Organic matrix-mediated biomineralization

Functions:

mechanical design – strength and toughness

mineral passivation – stabilization from dissolution/phase transformation

mineral nucleation – location and organization of nucleation sites

– structure and crystallographic orientation

boundary organization – partitioning with semi-permeable frameworks

The organic matrix is a preformed insoluble macromolecular framework that is a key mediator of controlled biomineralization.

Organic matrices as mechanical frameworks

0

100

200

0.01 0.02 0.03

Strain (l/l)

Str

ess/

MP

a

Antler

Femur

Nacre

Bone strength

normal no matrixtension 130 MPa 6 MPacompression 150 MPa 40 MPa

Young’s modulus 17 GPa 16 GPa(stress/strain = stiffness)

Organic frameworks play an important role in the mechanical design of biomineralized tissues such as bones, shells and teeth. Many of the general functions of these biominerals – movement, protection, cutting and grinding – are dependent on mechanical properties, such as strength and toughness, which are specifically associated with inorganic-organic composites.

Macromolecules and the organic matrix - a general model

Two-component model

Nucleating Functional surface acidic macromolecules

Hydrophobic Structuralframework cross-linked macromolecules

CaCO3

Ca phosphate soluble/insoluble macromolecules

silica

HCl/EDTA

HNO3/HF

N

H

C

H

C

O

CH2

CO O-

N

H

C

H

C

O

CH2

CH2

CO O-

N

H

C

H

C

O

CH2

CCOO-OC

H-O

N

H

C

H

C

O

CH2

OH

Asp Glu -Glu Ser PSer

N

H

C

H

C

O

CH2

O

P O-O

O

-

Acidic macromolecules

SYSTEM FRAMEWORK ACIDIC

Bone and dentine Collagen Glycoproteins (osteopontin, osteonectin)Proteoglycans (chondroitin sulfate)Gla-containing proteinsOsteocalcin

Tooth enamel Amelogenin Glycoproteins (enamelins)

Mollusc nacre -chitin Glycoproteins (nacrein, N66)Silk-like proteins (MSI 60)

N16/N14 Lustrin A

Crab cuticle -chitin Glycoproteins

Diatom shells Frustulins Glycoproteins (HEP200, silaffins)

Silica Sponges Silicatein ???

Plant silica Cellulose Proteins/carbohydrates

Macromolecules and organic matrix-mediated biomineralization

Matrix macromolecules in bone

collagen (90 wt%) + non-collagenous proteins and proteoglycans

synthesis of helical polypeptide chains

enzymatic modification of amino acids (proline and lysine hydroxylation)

self-assembly of triple-stranded helix filaments

secretion into the extracellular space 

enzymatic removal of short peptides from filament ends

self-assembly of collagen fibrils

formation of cross-links

mature collagen fibrils

biomineralization

Biosynthesis of collagen

osteoblast

extracellular space

Collagen – type I

1000 amino acids 30 % glycine (Gly) + 20% proline (Pro) + hydroxyproline (Hyp)

[Gly-X-Y]338 triplets often as [Gly-Pro-Hyp]

Pro

Pro

Pro

Pro

Gly

Gly

A B C

steric constraints helical backbone

small Gly triple superhelix

tropocollagen

coiled-coil 3.3 residues /turn

280 nm

1.5 nm

Tropocollagen interchain interactions:

steric, H-bonding (NH-OC, OH)

covalent crosslinks involving lysines

N C C

H H O

H

N

H2C

C

H

CH2

C

O

CH2

N

H2C

C

H

CH2

C

O

C

OH

H

Tropocollagen – assembly of collagen fibrils

N

C C

C

CNN N

Revised quarter-stagger model: five overlapping zones

Mismatch due to crosslinks near C and N ends.

Hole zones: 40 x 5 nm

grooves [001]

[110]

[110]-

Top face

CollagenGrooveDirection

Side face

End face

CollagenFibrilAxis

HAP crystals aligned in hole zone/grooves

Non-collagenous proteins in bone

MACROMOLECULES MOLECULAR COMPOSITION MASS (x 103 )

 Acidic glycoproteins

Osteonectin 44 (bovine) Asp/GluSialoprotein II 200 Asp[Glu]9 Phosphoprotein 40 Asp/Glu/PSerPhosphophoryns (dentine) 100 (human) [Ser-Asp]n [PSer]8

Proteoglycans (cartilage)Bone proteoglycans 350 chondroitin sulfate Cartilage proteoglycans 1,000 chondroitin/keratin sulfate

 Gla proteins

Osteocalcin 6 -carboxyGlu (x3)Matrix Gla protein 15 -carboxyGlu (x5)

 

N

H

C

H

C

O

CH2

CCOO-OC

H-O

-Glu

OH O

H HH

OHH

CH2OH

HH O

HH

NHCOCH3H

OH

CH2OSO3-

O

O

H O

HH

OHH

OH

COO-

HOH O

H HH

NHCOCH3H

CH2OSO3-

HOO

Chondroitin 6-sulfate Keratan sulfate

Tooth enamel proteins

Ameloblast

Ca2+ HPO42-

20nmnanosphere

Amelogenin monomer

c axis

Hydroxyapatite crystal+ enamelin sheath

amelogenins

180 amino acids (hydrophobic, Pro, Leu ..)25k monomer 20 nm nanospheres (gel)

spatial control of c axis growth

Only 5 % organic macromolecules

enamelins

60k highly acidic (Asp, Glu)

sheath around HAP crystals

Matrix macromolecules from shell nacre Aragonite

Acidicmacromolecules

Silk-fibroin-likehydrophobic proteins

-chitina

b

c

- chitin; R = -NHCOMe

Antiparallel -pleated sheet

MSI 60, N16; Ala, Gly-rich

-CO, -NH hydrogen bonds

Laminated hybrid structure

nacrein[Asp-Glu-PSer]

Macromolecules from silica biomineralization

Diatoms Frustulins HEP200 Silaffins (HF-extractable)

high Mr (75k) glycoproteins [Cys-Glu-Gly-Asp-Cys-Asp] + [Gly]n

25% Ser/Thr low Mr (4 to 17k)+ 20% Asp/Glu polylysine repeats

+ oligo-N-methylpropylamino

Sponge spicules Silicatein

x3 subunits; 20% Ser/Thrcatalytic (hydrolytic) properties in vitro

N

H

C

H

C

O

CH2

CH2

CH2

CH2

N

CH2 CH2 CH2 NH+]n

C

[

H2+

H3

Organic matrix-mediated nucleation

The activation energy for nucleation is lowered by specific interfacial interactions between functional groups on the organic matrix and ions in supersaturated solution.

no organic surface

organic matrix

G

G*N(2)

G*N(1)

rr*(1)r*

(2)

– control nucleation rate and number of sites

– organization of nucleation sites on organic surface

– structural selectivity of mineral polymorphs

– crystallographic alignment of nuclei on the organic surface. 

Organic matrix-mediated nucleation – structural control

1

2

2

2

22

2

11

1

1

1A A A

BB B

G I II III1 no matrix

2 matrix

A, B polymorphs

A kinetically favoured (no matrix)

Outcomes

I. promotion of non-specific nucleation - reduced activation energies for A and B, no change in the outcome of mineralization.

II. promotion of structure-specific nucleation of polymorph B - crystallographic recognition at matrix surface; activation energy of state 2B < 2A

III. promotion of a sequence of structurally non-specific to highly specific nucleation – variations in levels of recognition of nuclei A and B and reproducibility of matrix structure (genetic, metabolic, and environmental factors).

Interfacial molecular recognition

Latticegeometry

Charge Polarity Stereo-chemistry

Spacesymmetry

Topography

Inorganic nucleus

Organic matrix

Lowering of the activation energy for nucleation can arise from matching of charge, polarity, structure and stereochemistry at the interface between an inorganic nucleus and organic macromolecular surface.

The shape of the interface and the degree of chemical complementarity are important factors in this process.

Electrostatic accumulation – ionotropic model

Anionic surface ligands accumulate metal cations by electrostatic binding (ionotropy)

Site-directed ordering over nucleation scale by clustering – high spatial charge density

High capacity binding high localised supersaturation +

Low affinity binding migration of surface-bound ions to nucleus

Or, charge matching of preformed nuclei in regions of high spatial charge density

A B C

FeIII

FeII

O2

ferroxidase centre

2FeII + O2 + 4H2O 2FeOOH + H2O2 + 4H+

nucleation groove

Electrostatic accumulation – nucleation in ferritin

Surface topography

concave convex planar

 concave surfaces – high spatial charge density + good nucleation sites 3-D clustering of ions

convex surfaces – dissipated charge density poor nucleation sites limit on number of nucleation sites

planar substrates – localized charge distributions 2-D nucleation sitesstructural matching

 

Structural matching – the geometric model

- - - - -

-

+ +

-

+ +

- - -

-

+ +

-

+ +

- - -

+ +

- -

+ +

-

+ +

- - -

+ +

-

+ +

- -

x

y

Nucleating crystal

Organic matrix

Distances between regularly spaced binding sites on the surface of the organic matrix are commensurate with lattice spacings in particular crystal faces.

x

nacre

Structural matching in nacre

Tandem repeats of [Asp-X] explain the specific nucleation of the (001) face of aragonite on the surface of anti-parallel -pleated sheet proteins in shell nacre.

XRD and electron diffraction: a and b axes of -sheet and lattice are co-aligned

good matching along a directions; less so along b directions

Stereochemical matching in nacre

Importance of side group stereochemistry coordination environments, multidentate binding, cooperativitycharge balance

0.797

0.496

0.499

0.499 120°90°

(001) face; values in nm

Calcite ( ) vs aragonite ( ) (001) faces

Similar lattice geometry but different Ca2+ and CO3

2- stereochemistry

Calcite; CN = 6, planar CO32- all coaligned

Aragonite; CN = 9, planar CO32- x2 types

stereochemistry in crystal face

Oriented nucleation on soap films

surfactanthydrophobic tail

hydrophilic headgroup

Langmuir monolayers

air

supersaturated CaHCO3(aq)

Limiting area for single alkyl chain = 0.2 nm2

Oriented nucleation of calcium carbonate under Langmuir monolayers

CH3(CH2)16COOH CH3(CH2)19OSO3H

unit cell a axis to monolayer surface c axis to monolayer surface

NO oriented nucleation under CH3(CH2)17OH Ca2+ binding required !

A

B

carboxylate monolayer

sulfated monolayer

air

supersaturated CaHCO3 (aq)

Ca2+ binding

0.5 nm

0.5 nm

0.5 nm

0.5 nm

Matching of headgroup and orientation of CO3

2- anions in nucleatedcrystal face

Ca2+ binding

Matching of headgroup distance and Ca2+ spacing in nucleated crystal face