Role of Water in CaCO3 Biomineralization

Biomineralization occurs in aqueous environments. Despite the ubiquity and relevance of CaCO3 biomineralization, the role of water in the biomineralization process has remained elusive. Here, we demonstrate that water reorganization accompanies CaCO3 biomineralization for sea urchin spine generation in a model system. Using surface-specific vibrational spectroscopy, we probe the water at the interface of the spine-associated protein during CaCO3 mineralization. Our results show that, while the protein structure remains unchanged, the structure of interfacial water is perturbed differently in the presence of both Ca2+ and CO32– compared to the addition of only Ca2+. This difference is attributed to the condensation of prenucleation mineral species. Our findings are consistent with a nonclassical mineralization pathway for sea urchin spine generation and highlight the importance of protein hydration in biomineralization.


Protein Expression and Purification
The putative C-type lectin-like (CTL) domain of SpSM50 (Strongylocentrotus purpuratus, W4YSM4) was identified at the N-terminus by using the SMART server, 1 designated as SpSM50-CTL (15 kDa, pI: 6.02). The cDNA template of SpSM50 was provided by the Center for Regulatory Genomics, Beckman Institute and the Eric Davidson Lab, Division of Biology at Caltech. The target DNA sequence of SpSM50-CTL was inserted into a pET-3a-His vector with a N-terminal 6xHis-tag provided by Dr. Hao-Cheng Tang (Department of Biology, University of Konstanz, Germany). The restriction sites for enzymes BamHI and XhoI (New England Biolabs) were used in the forward and reverse primers, respectively. Following standard molecular biology procedures, 2  and ampicillin (100 µg/mL, ampicillin sodium salt, Carl Roth) at 37°C till the cell density attained an OD600 of 0.3-0.4. Afterward, the cell culture was incubated at 20°C. Upon reaching the absorbance of 0.6-0.7 O.D., isopropyl-ß-D-thiogalactopyranoside (IPTG, 0.1 mM) was introduced for induction.
The solubilized protein was purified by using Ni 2+ charged IDA (iminodiacetic acid) agarose resin (Protino® Ni-IDA Resin, Macherey-Nagel). The supernatant was loaded on a Ni 2+ -IDA resin column for immobilized metal affinity chromatography (IMAC), and the target protein was eluted with an imidazole gradient (from 20 mM to 200 mM). SDS polyacrylamide gel electrophoresis (SDS-PAGE) of the protein fractions was conducted using 12.5% acrylamide gels stained with Coomassie Blue. The purified protein was dialyzed against the following buffers at 4°C: (1) buffer A (1 M urea, 10 mM carbonate buffer or Tris buffer, pH 9.0) for 12 h and then (2) buffer B (10 mM carbonate buffer or Tris buffer, pH 9.0) for 8 h for three times. Protein concentration was measured by using NanoPhotometer (IMPLEN).

Experimental Section
5 µM (micromolar per liter) SpSM50-CTL protein solution in 10 mM carbonate or Tris buffer (both pH at 9.0) was placed in a 20 mL Teflon trough for 1 hour to reach equilibrium, which allows the proteins to assemble into monolayer at the air-buffer interface. CaCl 2 solution (10mM) was injected into the subphase of two buffer solutions. CaCO 3 mineralization was initiated for carbonate buffer while not for Tris buffer. The injection rate is very slow in accordance with the titration experiment; different volumes of CaCl 2 solution (10mM) were injected to obtain calcium concentrations ranging from 100 to 1250 µM.

Surface Pressure measurement
Surface pressure has been measured using a Langmuir tensiometer (Kibron, Finland). The Teflon trough was thoroughly cleaned sequentially with acetone, ethanol and milliQ, and dried under a nitrogen stream prior to measurements. The surface pressure (π) was normalized with pure water to 0 mN/m. The surface pressure was recorded together with the SFG experiment, and SFG spectra were collected when surface pressure reached equilibrium.

Titration Experiment
Detailed descriptions of the potentiometric titration setup were reported elsewhere. 3   throughout titration experiments.

Vibrational Sum Frequency Generation (SFG) Spectroscopy:
The vibrational SFG spectra were obtained by overlapping, in time and space, the visible and IR pulses.
A Ti:Sapphire amplified system (Spitfire Ace, Spectra Physics Inc.) delivers 35 fs long pulses at a central wavelength of ~800 nm and 1 kHz repetition rate. The beam is split into two parts: one is spectrally narrowed using a Fabry-Perot etalon to achieve a spectral resolution of 15 cm -1 (lambda = 800, E~25 mJ/pulse). The other part is used to generate tunable broadband IR pulses thanks to a parametric optical amplifier followed by a noncollinear difference frequency generation module (TOPAS Prime, LightConversion). The average power is 2 µJ/pulse at a wavelength of 6000 nm and 3 µJ/pulse at a wavelength of 3000 nm. Visible and IR beams are focused onto the sample using a 20 cm and 5 cm focal length (FL) lens, respectively. The polarization of both beams can be controlled (s or p) with a polarizer and a half waveplate. Beams are temporally and spatially overlapped at the sample position. The SFG signal is generated with visible and IR beam angles of 55° and 60° respective to the surface normal, and is collimated using a 20 cm FL lens, further focused into a spectrograph using a 5 cm FL achromatic lens, dispersed by a grating and collected by an intensified CCD camera. The polarization of the SFG signal can be well controlled like Visible and IR beams.
Each spectrum was acquired for 10 minutes, and the spectra were normalized by non-resonance reference spectra of a z-cut quartz crystal after background correction. Spectra in the amide I region were recorded in the SSP (sum, visible, and infrared) polarization combination, and spectra were Amide I SFG spectra in Figure 2 were fitted by Lorentzian peak shapes according to the following equation: In equation (1) above, the susceptibility ! consists of a non-resonant ( !" (!) ) and a resonant ( ! (!) ) term. !" and !" are the amplitude and phase of the non-resonant signal, respectively. ! is the amplitude of the resonant signal, ! is the resonant frequency, !" is the infrared frequency, and Γ ! is the width of transition.