 and were conducted by mixing equal volumes of 20 mM CaCl 2*2H2O (pH 5.5) and 20 mM NaHCO3 / Na2CO3 buffer (pH 9.75) to a final volume of 500 µL in sealed polypropylene tubes and incubating at room temperature for 1 hr.27-34 Aliquots of rSpSM50 stock solutions were added to the calcium solution prior to the beginning of the reaction, with final protein assay concentrations = 1.5 μM. The final pH of the reaction mixture was measured and found to be approximately 8.0 – 8.2.27-34 Mineral and protein deposits formed during the assay were captured on 5 x 5 mm Si wafer chips (Ted Pella, Inc.) that were placed at the bottoms of the vials. Upon completion of the mineralization assay period, the Si wafers were rinsed thoroughly with calcium carbonate saturated methanol and dried overnight at 37 oC prior to analysis. For TEM studies, a 10 µL aliquot of the mineralization assay supernatant was withdrawn at the completion of the assay period, spotted onto formvar-coated Au grids that were glow discharged for 30 sec to remove the contaminants present on the film before the sample preparation, then washed and dried as described above. Electron Microscopy. SEM imaging of the Si wafers extracted from the mineralization assays was performed using a Merlin (Carl Zeiss) field emission SEM (FESEM) using either an Everhart-Thornley type secondary electron detector (SE2) or an annular secondary electron detector (in lens) at an accelerating voltage of 1.5kV, a working distance of 4 mm, and a probe 11


Biochemistry

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 12 of 44

current of 300 pA. Prior to analysis, SEM samples were coated with iridium using a Cressington 208HR sputter coater with thickness controller attachment. Transmission electron microscopy (TEM) imaging and electron diffraction analyses of TEM were performed on a Philips CM20 transmission electron microscope equipped with a LaB6 filament electron beam source and 1024 x 1024 retractable CCD camera. All imaging and diffraction analyses were performed at 200 kV. A diffraction pattern of a polycrystalline gold standard was used as a calibration scale for all subsequently recorded diffraction patterns. The selected area diffraction (SAD) patterns were analyzed and indexed using CrysTBox software package. Focused ion beam sectioning and 3D tomographic reconstruction. Imaging of internal crystal morphology was performed using a Zeiss Auriga Small Dual-Beam FIB-SEM. For these analyses all samples were first coated with 4 nm iridium prior to SEM imaging, then coated with 50 nm of Au prior to performing FIB. A 30 kV 50 pA gallium ion beam was oriented perpendicular to the sample by tilting the sample stage to 54° and utilized to mill 10 nm serial cross sections.28,30,31 SEM images of cross-sectioned surfaces were then obtained using a 5.0 kV 200 nA electron beam and a secondary electron detector at a working distance of 5.0 mm. Images were taken shortly after cross sectioning to limit the exposure of the uncoated surfaces to the electron beam. For the 3D reconstruction, ~100 FIB cross-sectioned images were first manually segmented around the porosities and were used to construct a 3D model using the volume viewer tool in the Image J software. 28,30,31 Calcium potentiometric titrations of rSpSM50. Potentiometric titration experiments49-53 were conducted utilizing a computer-controlled system manufactured by Metrohm. The setup is composed of two devices (Titrando 809_1 and Titando 809_2) controlling three dosing devices

12


Page 13 of 44

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

Biochemistry

(800 Dosino) for dosing CaCl2 (10 mM), NaOH (10 mM) and HCl (10 mM), respectively. One Ca(II)-ion-selective electrode (Metrohm No. 6.0508.110) and one pH-electrode (Metrohm No. 6.0256.100) are utilized to monitor the calcium potential and pH, respectively. During a titration experiment, CaCl2 (10 mM) is continually dosed into carbonate buffer (10 mM, 7 mL) at a constant rate of 20 μL /min and the pH is kept constant at pH 8.5 by automatic titration of NaOH and HCl. Here, HCl titration is required for balancing the outgassing of CO 2, which starts to become significant below pH 9.00. For protein experiments, the potentiometric titrations are performed as described above, but in presence of 50 and 400 nM rSpSM50 protein. The ion products were mathematically corrected for CO2 out-diffusion in order to yield a post-nucleation solubility threshold in the reference experiments that is parallel to the x-axis, as rigorously established for 20 mL titrations at the same pH (unpublished). This correction is robust and did not affect the value of the solubility of the initially formed phases. It corrects the mass balance for the post-nucleation stage by adapting the quantification of CO 2 outgassing kinetics to the smaller buffer volume used herein (7 mL). Bioinformatics. To determine the location of disordered sequence regions within the SpSM50 sequence, we employed the DISOPRED3 54 and IUP_PRED55 prediction algorithms using default parameters. Subsequently, we utilized AGGRESCAN 56 and ZIPPER DB57 with default parameters to globally identify putative cross-beta strand sequence regions which exhibit association propensities (Figure 1). To determine a hypothetical global structure of SpSM50, we utilized the DISOclust (v1.1) - IntFOLD2 integrated protein structure and function prediction server (University of Reading, UK, using default parameters), which provides tertiary structure prediction/3D sequence homology modeling of protein sequences that contain folded and

13


Biochemistry

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 14 of 44

unfolded sequence elements.58

RESULTS

Analysis of rSpSM50 overexpression in E. coli.

SDS-PAGE analysis of rSpSM50 after

enterokinase tag cleavage and Ni(II) affinity chromatography reveals a primary Coomassie

Figure 2. (LEFT) MALDI-TOF-MS spectra of purified rSpSM50 protein in sinapinic acid (TOP) and DHA (BOTTOM). [M + H]+ = target protein, singly charged species. Peak identification and observed m/Z values are provided in each spectra. Note in each MALDI spectra the presence of a matrix-only baseline for comparison with the protein+matrix spectra. (RIGHT) 4-20% gradient SDS-PAGE gel analysis (Coomassie Blue staining) of tag-free, purified 1.5 µg SM50, shown alongside molecular weight standards (M r) and purified 2 µg bovine serum albumin (BSA). IMP = unidentified impurity protein that represents 10% of the total protein content in rSpSM50; M = monomeric rSpSM50.

stained band migrating at ~ 45 kDa, with a minor band evident at 37-39 kDa (Figure S2, Supporting Information). On the basis of densitometric scanning the purity of the major band was estimated to be ~ 90%, which is comparable to the purity reported for rSpSM30B/C 14


Page 15 of 44

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

Biochemistry

glycoprotein26 and several recombinant nacre proteins generated by the same recombinant approach.27-33 This rSpSM50 sample was subsequently analyzed using MALDI-TOF-MS to determine if, like other sea urchin spicule matrix proteins, higher-ordered complexes can form. 26 This analysis became more complex due to the fact that rSpSM50 is Pro-rich (Figure 1) and MALDIinduced N-Cα fragmentation on the N-terminal side of Pro residues can occur. 38,39 Potentially, this situation could lead to the presence of additional adduct species that do not correspond to either the singly or multiply charged protein adducts. To address this, we utilized two matrices: sinapinic acid (SA), which is considered the best matrix to preserve protein-protein interactions, and 2',5'-dihydroxyacetophenone

(DHA), a soft matrix that reduces fragmentation and

aggregation and provides a boost in sensitivity. 39-43 As shown in Figure 2, in the presence of SA, we detect the major [M + H] + species with a m/Z value of 44690 Da, which corresponds to the expected recombinant molecular weight, and the minor 10% impurity species (IMP) at 39378 Da. We also note that several broad adduct species A-E, ranging from 38 kDa to 77 kDa were detected but not present in the SDS-PAGE/Coomassie Blue gel (Figure 2); hence, these are not impurities. Turning to the DHA experiment, we see a different adduct pattern: the higher m/Z species generated in the presence of SA are not observed; instead, we observe the expected [M + H]+, IMP, and now a new set of adduct species (denoted as 1, 2 and 3) which possess m/Z values below that of the [M + H]+ species. Once again these species do not appear in the SDS-PAGE analyses and thus are not impurities. Collectively, we interpret these findings as follows: a) SA matrix allows rSpSM50 oligomerization and generates a specific fragmentation of SM50 proteinprotein complexes or aggregates that would hypothetically appear at 89 kDa (i.e., dimer) and

15


Biochemistry

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 16 of 44

higher m/Z values. Depending on the fragmentation pattern and aggregate mass these adducts are now detected at m/Z values above and below the target value of 44690 Da. b) Conversely, DHA does not promote aggregation nor much protein fragmentation. Consequently, the number of fragmentation adducts are fewer and primarily reflect fragmentation of the monomeric species. One further observation that we note is that each MALDI-TOF spectra exhibits a broad baseline (Figure 2) which is typical of the intensity fading effect that accompanies higher-order protein-protein complexes.43,44 Proteins that are aggregation-prone or experience intense intermolecular bonding oftentimes interfere with protein ionization from the matrix and induce this intensity fading effect (i.e., adduct peak broadening and intensity diminishment increase as a function of higher m/Z)43,44 which complicates the resolution of higher-order protein adduct species. In conclusion, we confirm the correct rSpSM50 polypeptide and the presence of the minor impurity via SDS-PAGE and MALDI-TOF-MS, and indirectly demonstrate that this protein is aggregation-prone. rSpSM50 self-associates to form protein hydrogel particles in solution and films on surfaces . As shown in Figure 1, bioinformatics assessments (AGGRESCAN56, ZIPPER DB57, IUP_PRED55, DISOPRED354) indicate the SpSM50 sequence contains 5 amyloid-like cross-beta strand aggregation prone regions, 4 of which occur within the conserved CTLL domain (V8-S15, P43L49, Q56-Q63, N99-G100). In addition, this protein is highly disordered, with the CTLL domain possessing 3 short disordered regions (A44-V52, I61-A66, E75-N76) and the MAQPG domain and C-terminal segment (F101-A428) being completely disordered. As discussed for the recombinant spicule matrix glycoprotein, rSpSM30B/C, 26 and several other nacre-associated proteins,27,29,31-35 the presence of aggregation-prone and disordered domains is directly correlated 16


Page 17 of 44

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

Biochemistry

to aggregation and hydrogelation59,60 within ionic environments such as those found within in vitro mineralization assays. To confirm this for SM50, we performed AFM (Figure 3), light microscopy, and flow cytometry experiments26,46-48 (Figure 4) on rSpSM50 within pH 8.0 environments in the presence and absence of Ca(II) ions. The goal of these experiments was to ascertain how rSpSM50 would respond to low (10 mM HEPES) and high (10 mM HEPES, 10 mM CaCl 2) ionic strength environments that utilize the same pH and Ca(II) concentrations as in vitro mineralization assays.26-33 On mica surfaces, we find that at 300 nM rSpSM50 forms a small number of large complexes at pH 8.0, and, as indicated by the surface roughness parameter, R q, relative to plain mica we see that the rSpSM50 R q value is higher and thus indicative of protein film formation under these conditions (Figure 3, dataset). 26-33 In the presence of Ca(II) ions, these complexes do not increase in number but do increase in dimension (by a factor of 2) and the corresponding protein films become thicker (by a factor of 10). Thus, Ca(II) ions increase the aggregation propensity of rSpSM50 at pH 8.0.. We note that rSpSM30B/C 25 and other mollusk shell nacre proteins27-33 also exhibited aggregation enhancement on mica surface in the presence of Ca(II). We attribute these increases to either a Ca(II) specific interaction between protein molecules, or, to the general phenomenon of non-specific electrostatic interactions and charge neutralization that occur between Asp, Glu residues on rSpSM50 (Figure 1) and Ca(II) ions. 26-33,59,60 Given that the CTLL domain contains 8 of the 12 total Asp, Glu anionic residues in this protein (Figure 1), we speculate that this domain may trigger additional aggregation of rSpSM50 when Ca(II) or other ions are introduced. These AFM findings are confirmed by light microscopy imaging and flow cytometry 17


Biochemistry

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 18 of 44

analysis,46-48 which show that rSpSM50 at pH 8.0 forms mesoscale assemblies that appear as porous hydrogel particles (Figure 4). This is similar to what has been reported for the nacre proteins AP759 and n16.360 as well as the recombinant spicule matrix glycoprotein, rSpSM30B/C.26 These rSpSM50 hydrogels become more complex in appearance when Ca(II) is present, where we see what appears to be either external blebs or internal voids that have increased in size. This finding suggesting that there are dimensional and internal changes that Figure 3. Tapping mode AFM imaging of 300 nM rSpSM50 protein phase in 10 mM HEPES (pH 8.0) and 10 mM

HEPES/10 mM CaCl2 (pH 8.0). AFM tapping mode amplitude images (top, scalebars = 1 µm) are plotted at 3 µm x 3 µm as are the z-plots (bottom). “AFM dataset” refers to statistical measurements of mean particle heights (H), diameters (D), and surface roughness factor (R q ) ± S.D., taken for 10 particles in each buffer condition. The R q values for plain mica in each buffer condition were subtracted from protein values.

occur to these hydrogels in response to Ca(II) ion introduction. To verify this, we analyzed the rSpSM50 hydrogel particles using flow cytometry, which allows us to determine the particle size distributions (FSC, forward scattered component, x-axis) versus

granularity or internal

complexity (SSC, side-scattered component, y-axis) of the particles. 46-48;59,60 As shown in the lower panel of Figure 4, the 2D density plots show that rSpSM50 hydrogel particles experience

18


Page 19 of 44

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

Biochemistry

significant shifts in particle size distribution and internal complexity or structure

46-48;59,60

in the

presence of 10 mM Ca(II), which correlates with our light microscopy findings as shown in the upper panel of Figure 4. Thus, rSpSM50 is a hydrogelator that forms mesoscale porous-

Figure 4. TOP: Light microscope images taken at 100x magnification of 10 µM rSpSM50 in 10 mM HEPES, pH 8.0, and 10 mM HEPES / 10 mM CaCl 2, pH 8.0. Scalebar = 10 microns. BOTTOM: Flow cytometry 2-D density plots of FSC as a function of SSC. Here, forward scattered light (FSC, x-axis) determines particle size distributions; and side-scattered light (SSC, y-axis) measures refracted and reflected light that occurs at any interface within the particles where there is a change in refractive index (RI) that results from variations in particle granularity or internal structure.46-48

appearing particles in solution (Figure 4) and protein particles and films on charged surfaces (Figure 3) at pH 8.0, and these species increase in dimension and internal complexity in a Ca(II) environment. rSpSM50 modifies the surface of calcite crystals and introduces intracrystalline voids within calcite and vaterite. The next step in our investigation was to ascertain how rSpSM50 hydrogel particles affect calcium carbonate crystal formation in vitro. Using our standardized calcite-based 19


Biochemistry

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 20 of 44

mineralization 60 min assay, which allows protein aggregation to occur in synchrony with the nucleation process,26-33 we tracked the formation and morphology of crystals that deposited onto

Figure 5. SEM images of mineral deposits captured on Si wafers within in vitro assays containing 1.5 µM rSpSM50 (A-E) and without protein (F). (A-C) rhombohedral calcite crystals featuring extensive protein hydrogel deposition (arrows) and evidence of organized nanotexturing on exposed crystal surfaces. (D) Vaterite crystal featuring evidence protein hydrogel deposition (white arrows) and (E) higher magnification image revealing the presence of porosities or void chambers near the surface of these vaterite crystals. Note that these voids or porosities have outlines that correspond to the amorphous morphology of the surrounding protein hydrogel deposits. MicroRaman microscopy studies confirm the presence of both calcite and vaterite crystals in these assays (Figure S2, Table S2, Supporting Information).

Si wafers (Figure 5). We observed significant protein hydrogel deposition (Figure 5A) and two crystalline polymorphs forming in the presence of these hydrogels: 1) highly modified rhombohedral calcite crystals that contain organized nanotexturing and evidence of protein hydrogel adsorption (Figure 5B,C; note that nanotextured calcite surfaces were also generated by a number of nacre-specific proteins).27-33 2) vaterite crystals that contain evidence of proteininduced porosities or voids (Figure 5D,E), i.e., nanoporous vaterite. As discussed in previous intracrystalline studies, presumably, these voids represent areas where the hydrogels deposited onto forming crystals and interfered with crystal growth.27-32 The presence of both polymorphs in 20


Page 21 of 44

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

Biochemistry

Figure 6. SEM images of FIB-sectioned calcite crystals. (A) (-) rSpSM50 control calcite crystal, featuring minimal porosities; (B,C) (+) rSpSM50 calcite crystal sections where one can clearly denote multiple subsurface void or porosities (white arrows). (D-F) Representative 3D tomographic reconstruction of void regions superimposed upon corresponding FIB/SEM serial sections (D = slice 10; E = slice 30; F = slice 60 of 60 sections) of calcite crystals grown in the presence of rSpSM50.

these rSpSM50 assays were confirmed by microRaman spectroscopy (Figure S2, Table S2, Supporting Information); given that this is a calcite-based assay system we did not observe significant vaterite formation in control assays (Figure 5F). This suggests that rSpSM50 hydrogels may promote or stabilize vaterite under the mineralization conditions utilized in this study. Based upon the presence of voids or porosities detected at the surface of vaterite crystals, and the fact that SpSM50 exists as an intracrystalline species, 4-8,11-19 we were curious to learn if the nanotextured calcite crystal also contained intracrystalline voids or porosities as well. Thus, we performed FIB tomography (repetitive serial FIB sectioning and SEM imaging as described in Experimental Methods on a representative control (Figure 6A) and rSpSM50-modified (Figure 21


Biochemistry

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

6B,C) calcite crystals obtained from the 60 min assay. From 60 serial sections (10 nm thick) we generated both an internal 3-D reconstruction of the internal region of the rSpSM50-modified crystal and superimposed these images on the corresponding SEM image (Figure 6D-F). This tomographic image provides us with interior views of subsurface calcite crystal modifications induced by rSpSM50. As one can see,

compared to (-) rSpSM50 control assays, serial

sectioning of (+) rSpSM50 crystals reveals the presence of intracrystalline voids or porosities (Figure 6A-C). With the 3D reconstruction we can see that the majority of these voids are interconnected species within the calcite crystal, similar to the findings reported for calcite crystals grown in the presence of nacre protein hydrogels. 28,30,31 Thus, rSpSM50 self-associates under mineralization conditions to form hydrogels that deposit onto forming crystals. These hydrogels create surface nanotexturing and subsurface intracrystalline nanochambers, and in the process, generate a porous inorganic material, similar to mineralized sea urchin spicules.4-8,11-19

rSpSM50 hydrogels organize calcite and vaterite mineral nanoparticles. Next, we investigated the formation of hydrogel particles that occur within the same assay supernatants at 60 min via supernatant sampling and TEM imaging (Figure 7). Compared to (-) rSpSM50 control scenarios, where typical calcite crystals were discovered (Figure 7A), (+) rSpSM50 samples contained protein hydrogels (albeit alcohol-dehydrated) that contain mineral nanoparticles within the hydrogel framework (Figure 7B-D). In some instances these hydrogels possessed unusual calcite nanoparticles that appears sickle-like, elongated, and faceted (Figure 7C; Figure S3, Supporting Information), whereas the vaterite nanoparticles appeared condensed with some evidence of crystal elongation (Figure 7D; Figures S3, S4, Supporting Information). We note that faceted calcite nanoparticles were also generated by another recombinant S. purpuratus protein, 22


Page 22 of 44

Page 23 of 44

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

Biochemistry

rSpSM30B/C,26 but to date faceted calcite nanoparticles have not been observed in the presence of nacre protein hydrogels.27-34 Selected area diffraction (SAD) measurements confirm that these rSpSM50 hydrogels contain either single calcite (Figure 7C) or polycrystalline vaterite (Figure 7D) nanoparticles (Figures S3, S4, Supporting Information), which corroborates with our SEM and microRaman findings (Figure 5; Figure S2, Table S2, Supporting Information). Thus, like other nacre protein and spicule matrix hydrogels, rSpSM50 hydrogels act as nanoparticle organizers within in vitro mineralization assay environments and permits the formation of two calcium carbonate polymorphs. This organizational and assembly capability was also noted for the 26-AA SM50M peptidomimetic aggregate in recent studies.25

Figure 7. TEM images and corresponding SAD patterns of supernatant samples taken from (A) (-) rSpSM50 and (B-D) (+) rSpSM50 60 min mineralization assays. In (C), note elongated, faceted crystals. In (D), note elongated crystals; an enlarged view of these crystals can be found in Figure S4, Supporting Information. For fully indexed SAD patterns please consult Figure S3, Supporting Information.

rSpSM50 hydrogels modulate the early events in non-classical calcium carbonate nucleation in bulk solution. It has been established that nacre proteins, 27-32 the sea urchin recombinant spicule matrix protein rSpSM30B/C,26 and select domain fragments of the SM50 protein 11 modulate the early events in the non-classical nucleation pathway of calcium carbonates, i.e., the formation of pre-nucleation clusters (PNCs) and their assembly into amorphous calcium carbonate (ACC).27,29,32,34

To determine if this function is also common to rSpSM50, we performed

23


Biochemistry

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 24 of 44

standardized Ca(II) potentiometric titration experiments (Figure 8; Table S3, Supporting Information).49-53 These experiments were conducted at pH 8.5 to explore the effects of rSpSM50 hydrogel particles on the early stages of calcium carbonate formation under relevant conditions. By accounting for CO2 outgassing and maintaining a constant pH level, the influence of the protein on pre-nucleation cluster formation, on the kinetic stability of the metastable system against nucleation, and on the thermodynamic stability of the initially precipitated ACC can be quantified.49-53 Note that the potentiometric experiments, being electrode-based, can only report on nucleation events that occur in bulk solution and not within the hydrogels themselves.59,60 The standard potentiometric curve provides information on the formation and stabilities of PNCs in bulk solution.49-53 As free Ca(II) is added to the carbonate solution, ion complexes (i.e., PNCs) form and this is represented by the initial linear region of the titration curve. Where the measured free Ca(II) decreases upon further addition of CaCl 2 (i.e., the peak region), this marks the start of solid phase nucleation (e.g., ACC) from PNCs. Recent work showed that ACC formation is initiated by the liquid-liquid separation of PNC precursors, and proceeding via their aggregation and solidification.50 The slope of the prenucleation regime (i.e., the initial linear region) provides indirect evidence of the interaction between additive molecules and solute PNCs, leading to their thermodynamic stabilization (i.e., SlopeAdditive < SlopeRef) or destabilization (SlopeAdditive > SlopeRef).49-53 As shown in Figure 8A, similar to charged polymers

44-48

and other

nacre27-32 and sea urchin matrix proteins,26 at 50 nM and 400 nM rSpSM50 concentrations we note no significant change of the pre-nucleation slope in comparison with the reference experiment, showing that rSpSM50 hydrogels do not affect the thermodynamic stability of PNCs in bulk solution at the applied concentrations. However, the nucleation of a solid, which

24


Page 25 of 44

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

Biochemistry

Figure 8. Development of (A) free Ca (II) ion concentration and (B) calcium carbonate ion product in potentiometric titrations without and with 50 nM and 400 nM rSpSM50, as indicated, in 10 mM carbonate buffer at pH 8.5 as a function of time. In each plot the reference curve refers to experiments conducted in the absence of protein. Experiments were performed in duplicate, and the dashed horizontal line gives the solubility threshold of the initially precipitated ACC in the reference experiment (see Table S3, Supporting Information).

becomes apparent by the drop in free calcium (Figure 8A) and ion product (Figure 8B), is postponed corresponding to a retarding factor of 1.6 and 4.7, respectively (Table S3, Supporting Information). This retardation effect is due to the presence of the protein hydrogels (Figures 3,4), inhibiting the aggregation and solidification of the calcium carbonate precursors,50,52 whereas the critical supersaturation conditions in bulk free solution is significantly higher than in the reference experiment without the protein (Figure 8B).58 In the presence of 50 nM rSpSM50, the solubility of the initially precipitated ACC phase is higher than in the reference scenario (Figure 8B, Table S3, Supporting Information; note higher final baseline compared to reference at dotted line, where initial solubility threshold for 50 nM rSpSM50 = 3.69 E-8 M2 and reference threshold = 3.02 E-8 M2, indicative of protocalcite ACC). This suggests shows that the rSpSM50 hydrogels promote the formation of more

25


Biochemistry

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

soluble forms of calcium carbonate which initially forms in bulk solution.48-52 This could be due to the kinetic stabilization of transient forms that also occur in the reference scenario, or the induction of thermodynamically less stable short-range orders that otherwise do not occur without the protein. For the 400 nM sample, we note the continuously decreasing solubility that occurs relative to the reference state (Figure 8B); these features indicate that a more soluble form of ACC is transient, and rather quickly transforms into the same form that is also observed in the reference experiment, and then into crystals. The latter transformation, however, is not seen in the progression of the solubility threshold (Figure 8B), because it is governed by the presence of the soluble ACC forms as long as they are present, and is thus insensitive towards the presence of the first crystals.48-52 Note that when this process is independent of the protein concentration, the thermodynamically more soluble ACC would have formed initially also at the higher protein concentration, but would have already transformed by the time the solubility threshold is eventually established. Hence, a transient form of ACC precipitates in bulk solution in the presence of rSpSM50 hydrogels, and this ACC is more soluble than the ACC formed initially in the control experiment, i.e., essentially, rSpSM50 hydrogels thermodynamically destabilize ACC in bulk solution, either through direct interactions with the mineral phase or via other mechanisms. It can be speculated that this ACC has a different short-range structure that might relate to that of proto-vaterite ACC, which would potentially explain the detected presence of vaterite in our assays (Figures 5,7). The molecular basis of rSpSM50 assembly. The putative molecular features which enable rSpSM50 to form protein hydrogels can be better understood using the DISOclust IntFOLD2 predicted 3D modeling prediction program, which uses sequence homology modeling to predict

26


Page 26 of 44

Page 27 of 44

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

Biochemistry

conformational preferences for both folded and unfolded regions (Figure 9). 58 Here, we observe the putative Q1-F101 CTLL domain as an accessible C-type folded domain 61-63 at the N-terminal portion of the protein, with the F101-Q392 MAQPG region existing as an unfolded, open, accessible structure, similar to what was reported in previous SM50 bioinformatics studies. 11 Overall, the SpSM50 molecule appears as an extended protein with a small globular N-terminal region, not unlike that predicted for other hydrogelation proteins such as the model spicule matrix protein, SpSM30B/C,26 and the Type A von Willebrand-containing nacre framework protein, Pif97.27 The significance of an extended protein molecule is the presence of accessible regions for protein-mineral and/or protein-protein interactions.27-34;53,54 Upon further inspection and referencing to Figure 1, we believe that this accessible protein molecule has two putative sites for multipurpose molecular interactions: the CTLL domain and the MAQPG domain. We consider the CTLL domain as an interactive region for the following reasons: the combined presence of an interactive alpha-helical fold, 61-63 disordered and cross-beta strand regions (V8-S15; P43-L49; Q36-Q63)(Figure 1), and 8 anionic residues. This combination creates a putative site for protein-protein or protein-mineral interactions (Figures 3-6).11 The other interactive site would be the MAQPG sequence region F101 – Q392; this is a highly disordered or unfolded region (Figure 1) which would destabilize the internal structure of SM50 and increase its overall self-association propensity, 27-34;53,54 which is consistent with experimental studies conducted with this domain. 11 We should mention that in addition to the high degree of disorder, there is one other property of the MAQPG region that is unique: the presence of Arg (Figure 1), which would create a highly cationic or hydrogen-bonding region within SM50. These unique residues may also play a role in protein-protein or protein-

27


Biochemistry

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

mineralinteractions on some level. In conclusion, our bioinformatic studies indicate that

Figure 9. Multiple views of DISOclust IntFOLD2 predicted 3D modeling predicted structure of the SpSM50 protein. In ribbon representation (backbone only). N- and C-termini are denoted, as is the location of the conserved CTLL domain (Q1 – G100, circled). The MAQPG domain (F101 – Q392) resides outside the circled region. Color designations of backbone regions: red = anionic; blue = cationic; green = polar; white = hydrophobic. The top five models for the conserved CTLL domain region had confidence and P value scores of 5.418 E-1 and global model quality scores of 0.049, which are not very high, and this fold exhibited the best fit to three different CTLL template crystal structures: 1) 1vt4I3 (Drosophilla apoptosome Apaf-1 related killer protein);61 2ox9A (Mouse scavenger receptor c-type lectin carbohydrate-recognition domain), 62 and 4uWW (struthiocalcin-1, an intramineral protein from Struthio camelus eggshell).63 The overall poor fit can be attributed to the existence of 3 disordered regions within the CTLL domain of SM50 (A44 – V52; I61 – A66; E75 - E77, Figure 1) which are not found in the other template CTLL structures.61-63

the molecular properties of the CTLL and MAQPG domains potentially create a highly reactive rSpSM50 protein molecule.

DISCUSSION

The intracrystalline sea urchin spicule matrix protein, SpSM50, is a major spicule matrix 28


Page 28 of 44

Page 29 of 44

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

Biochemistry

component and has been hypothesized as an important protein not only for establishing the ECM matrix for mineral deposition and maturation but also for controlling the mineralization process itself. Using the recombinant rSpSM50 protein (Figure 2), we verify these hypotheses to be true for in vitro conditions.

Our study shows that this recombinant protein possesses a high

aggregation propensity, owing to the presence of cross-beta strand amyloid-like domains within the CTLL region and a major disordered domain in the MAQPG sequence (Figure 1). Thus, this combination of aggregation propensity and open, unfolded structure create a highly reactive protein molecule (Figure 9) that can assemble to form nanoscale protein particles and films on charged surfaces (Figure 3) and mesoscale hydrogels in solution (Figure 4). These hydrogels are dimensionally and internally heterogeneous and these parameters are enhanced by the presence of Ca(II)(Figures 3,4). These findings correlate with the reported aggregation-prone behavior of the SpSM50 protein11,21 and suggest that in the spicule the ubiquitous SM50 protein could create an ECM consisting of a hydrogel-like environment. Such a hydrogel environment could limit both

Ca(II)

and

carbonate/bicarbonate

ion

diffusion,

create

limited

volumes,

and

compartmentalize nucleation deposits, thereby controlling important nucleation processes such as ACC formation,22-24 polymorph selection,64 nanoparticle mineral assembly25,64 and transformation65 in the spicule. The rSpSM50 protein hydrogel exerts a definite effect on the early stages of the calcium carbonate nucleation process in bulk solution (Figure 8A,B; Table S3, Supporting Information). There is a protein-concentration-dependent kinetic stabilization of calcium carbonate solutions against nucleation but no effect on PNC stabilities. Thus, similar to rSpSM30B/C, rSpSM50 hydrogels neither stabilize nor destabilize these mineral particles. With regard to ACC

29


Biochemistry

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

solubilities in bulk solution, our experiments show that rSpSM50 hydrogels thermodynamically destabilize ACC (Figure 8B, Table S3, Supporting Information), but apparently allow ACC nucleation and subsequent crystal formation to take place within the hydrogels themselves (Figure 7). Using identical techniques, earlier studies with the model spicule glycoprotein rSpSM30B/C showed the opposite effect on ACC in bulk solution: 26 these hydrogels thermodynamically stabilize ACC while allowing nucleation and calcite crystal formation within the gel matrix itself. Together, these findings reveal an intriguing scenario within the spicule during spiculogenesis: the presence of protein hydrogels that either stabilize (SpSM30B/C) or destabilize (SpSM50) ACC in bulk solution but foster nucleation and crystal formation within the hydrogel environments. These phenomena may be relevant for managing the ACC formation and transformation processes that take place during spiculogenesis4-8,11-19 We note that our rSpSM50 findings are in contrast to reported studies of another recombinant SpSM50, which apparently stabilized ACC in vitro. 24 Note, however, that these previous ACC studies examined the effect of recombinant SpSM50 within a droplet of water in contact with a calcite single crystal (i.e., dissolution and reprecipitation experiments with no control over ion concentrations, pH, or ACC formation), whereas our studies examined de novo ACC formation in bulk solution under controlled pH and ion concentrations in the presence and absence of rSpSM50 hydrogels (i.e., non-classical nucleation). 49-53 Hence, we believe that these earlier studies are reflective of induced dissolution conditions and subsequent SM50 stabilization of ACC in the presence of a crystalline mineral phase. 24 Clearly, in situ studies involving SpSM50 and ACC formation are necessary in order to understand the true role that this protein has on ACC formation and transformation within the spicule matrix.

30


Page 30 of 44

Page 31 of 44

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

Biochemistry

The ability of the rSpSM50 hydrogel to control the later events in mineralization (i.e., crystal formation) were also witnessed in our studies (Figures 5-7; Figures S2, S3, Table S2, Supporting Information). The rSpSM50 hydrogels deposit onto pre-existing calcite and vaterite crystals, leading to surface nanotexturing and altered crystal morphology (Figure 5). In addition to crystal modifications, the rSpSM50 hydrogels also nucleate and organize calcite and vaterite mineral nanoparticles (Figure 7). These phenomena were also reported for a number of nacreassociated proteins,27-32 the sea urchin spicule matrix model glycoprotein, rSpSM30B/C, 26 and the 26 AA SM50 polypeptide aggregates. 25 However, what is distinctive about rSpSM50 is the following: (1) vaterite formation takes place alongside calcite nucleation, (Figures 5, 7; Figures S2, S3, Table S2, Supporting Information), which is not common to either nacre-associated or rSpSM30B/C proteins under conditions identical to that utilized in the present study. 26-32 The presence of two polymorphs may reflect the fact that rSpSM50 exerts control over ACC stabilities (Figure 8) and may kinetically stabilize proto-vaterite ACC, which in our assay systems either transforms into proto-calcite ACC (and then to calcite) or directly into vaterite crystals (Figures 5,7). 2) The rSpSM50 hydrogels foster the formation of sickle-shaped, faceted calcite crystals within the hydrogels themselves, which is not common to nacre-associated proteins27-32 but has been observed for rSpSM30B/C.26 This suggests that there is some shared chemical and/or physical property of both rSpSM30B/C and rSpSM50 hydrogels that fosters faceted crystal growth. At this time we do not know what this property is and we intend to investigate this in future studies of both proteins. One of the intriguing aspects of spicule matrix proteins is their ability to intercalate or become occluded within the mineral phase of the spicule, leading to the induction of fracture-

31


Biochemistry

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 32 of 44

resistance.4-8,11-19 Although fracture resistance could not be assessed in our current study, the occlusion or entrapment of rSpSM50 hydrogels was clearly observed in our SEM and FIB imaging studies (Figures 5,6), i.e., the creation of nanoporous calcite and vaterite crystals. These intracrystalline features are “Swiss-cheese”66 in appearance, randomly distributed, and include individual nanoporosities as well as interconnected channels; similar intracrystalline features have also been noted in other in vitro nacre protein studies.27,28,30,31 As postulated for nacre proteins, we believe that the formation of porous regions and interconnected channels occur as a result of rSpSM50 protein hydrogels depositing onto existing crystals surfaces, with subsequent overgrowth of the mineral phase that leads to subsurface hydrogel entrapment. 27,28,30,31 This is consistent with the findings of several in vitro studies which explored the phenomena of intracrystalline inclusions and macromolecular incorporation in calcium carbonates. 66-75 Some of these studies involved the use of sponge-like templates 70,73,74 and hydrogels,66,68,75 both of which create nanoporous calcite. The mechanism for intracrystalline inclusion of templates is straightforward: porosities form as the calcite overgrowth phase expands over and into the accessible porous regions of the template.70,73,74 However, for hydrogels the porosity formation and macromolecular incorporation scenarios are much more complex 66,68 and several mechanisms for these phenomena have been postulated, including force competition, mass competition, hydrodynamic force, and gel resistance to crystallization pressure. 75 We postulate that one or more of these hydrogel-based mechanisms are at work in the formation of rSpSM50induced intracrystalline porosities in vitro (Figures 5,6), and may be the basis for SpSM50 and other spicule matrix protein inclusions within mineralizing sea urchin spicules. 4-8,11-19 We await future experimentation that will verify these possibilities in the sea urchin spicule.

32


Page 33 of 44

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

Biochemistry

ACKNOWLEDGMENTS

AFM imaging was conducted at the Molecular Cytology Core

Facility, Memorial Sloan-Kettering Cancer Center, New York. Flow cytometry studies were conducted at the Department of Microbiology, Columbia University, New York. This report represents contribution number 83 from the Laboratory for Chemical Physics, New York University. Funding sources: Portions of this research (recombinant protein synthesis, LM, flow cytometry, AFM, SEM, FIB, TEM) were supported by the Life Sciences Division, U.S. Army Research Office, under award W911NF-16-1-0262 (JSE). The potentiometric experiments were supported by the Zukunftskoleg of the University of Konstanz (DG).

Supporting Information Available. Primary sequence of rSpSM50 subcloned sequence including tag location (Figure S1); N-terminal sequencing of the first 8 residues of rSpSM50 (Table S1); Experimental procedures and microRaman spectra of Si-wafer collected mineral deposits (Fig S2; Table S2); Fully indexed SAD patterns of control and rSpSM50-containing mineral nanoparticles (Fig S3); Large scale TEM image of vateite deposits within rSpSM50 hydrogels (Figure S4); Ca(II) potentiometric titration data obtained for reference and rSpSM50 samples (Table S3). This material is available free of charge via the Internet at http://pubs.acs.org.

33


Biochemistry

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 34 of 44

REFERENCES 1 Studart, A.R. (2012) Towards high-performance bioinspired composites. Adv. Materials 24, 5024-5044. 2 Cameron, R.A., Samanta, M., Yuan, A., He, D., Davidson, E. (2009) SpBase: the sea urchin genome database and web site. Nucleic Acids Research. http://www.spbase.org/ 3 Sea urchin genome sequencing consortium et. al., (2006) The genome of the sea urchin Strongylocentrotus purpuratus. Science 314, 941-952. 4 Mann, K., Poustka, A.J., Mann, M. (2008) The sea urchin (Strongylocentrotus purpuratus) test and spine proteomes. Proteome Science 6, 1-10. 5 Mann, K., Wilt, F.H., Poustka, A.J. (2010) Proteomic analysis of sea urchin (Strongylocentrotus purpuratus) spicule matrix. Proteome Science 8, 1-12. 6 Seto, J., Ma, Y., Davis, S.A., Meldrum, F., Schilde, U., Gourrier, A., Jaeger, C., Cölfen, H. (2012) Structure-property relationships of a biological mesocrystal in the adult sea urchin spine. Proc. Natl. Acad. Sci USA 109, 3699-3704. 7 Berman, A., Addadi, L., Kvick, A., Leiserowitz, L., Nelson, M., Weiner, S. (1990) Intercalation of sea urchin proteins in calcite: Study of a crystalline composite material. Science 250, 664667. 8 Aizenberg, J., Hanson, J., Koetzle, T.F., Weiner, S., Addadi, L. (1997) Control of macromolecular distribution within synthetic and biogenic single calcite crystals. J. Am. Chem. Soc. 119, 881-886.

34


Page 35 of 44

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

Biochemistry

9 Sharmankina, V.V., Kiselev, K.V. (2016) Expression of SM30(A-F) genes encoding spicule matrix proteins in intact and damaged sea urchin Strongylocentrotus intermedius.

Russ. J.

Genetics 52, 298-303. 10 Wilt, F., Killian, C.E., Croker, L., Hamilton, P. (2013) SM30 protein function during sea urchin larval spicule formation. J. Struct. Biol. 183, 199-204. 11 Rao, A., Seto, J., Berg, J.K., Kreft, S.G., Scheffner, M., Cölfen, H. (2013) Roles of larval sea urchin spicule SM50 domains in organic matrix self-assembly and calcium carbonate mineralization. J. Struct. Biol. 183, 205-215. 12 Wu, C.-H., Park, A., Joester, D. (2011) Bioengineering single crystal growth. J. Am. Chem. Soc. 133, 1658–1661. 13 Seto J., Zhang, Y., Hamilton, P., Wilt, F.H. (2004) The localization of occluded matrix proteins in calcareous spicules of sea urchin larvae. J. Structural Biology 148, 23-30. 14 Livingston, B.T., Killian, C.E., Wilt, F., Cameron, A., Landrum, M.J., Ermolaeva, O., Sapojnikov, V., Maglott, D.R., Buchanan, A.M., Ettensohn, C.A. (2006) Genome-wide analysis of biomineralization-related proteins in the sea urchin Strongylocentrotus purpuratus. Devel. Biol. 300, 335-348. 15 Killian, C.E., Wilt, F.H. (1996) Characterization of the proteins comprising the integral matrix of Strongylocentrotus purpuratus embryonic spicules. J. Biol. Chem. 271, 9150–9159. 16 Killian, C.E., Wilt, F.H. (2008) Biomineralization of the echinoderm endoskeleton. Chem. Rev. 108, 4463–4474.

35


Biochemistry

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

18 Kitajima, T., Urakami, H. (2000) Differential distribution of spicule matrix proteins in the sea urchin embryo skeleton. Dev. Growth Differ. 42, 295–306. 19 Urry, L.A., Hamilton, P.C., Killian, C.E., Wilt, F.H. (2000) Expression of spicule matrix proteins in the sea urchin embryo during normal and experimentally altered spiculogenesis. Dev. Biol. 225, 201–213. 20 Wilt, F.H., Ettensohn, C.E. (2007) Morphogenesis and biomineralization of the sea urchin larval endoskeleton. In: Baeuerlein, E. (Ed.), Handbook of Biomineralization. Wiley-VCH, Weinheim, pp. 183–210. 21 Wilt, F., Croker, L., Killian, C.E., McDonald, K. (2008) The role of LSM34/SpSM50 in endoskeletal spicule formation in sea urchin embryos. Invert. Biol. 127, 452–459. 22 Tester, C.C., Wu, C.-H., Krejci, M. R., Mueller, L., Park, A., Lai, B., Chen, S., Sun, C., Joester, D. (2013) Time-resolved evolution of short- and long-range order during the transformation of ACC to calcite in the sea urchin embryo. Adv. Fun. Mat. 23, 4185-4194. 23 Politi, Y, Metzler, R.A., Abrecht, M., Gilbert, B., Wilt F.H., Sagi, I, Addadi, L., Weiner, S., Gilbert, P. (2008) Transformation mechanism of amorphous calcium carbonate into calcite in the sea urchin larval spicule. Proc Natl Acad Sci U S A. 105, 17362-17366. 24 Gong, Y.U.T., Killian, C.E., Olson, I.C., Appathurai, N.P., Amasino, A.L., Martin, M.C., Holt, L.J., Wilt, F.H., Gilbert, P. (2012) Phase transitions in biogenic amorphous calcium carbonate. Proc Natl Acad Sci USA. 109, 6088-6093.

36


Page 36 of 44

Page 37 of 44

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

Biochemistry

25 Mao, Y., Satchell, P.G., Luan, X., Diekwisch, T.G.H. (2016) SM50 repeat polypeptides selfassemble into discrete matrix subunits and promote appositional calcium carbonate crystal growth during sea urchin tooth biomineralization. Annals of Anatomy 203, 38-46. 26 Jain, G., Pendola, M., Rao, A., Cölfen, H., Evans, J.S. (2016) A model sea urchin spicule matrix protein self-associates to form mineral-modifying protein hydrogels. Biochemistry 55, 4410-4421. 27 Chang, E.P., Evans, J.S. (2015) Pif97, a von Willebrand and Peritrophin biomineralization protein, organizes mineral nanoparticles and creates intracrystalline nanochambers. Biochemistry 54, 5348-5355. 28 Chang, E.P., Williamson G., Evans, J.S. (2015) Focused ion beam tomography reveals the presence of micro-, meso-, and macroporous intracrystalline regions introduced into calcite crystals by the gastropod nacre protein AP7. Crystal Growth and Design 15, 1577-1582. 29 Perovic, I., Chang, E.P., Verch, A., Rao, A., Cölfen, H., Kroeger, R., Evans, J.S. (2014) An oligomeric C-RING nacre protein influences pre-nucleation events and organizes mineral nanoparticles. Biochemistry 53, 7259-7268. 30 Chang, E.P., Russ, J.A., Verch, A., Kroeger, R., Estroff, L.A., Evans, J.S. (2014) Engineering of crystal surfaces and subsurfaces by an intracrystalline biomineralization protein. Biochemistry 53, 4317-4319. 31 Chang, E.P., Russ, J.A., Verch, A., Kroeger, R., Estroff, L.A., Evans, J.S. (2014) Engineering of crystal surfaces and subsurfaces by framework biomineralization protein phases. Cryst.Eng. Commun. 16, 7406-7409. 37


Biochemistry

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 38 of 44

32 Perovic, I., Chang, E.P., Lui, M., Rao, A., Cölfen, H., Evans, J.S. (2014) A framework nacre protein, n16.3, self-assembles to form protein oligomers that participate in the post-nucleation spatial organization of mineral deposits. Biochemistry 53, 2739-2748. 33 Perovic, I., Mandal, T., and Evans, J.S. (2013) A pseudo EF-hand pearl protein self-assembles to form protein complexes that amplify mineralization. Biochemistry 52, 5696-5703. 34 Evans, J.S. (2013) “Liquid-like” biomineralization protein assemblies:

A key to the

regulation of non-classical nucleation. Crystal Engineering Communications 15, 8388 – 8394. 35 Evans, J.S. (2012) Identification of intrinsically disordered and aggregation - promoting sequences within the aragonite-associated nacre proteome. Bioinformatics 28, 3182-3185. 36 Terpe, K. (2003) Overview of tag protein fusions:

From molecular and biochemical

fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 60, 523-533. 37 Catanzariti, A.M., Soboleva, T.A., Jans, D.A., Board, P.G., Baker, R.T. (2004) An efficient system for high-level expression and easy purification of authentic recombinant proteins. Protein Science 13, 1331-1339. 38 Takayama, M. (2016) MALDI in-source decay of protein: The mechanism of c-Ion formation. Mass Spect. 5, 1-12. 39 Liu, Z., Schey, K.L. (2005) Optimization of a MALDI-TOF-TOF mass spectrometer for intact protein analysis. J. Am. Mass Spectrom. 16, 482-490. 40 Seeley, E.H., Caprioli, R.M. (2008) Molecular imaging of proteins in tissues by mass spectrometry. Proc. Natl. Acad. Sci USA 105, 18126-18131.

38


Page 39 of 44

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

Biochemistry

41 Asara, J.M. Allison, J. (1999) Enhanced detection of phosphopeptides in matrix-assisted laser desorption/ionization mass spectrometry using ammonium salts. J. Am. Soc. Mass Spec 10, 3542. 42 Stringano, E., Cramer, R., Hayes, W., Smith, C., Gibson, T., Mueller-Harvey, I. (2011) Deciphering the Complexity of Sainfoin (Onobrychis viciifolia) Proanthocyanidins by MALDITOF mass spectrometry with a judicious choice of isotope patterns and matrixes. Anal. Chem. 83, 4147–4153. 43 Yanes, O., Villanueva, J., Querol, E., Aviles, F.X. (2007) Detection of non-covalent protein interactions by ‘intensity fading’ MALDI-TOF mass spectrometry: applications to proteases and protease inhibitors. Nature Protocols 2, 119-130. 44 Song, F. (2007) A study of non-covalent protein complexes by matrix-assisted laser desorption/ionization. Am. Soc. Mass Spect. 18, 1286-1290. 45 Niedermeyer, T.H.J, Strohalm, P. (2012) mMass as a software tool for the annotation of cyclic peptide tandem mass spectra. PloS One 7, e44913, DOI:10.1371/journal.pone.0044913. 46 Cui, J., Bjornmalm, M., Liang, K., Xu, C., Best, J.P., Zhang, X., Caruso, F. (2014) Super soft hydrogel paticle with tunable elasticity in a microfluidic blood capillary model. Adv. Materials 26, 7295. 47 Cho, S.H., Godin, J.M., Chen, C.H., Qiao, W., Lee, H., Lo,Y.H., (2010) Recent advancements in optofluidic flow cytometer. Biomicrofluidics 4, 043001. 48 Henel, G., Schmitz, J.L., (2007) Basic theory and clinical applications of flow cytometry. Labmedicine 38, 428.

39


Biochemistry

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 40 of 44

49 Gebauer, D., Volkel, A., Cölfen, H., (2008) Stable prenucleation calcium carbonate clusters. Science 322, 1819-1822. 50 Sebastiani, F., Wolf, S.L.P., Born, B., Luong, T.Q., Cölfen, H., Gebauer, D., Havenith, M. (2017) Water dynamics from THz spectroscopy reveal the locus of a liquid-liquid binodal limit in aqueous calcium carbonate solutions. Angew. Chem. 56, 490-495. 51 Gebauer, D., Cölfen, H., Verch, A., Antonietti, M. (2008) The multiple roles of additives in CaCO3 crystallization: A quantitative case study. Adv. Mat. 21, 435-439. 52 Kellermeier, M., Gebauer, D., Melero-Garcia, E., Dreschler, M., Talmon, Y., Kienle, L., Cölfen, H., Garcia-Ruiz, J.M., Kunz, W. (2012) Colloidal stabilization of calcium carbonate prenucleation cluster with silica. Adv. Funct. Mat. 22, 4301-4305. 53 Kellermeier, M., Cölfen, H., Gebauer, D., (2013) Investigating the early stages of mineral precipitation by potentiometric titration and analytical ultracentrifugation. Research Methods in Biomineralization Science 532, 45-69. 54 Kozlowski, L.P., Bujnicki, J.M. (2012) MetaDisorder: A meta-server for the prediction of intrinsic disorder in proteins. BMC Bioinformatics 13, 111-122. 55 Dosztányi, Z., Csizmók, V., Tompa, P., Simon, I. (2005) IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content, Bioinformatics 21, 3433-3434. 56 Conchillo-Sole, O., de Groot, N.S., Aviles, F.X., Vendrell, J., Daura, X., Ventura, S. (2007) AGGRESCAN: a server for the prediction and evaluation of “hot spots” of aggregation in polypeptides. BMC Bioinformatics 8, 65-82.

40


Page 41 of 44

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

Biochemistry

57 Goldschmidt, L., Teng, P.K., Riek, R., Eisenberg, D. (2010) The amylome, all proteins capable of forming amyloid-like fibrils. Proc. Natl. Acad. Sci USA 107, 3487-3492. 58 Roche, D. B., Buenavista, M. T., Tetchner, S. J., McGuffin, L. J. (2011) The IntFOLD server: an integrated web resource for protein fold recognition, 3D model quality assessment, intrinsic disorder prediction, domain prediction and ligand binding site prediction. Nucleic Acids Res. 39, W171-176. 59 Pendola, M., Jain, G., Davidyants, A., Huang, Y.C., Gebauer, D., Evans, J.S. (2016) A nacre protein forms mesoscale hydrogels that “hijack” the biomineralization process within a seawater environment. Cryst. Eng. Commun. 18, 7675-7679 60 Perovic, I., Davidyants, A., Evans, J.S. (2016) Aragonite-associated mollusk shell protein aggregates to form mesoscale “smart” hydrogels. ACS Omega 1: 886-896. 61 Yuan, S., Yu, X., Topf, M, Dorstyn, L., Kumar, S., Ludtke, S.J., Akey, C.W. Structure of the Drosophila apoptosome at 6.9 Angstrom resolution. Structure 2011, 19, 128-140. 62 Feinberg, H., Taylor, M.E., Weis, W.I. (2007) Scavenger receptor C-type lectin binds to the leukocyte cell surface glycan Lewis x by a novel mechanism. J Biol Chem. 282, 17250-17258. 63 Ruiz-Arellano, R.R., Medrano, F.J., Moreno, A.,

Romero, A. (2015). Structure of

struthiocalcin-1, an intramineral protein from Struthio camelus eggshell, in two crystal forms. Acta Crystallogr D Biol Crystallogr, 71, 809-818. 64 De Yoreo, J.J. , Gilbert, P.U.P.A., Sommerdijk, N.A.J.M, Penn, R.L., Whitelam, S., Joester, D., Zhang,H., Rimer, J.D., Navrotsky, A., Banfield, J.F., Wallace, A.F., Michel, F.M., Meldrum, F.C., Cölfen, H., Dove, P.M. (2015) Crystallization by particle attachment in synthetic, biogenic, and geologic environments. Science 349, 498-508.

41


Biochemistry

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 42 of 44

65 Politi, Y., Arad, T., Klein, E., Weiner, S., Addadi, L., (2004) Sea urchin spine calcite forms via a transient amorphous calcium carbonate phase. Science 306, 1161-1163. 66 Lin, H.Y., Xin, H.L.,Kunitake, M.E., Keene, E.C., Muller, D.A.,Estroff, L.A. (2011) Calcite prisms from mollusk shells (Atrina Rigida): Swiss-cheese-like organic–inorganic single-crystal composites. Adv. Materials 21, 2028-2034. 67 Kim, Y.Y., Ganesan, K., Yang, P., Kulak, A.N., Borukhin, S., Pechook, S., Ribeiro, L., Kröger, R., Eichhorn, S.J., Armes, S.P., Pokroy, B., Meldrum, F.C. (2011) An artificial biomineral formed by incorporation of copolymer micelles in calcite crystals. Nature Materials 10, 890-896. 68 Li, H., Lin, H.L., Muller, D.A., Estroff, L.A., (2009) Visualizing the 3D internal structure of calcite single crystals grown in agarose hydrogels. Science 326, 1244-1247. 69 Goodwin, A.L., Michel, F.M., Phillips, B.L., Keen, D.A., Dove, M.T., Reeder, R.J. (2010) Nanoporous structure and medium-range order in synthetic amorphous calcium carbonate. Chem. Mat. 22, 3197-3205. 70 Hetherington, N.B.J., Kulak, A.N., Kim, Y.Y., Noel, E.H., Snoswell, D., Butler, M., Meldrum, F.C., (2011) Porous single crystals of calcite from colloidal crystal templates: ACC is not required for nanoscale templating. Adv. Materials 21, 948-954. 71 Fratzl, P.; Fischer, F.D.; Swoboda, J.; Aizenberg, J., (2010) A kinetic model of the transformation of a micropatterned amorphous precursor into a porous single crystal. Acta Biomaterialia 6, 1001-1005. 72 Yue, W., Park, R.J., Kulak, A.N., Meldrum, F.C., (2006) Macroporous inorganic solids from a biomineral template. Macroporous inorganic solids from a biomineral template J. Cryst. Growth 294, 69-77.

42


Page 43 of 44

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

Biochemistry

73 Park, R.J., Meldrum, F. C. (2002) Synthesis of Single Crystals of Calcite with Complex Morphologies. Adv. Materials 14, 1167-1169. 74 Wucher, B., Yue, W., Kulak, A.N., Meldrum, F.C. (2007) Designer crystals: Single crystals with complex morphologies. Chem. Mat. 119, 1111-1119. 75 Asenath-Smith, E., Li, H.; Keene, E.C.,Wei Seh, Z.,Estroff, L.A. (2012) Crystal growth of calcium carbonate in hydrogels as a model of biomineralization. Adv. Materials. 22, 2891-2914.

Table of Contents Figure

43


Biochemistry

1 2 3 4

Page 44 of 44

ACS Paragon Plus Environment 1 μm

A Model Sea Urchin Spicule Matrix Protein ... - ACS Publications

Recommend Documents