MTYMCSILICLVLILCARGAEA

1. Michenfelder M, Fu G, Lawrence C, Weaver JC, Wustman BA, Taranto L, Evans JS, Morse DE. "Characterization of two molluscan crystal-modulating biomineralization proteins and identification of putative mineral binding domains." Biopolymers. 2003; 70(4): 522-33. PubMed: 14648763
   
   

UniProt describes a potential signal peptide at aa 1-21, Michenfelder et al (2003) describe it at aa 1-22.

DDNGNYGNGMASVRTQGNTYDDLASLISYL

1. Circular dichroism (CD) spectroscopy, far-UV (298 K; pH: 7.4; AP7-1 peptide 8 uM; HCl or NaOH)


2. Nuclear magnetic resonance (NMR) (293 K; pH: 7.4; 90% H2O, 10% D2O; AP7-1 peptide 850 uM)

1. Amos FF, Evans JS. "AP7, a partially disordered pseudo C-RING protein, is capable of forming stabilized aragonite in vitro." Biochemistry. 2009; 48(6): 1332-9. PubMed: 19159266
   
   
2. Kim IW, Collino S, Morse DE, Evans JS. [http://pubs.acs.org/doi/abs/10.1021/cg060056q]. "A Crystal Modulating Protein from Molluscan Nacre That Limits the Growth of Calcite in Vitro." ACS Crystal Growth & Design. 2006; 6(5): 1078-1082.
   
   
3. Michenfelder M, Fu G, Lawrence C, Weaver JC, Wustman BA, Taranto L, Evans JS, Morse DE. "Characterization of two molluscan crystal-modulating biomineralization proteins and identification of putative mineral binding domains." Biopolymers. 2003; 70(4): 522-33. PubMed: 14648763
   
   
4. Wustman BA, Morse DE, Evans JS. "Structural characterization of the N-terminal mineral modification domains from the molluscan crystal-modulating biomineralization proteins, AP7 and AP24." Biopolymers. 2004; 74(5): 363-76. PubMed: 15222016
   
   

Michenfelder et al (2003) and Wustman et al (2004) used a synthetic construct of mature AP7 N-terminal (residues 23-52 of full-length protein) which they call AP7-1. Detection method used by Michenfelder et al was far-UV CD; Wustman et al used NMR.

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Michenfelder et al (2003) identified the N-terminal as a putative calcite binding domain, showing it to "bind strongly to calcite growth steps,...inhibiting* calcite growth." The binding sites would be D23-D24 and D43-D44.

They also describe the presence of two Cys-His-containing motifs that are "similar to those found in Zn(II) finger binding proteins," but note that it is unknown if AP7 binds zinc. Further, they discuss that disulfide linkages in this region are 'unlikely,' based on MALDI-TOF and SDS-PAGE analyses.

It was found that AP7 and AP24 work in complex for optimal calcite inhibition*.

*NOTE: Author DE Morse suggests "modulate" as more fitting term than "inhibit." (see Fu et al, 2005; Fu et al, 2006).

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Wustman et al (2004) confirmed the unfolded structure of Region 2, but found it may contain residual beta-strand or polyproline II-like structure at A33-M32, S34-V35 and S50-I49.

In addition, they confirmed that this region is likely a "calcium carbonate mineral modification domain."

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Additional molecular characterization of this region is reported in Kim IW, Morse DE, Evans JS. Langmuir 2004, 20(26), pp11664-73; PMID 15595796.

TRH

1. Nuclear magnetic resonance (NMR) (293 K; pH: 7.5; 90% H2O, 10% D2O; AP7C 450 uM; DTT 50 uM)


2. Circular dichroism (CD) spectroscopy, far-UV (293 K; pH: 7.5; AP7C 8 uM; Tris-HCl 100 uM; ZnCl2 stock soln in range of AP7C:ZnCl ratios (2:1, 1:1, 1:2, 1:4, 1:10, 1:20))

1. Amos FF, Evans JS. "AP7, a partially disordered pseudo C-RING protein, is capable of forming stabilized aragonite in vitro." Biochemistry. 2009; 48(6): 1332-9. PubMed: 19159266
   
   
2. Collino S, Kim IW, Evans JS. "Identification and structural characterization of an unusual RING-like sequence within an extracellular biomineralization protein, AP7." Biochemistry. 2008; 47(12): 3745-55. PubMed: 18298090
   
   
3. Kim IW, Collino S, Morse DE, Evans JS. [http://pubs.acs.org/doi/abs/10.1021/cg060056q]. "A Crystal Modulating Protein from Molluscan Nacre That Limits the Growth of Calcite in Vitro." ACS Crystal Growth & Design. 2006; 6(5): 1078-1082.
   
   

Collino et al (2008) characterize the structure of AP7 C-terminal domain (CTD) (aa T53-N88, DP Regions 3-8) as a short disordered region (T53-H55, Rgn 3), three transient helical segments (S56-R59, C64-G74, M82-N88; Rgns 4,6,8, respectively) interspersed with a type I beta-turn (P60-E63, Rgn 5) and an extended loop (E75-D81, Rgn 7).

They also describe proline-induced motion at H55, R58, Y68 and S69. Proline hinges are found at P60 and P73.

Additionally, they describe "significant cationic character" at T53, H55, R58, R59 and H62.

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The primary citation for PDB 2JYP is Collino et al (2008), PubMed: 18298090.

SFRR

1. Nuclear magnetic resonance (NMR) (293 K; pH: 7.5; 90% H2O, 10% D2O; AP7C 450 uM; DTT 50 uM)


2. Circular dichroism (CD) spectroscopy, far-UV (293 K; pH: 7.5; AP7C 8 uM; Tris-HCl 100 uM; ZnCl2 stock soln in range of AP7C:ZnCl ratios (2:1, 1:1, 1:2, 1:4, 1:10, 1:20))

1. Amos FF, Evans JS. "AP7, a partially disordered pseudo C-RING protein, is capable of forming stabilized aragonite in vitro." Biochemistry. 2009; 48(6): 1332-9. PubMed: 19159266
   
   
2. Collino S, Kim IW, Evans JS. "Identification and structural characterization of an unusual RING-like sequence within an extracellular biomineralization protein, AP7." Biochemistry. 2008; 47(12): 3745-55. PubMed: 18298090
   
   
3. Kim IW, Collino S, Morse DE, Evans JS. [http://pubs.acs.org/doi/abs/10.1021/cg060056q]. "A Crystal Modulating Protein from Molluscan Nacre That Limits the Growth of Calcite in Vitro." ACS Crystal Growth & Design. 2006; 6(5): 1078-1082.
   
   

Collino et al (2008) characterize the structure of AP7 C-terminal domain (CTD) (aa T53-N88, DP Regions 3-8) as a short disordered region (T53-H55, Rgn 3), three transient helical segments (S56-R59, C64-G74, M82-N88; Rgns 4,6,8, respectively) interspersed with a type I beta-turn (P60-E63, Rgn 5) and an extended loop (E75-D81, Rgn 7).

They also describe proline-induced motion at H55, R58, Y68 and S69. Proline hinges are found at P60 and P73.

Additionally, they describe "significant cationic character" at T53, H55, R58, R59 and H62.

---

Collino et al (2008) described a RING-like region at R58-I70 (Rgns 4,5,6), a "putative interaction site with other nacre proteins such as AP24." This RING-like region has partial homology to zinc-binding RING motif, but does not require zinc to function. It can bind Zn(II), Co(II) and other multivalent metal ions in mono-, bi- or tridentate fashion.

Amos and Evans (2009) describe this as a "pseudo-C-RING," displaying homology to RING C-subclass. They also note that the pseudo-C-RING does not exhibit the tetra-coordinate binding of a typical zinc finger.

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The primary citation for PDB 2JYP is Collino et al (2008), PubMed: 18298090.

PFHE

1. Nuclear magnetic resonance (NMR) (293 K; pH: 7.5; 90% H2O, 10% D2O; AP7C 450 uM; DTT 50 uM)


2. Circular dichroism (CD) spectroscopy, far-UV (293 K; pH: 7.5; AP7C 8 uM; Tris-HCl 100 uM; ZnCl2 stock soln in range of AP7C:ZnCl ratios (2:1, 1:1, 1:2, 1:4, 1:10, 1:20))

1. Amos FF, Evans JS. "AP7, a partially disordered pseudo C-RING protein, is capable of forming stabilized aragonite in vitro." Biochemistry. 2009; 48(6): 1332-9. PubMed: 19159266
   
   
2. Collino S, Kim IW, Evans JS. "Identification and structural characterization of an unusual RING-like sequence within an extracellular biomineralization protein, AP7." Biochemistry. 2008; 47(12): 3745-55. PubMed: 18298090
   
   
3. Kim IW, Collino S, Morse DE, Evans JS. [http://pubs.acs.org/doi/abs/10.1021/cg060056q]. "A Crystal Modulating Protein from Molluscan Nacre That Limits the Growth of Calcite in Vitro." ACS Crystal Growth & Design. 2006; 6(5): 1078-1082.
   
   

Collino et al (2008) characterize the structure of AP7 C-terminal domain (CTD) (aa T53-N88, DP Regions 3-8) as a short disordered region (T53-H55, Rgn 3), three transient helical segments (S56-R59, C64-G74, M82-N88; Rgns 4,6,8, respectively) interspersed with a type I beta-turn (P60-E63, Rgn 5) and an extended loop (E75-D81, Rgn 7).

They also describe proline-induced motion at H55, R58, Y68 and S69. Proline hinges are found at P60 and P73.

Additionally, they describe "significant cationic character" at T53, H55, R58, R59 and H62.

---

Collino et al (2008) described a RING-like region at R58-I70 (Rgns 4,5,6), a "putative interaction site with other nacre proteins such as AP24." This RING-like region has partial homology to zinc-binding RING motif, but does not require zinc to function. It can bind Zn(II), Co(II) and other multivalent metal ions in mono-, bi- or tridentate fashion.

Amos and Evans (2009) describe this as a "pseudo-C-RING," displaying homology to RING C-subclass. They also note that the pseudo-C-RING does not exhibit the tetra-coordinate binding of a typical zinc finger.

---

The primary citation for PDB 2JYP is Collino et al (2008), PubMed: 18298090.

CALCYSITDPG

1. Nuclear magnetic resonance (NMR) (293 K; pH: 7.5; 90% H2O, 10% D2O; AP7C 450 uM; DTT 50 uM)


2. Circular dichroism (CD) spectroscopy, far-UV (293 K; pH: 7.5; AP7C 8 uM; Tris-HCl 100 uM; ZnCl2 stock soln in range of AP7C:ZnCl ratios (2:1, 1:1, 1:2, 1:4, 1:10, 1:20))

1. Amos FF, Evans JS. "AP7, a partially disordered pseudo C-RING protein, is capable of forming stabilized aragonite in vitro." Biochemistry. 2009; 48(6): 1332-9. PubMed: 19159266
   
   
2. Collino S, Kim IW, Evans JS. "Identification and structural characterization of an unusual RING-like sequence within an extracellular biomineralization protein, AP7." Biochemistry. 2008; 47(12): 3745-55. PubMed: 18298090
   
   
3. Kim IW, Collino S, Morse DE, Evans JS. [http://pubs.acs.org/doi/abs/10.1021/cg060056q]. "A Crystal Modulating Protein from Molluscan Nacre That Limits the Growth of Calcite in Vitro." ACS Crystal Growth & Design. 2006; 6(5): 1078-1082.
   
   

Collino et al (2008) characterize the structure of AP7 C-terminal domain (CTD) (aa T53-N88, DP Regions 3-8) as a short disordered region (T53-H55, Rgn 3), three transient helical segments (S56-R59, C64-G74, M82-N88; Rgns 4,6,8, respectively) interspersed with a type I beta-turn (P60-E63, Rgn 5) and an extended loop (E75-D81, Rgn 7).

They also describe proline-induced motion at H55, R58, Y68 and S69. Proline hinges are found at P60 and P73.

Additionally, they describe "significant cationic character" at T53, H55, R58, R59 and H62.

---

Collino et al (2008) described a RING-like region at R58-I70 (Rgns 4,5,6), a "putative interaction site with other nacre proteins such as AP24." This RING-like region has partial homology to zinc-binding RING motif, but does not require zinc to function. It can bind Zn(II), Co(II) and other multivalent metal ions in mono-, bi- or tridentate fashion.

Amos and Evans (2009) describe this as a "pseudo-C-RING," displaying homology to RING C-subclass. They also note that the pseudo-C-RING does not exhibit the tetra-coordinate binding of a typical zinc finger.

---

The primary citation for PDB 2JYP is Collino et al (2008), PubMed: 18298090.

ERQRCID

1. Nuclear magnetic resonance (NMR) (293 K; pH: 7.5; 90% H2O, 10% D2O; AP7C 450 uM; DTT 50 uM)


2. Circular dichroism (CD) spectroscopy, far-UV (293 K; pH: 7.5; AP7C 8 uM; Tris-HCl 100 uM; ZnCl2 stock soln in range of AP7C:ZnCl ratios (2:1, 1:1, 1:2, 1:4, 1:10, 1:20))

1. Amos FF, Evans JS. "AP7, a partially disordered pseudo C-RING protein, is capable of forming stabilized aragonite in vitro." Biochemistry. 2009; 48(6): 1332-9. PubMed: 19159266
   
   
2. Collino S, Kim IW, Evans JS. "Identification and structural characterization of an unusual RING-like sequence within an extracellular biomineralization protein, AP7." Biochemistry. 2008; 47(12): 3745-55. PubMed: 18298090
   
   
3. Kim IW, Collino S, Morse DE, Evans JS. [http://pubs.acs.org/doi/abs/10.1021/cg060056q]. "A Crystal Modulating Protein from Molluscan Nacre That Limits the Growth of Calcite in Vitro." ACS Crystal Growth & Design. 2006; 6(5): 1078-1082.
   
   

The primary citation for PDB 2JYP is Collino et al (2008), PubMed: 18298090.

MYCSYTN

1. Nuclear magnetic resonance (NMR) (293 K; pH: 7.5; 90% H2O, 10% D2O; AP7C 450 uM; DTT 50 uM)


2. Circular dichroism (CD) spectroscopy, far-UV (293 K; pH: 7.5; AP7C 8 uM; Tris-HCl 100 uM; ZnCl2 stock soln in range of AP7C:ZnCl ratios (2:1, 1:1, 1:2, 1:4, 1:10, 1:20))

1. Amos FF, Evans JS. "AP7, a partially disordered pseudo C-RING protein, is capable of forming stabilized aragonite in vitro." Biochemistry. 2009; 48(6): 1332-9. PubMed: 19159266
   
   
2. Collino S, Kim IW, Evans JS. "Identification and structural characterization of an unusual RING-like sequence within an extracellular biomineralization protein, AP7." Biochemistry. 2008; 47(12): 3745-55. PubMed: 18298090
   
   
3. Kim IW, Collino S, Morse DE, Evans JS. [http://pubs.acs.org/doi/abs/10.1021/cg060056q]. "A Crystal Modulating Protein from Molluscan Nacre That Limits the Growth of Calcite in Vitro." ACS Crystal Growth & Design. 2006; 6(5): 1078-1082.
   
   

The primary citation for PDB 2JYP is Collino et al (2008), PubMed: 18298090.

1. Amos FF, Evans JS. "AP7, a partially disordered pseudo C-RING protein, is capable of forming stabilized aragonite in vitro." Biochemistry. 2009; 48(6): 1332-9. PubMed: 19159266
   
   
2. Collino S, Kim IW, Evans JS. "Identification and structural characterization of an unusual RING-like sequence within an extracellular biomineralization protein, AP7." Biochemistry. 2008; 47(12): 3745-55. PubMed: 18298090
   
   
3. Fu, G., S.R. Qiu, C.A. Orme, D.E. Morse, and J.J. DeYoreo. "Acceleration of Calcite Kinetics by Abalone Nacre Proteins (+cover)." Advanced Materials. 2005; 17(22): 2678-2683.
   
   
4. Fu G, Valiyaveettil S, Wopenka B, Morse DE. "CaCO3 biomineralization: acidic 8-kDa proteins isolated from aragonitic abalone shell nacre can specifically modify calcite crystal morphology." Biomacromolecules. 2006; 6(3): 1289-98. PubMed: 15877344
   
   
5. Kim IW, Collino S, Morse DE, Evans JS. [http://pubs.acs.org/doi/abs/10.1021/cg060056q]. "A Crystal Modulating Protein from Molluscan Nacre That Limits the Growth of Calcite in Vitro." ACS Crystal Growth & Design. 2006; 6(5): 1078-1082.
   
   
6. Michenfelder M, Fu G, Lawrence C, Weaver JC, Wustman BA, Taranto L, Evans JS, Morse DE. "Characterization of two molluscan crystal-modulating biomineralization proteins and identification of putative mineral binding domains." Biopolymers. 2003; 70(4): 522-33. PubMed: 14648763
   
   
7. Wustman BA, Morse DE, Evans JS. "Structural characterization of the N-terminal mineral modification domains from the molluscan crystal-modulating biomineralization proteins, AP7 and AP24." Biopolymers. 2004; 74(5): 363-76. PubMed: 15222016
   
   

According to Amos and Evans (2009), AP7 promotes in vitro aggregation of both amorphous and crystalline types, with aragonite stabilization of the crystalline aggregate. This stabilization did not require metal coordination, and it appears to be concentration-dependent.

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Verification request sent 2-21-2012 (PMID: 19159266 and PMID: 14648763 - for region 1)