1

Synergistic biomineralization phenomena created by a combinatorial nacre protein model system.

Eric P. Chang,† Teresa Roncal-Herrero,‡ Tamara Morgan,§ Katherine E. Dunn,§ Ashit Rao,¥

Jennie A.M.R. Kunitake,& Susan Lui,† Matthew Bilton,‡ Lara A. Estroff,& Roland Kröger,‡

Steven Johnson,§ Helmut Cölfen,¥ John Spencer Evans†*

†Center for Skeletal Biology, Laboratory for Chemical Physics, New York University College ofDentistry, New York, NY 10010 USA. jse1@nyu.edu

‡Department of Physics, University of York, Heslington, York, United Kingdom.

§Department of Electronics, University of York, Heslington, York, United Kingdom.

¥Department of Chemistry, Universitat Konstanz, Konstanz, Germany.

&Department of Materials Engineering, Cornell University, Ithaca, NY, USA.

*To whom correspondence should be addressed: Dr. John Spencer Evans, Center for Skeletal Sciences,Laboratory for Chemical Physics, New York University College of Dentistry, 345 E. 24 th Street, NewYork, NY 10010 Telephone: (347) 753-1955; FAX: (212) 995-4087. E-mail: jse1@nyu.edu.

2

TABLE OF CONTENTS

I. Primary sequences of AP7 and PFMG1 (Fig S1)II. Additional SEM images of crystals generated by 1:1 AP7 : PFMG1 (10 µM : 10 µM)(Fig

S2)III. Experimental methods, microRaman spectra (Fig S3), and assignments (Table S1) for

calcium carbonate crystals formed in the presence of 1:1 cAP7 : rPFMG1IV. Calcium potentiometric titration data for cAP7, rPFMG1, and cAP7:rPFMG1 mixtures.

(Table S1, Fig S4, S5)V. QCM-D data for cAP7 : rPFMG1 = 1:1 (Fig S6, S7)

I. Primary sequences of AP7 and PFMG1

Figure S1. Mature, signal sequence deleted primary amino acid sequences of P. fucata PFMG1 pearlnacre protein and H. rufescens AP7 intracrystalline nacre shell protein. Gray lettering denotes predictedlocation of intrinsically disordered sequences; black highlighted regions denote predicted aggregation-prone cross-beta strand sequence regions.14,18 Note that rPFMG1 has a Gly residue at position 1 (post-TEV cleavage) and thus is 117 AA in length. The C-RING domain of AP7 is at 31-66 and the pseudo-EFhand domain of PFMG1 is at 60-110.

II. Additional SEM images of crystals generated by 1:1 AP7 : PFMG1 (10 µM : 10 µM)

Figure S2. SEM images of crystals grown in the presence of 1:1 cAP7 : rPFMG1 (10 µM : 10µM). (A) Example of a multicluster “pinecone”; (B) Alternate pinecone morphology; (C) Highermagnification image of (B), showing nanotextured/nanopatterned surfaces.

3

III. Experimental methods, microRaman spectra and assignments for calciumcarbonate crystals formed in the presence of 1:1 cAP7 : rPFMG1

Figure S3. MicroRaman spectra and corresponding light microscope (LM) images of calciumcarbonate mineral deposits retrieved from 60 min microassays. (A) Protein-deficient negativecontrol; (B-D) 1:1 cAP7 : rPFMG1 representative mineral deposits taken of crystals at center ofLM image. “Si” refers to background signal from Si wafer supports; “c” = calcite, assigned fromRaman bands and the lattice and ν1-4 data in Table S1.

Experimental: The procedure for microRaman analyses involved the use of Si waferscontaining washed and dried precipitated assay deposits taken from protein deficient and 1:1cAP7 : rPFMG1 (10 µM each) 60 min microassays. These deposits were analyzed using aRenishaw InVia Raman microscope at Cornell University. Spectra were acquired under a 100Xmicroscope objective with a laser excitation wavelength of 785 nm at 50% power (~ 4 mW), a1200 line/mm grating, and a spot size of less than 1 micron.

4

Table S1: Raman band assignments for CaCO3 polymorphs

Mode Calcite (cm-1) Vaterite (cm-1) Aragonite (cm-1)

Lattice 156, 283 118, 268, 301 154, 208, 273

ν1 1086 1074, 1089 1086

ν2 --- 874 854

ν3 1435 1445, 1485 1462, 1574

ν4 713 738, 750 704, 717

Overtones 1749 1749 ---

Adapted from reference 1. Legend to table: ν1 = symmetric stretch; ν2 = out-of-plane bending;ν3 = asymmetric stretch; ν4 = in-plane bending

IV. Calcium potentiometric titration data for cAP7, rPFMG1, and cAP7 : rPFMG1mixtures.

Table S2: Potentiometric Ca(II) titration data for cAP7, rPFMG1, and cAP7:rPFMG1.

Sample Slope Nucleation Time (s) Solubility ProductReference 3.21E-010 6022 3.17E-008

cAP7 (1 µM) 3.10E-010 8240 3.25E-008cAP7 (10 µM) 3.05E-010 10500 3.10E-008

rPFMG1 (1 µM) 1.92E-010 8945 4.58E-008rPFMG1 (10 µM) 1.81E-010 10780 8.25E-008

cAP7:rPFMG1 = 1:10 1.67E-010 9980 5.46E-008cAP7:rPFMG1 = 10:1 1.88E-010 9400 3.22E-008cAP7:rPFMG1 = 1:1 (1 µM)) 1.51E-010 10570 3.39E-008cAP7:rPFMG1 = 1:1 (10 µM) 1.47E-010 15700 3.48E-008Reference is the protein-deficient control titration. The standard potentiometric curve provides information on theformation and stabilities of PNCs in solution. As free Ca2+ 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 2+

decreases upon further addition of CaCl2 (i.e., the peak region), this marks the start of solid phase nucleation (e.g.,ACC) from PNCs. With respect to PNC stability, the slope of the prenucleation regime (i.e., the initial linear region)provides indirect evidence of the interaction between additive molecules and solute ion associates, leading to PNCstabilization (i.e., SlopeAdditive < SlopeRef) or destabilization (SlopeAdditive > SlopeRef). Please refer to Figures S3, S4 forgraphical representation of these terms.

5

Figure S4. Development of free Ca(II) ion concentration (TOP) and calcium carbonate solubilityproduct (BOTTOM) in potentiometric titrations of 400 nM and 4 µM cAP7 and rPFMG1proteins in 10 mM carbonate buffer, pH 9.0 as a function of time. In each plot the referencecurve refers to parallel experiments conducted in the absence of protein. Ref = protein-deficientcontrol.

Figure S5. Development of free Ca(II) ion concentration (TOP) and calcium carbonate solubilityproduct (BOTTOM) in potentiometric titrations of cAP7 and rPFMG1 protein mixtures in 10mM carbonate buffer, pH 9.0 as a function of time. In each plot the reference curve refers toparallel experiments conducted in the absence of protein. Ref = protein-deficient control. Forthe 1:1 scenario, two concentration states were examined (i.e., 1 µM : 1 µM and 10 µM : 10µM). In top graph, arrow denotes second nucleation event for 1:1 scenario.

6

V. QCM-D data for cAP7 : rPFMG1 = 1:1 (Figure S5, S6)

Figure S6. Real-time QCM-D data showing functionalization of Au sensor surface with Poly(L-Lysine)(PL) followed by immobilization of rPFMG1 (10 µM solution) and then the introductionof an additional component. The raw data shows decrease in resonant frequency (Δf) and thus anincrease in mass when exposing this layer of adsorbed rPFMG1 to (A) 10 µM cAP7 in 10 mMCaCl2; (B) 10 mM CaCl2 solution only; (C) 10 µM cAP7 in Milli-Q water. Arrows denotetimepoint of sample injection.

7

Figure S7. Real-time QCM-D data showing (A) functionalization of Au sensor surface withpoly-lysine followed by immobilization of AP7 (10 µM in Milli-Q water); (B) exposure of thiscAP7 surface to 10 mM CaCl2 solution; (C) parallel experiment involving exposure of the cAP7surface to 10 µM rPFMG1 in Milli-Q water; (D) parallel experiment involving exposure of thecAP7 surface to 10 µM rPFMG1 in 10 mM CaCl2. Arrows denote timepoint of sample injection.In (D) sample exchange was performed twice.

References

1. M . Ndao, E. Keene, F.A. Amos, G. Rewari, C.B. Ponce, L.A. Estroff, J.S. Evans, Biomacromolecules 2010, 11, 2539.

8