Download Macromolecules and the organic matrix

Organic matrix-mediated biomineralization
The organic matrix is a preformed insoluble macromolecular framework that
is a key mediator of controlled 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
Organic matrices as mechanical frameworks
Stress/MPa
Antler
200
Femur
tension
compression
Nacre
100
0
Bone strength
normal
130 MPa
150 MPa
Young’s modulus
17 GPa
(stress/strain = stiffness)
0.01
0.02
no matrix
6 MPa
40 MPa
16 GPa
0.03
Strain (l/l)
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
CaCO3
Two-component model
HCl/EDTA
Ca phosphate
soluble/insoluble macromolecules
HNO3/HF
silica
Nucleating
surface
Functional
acidic macromolecules
Hydrophobic
framework
Structural
cross-linked macromolecules
H H O
H H O
H H O
H H O
H H O
N C C
N C C
N C C
N C C
N C C
CH2
CH2
C
O
CH2
-
O
C
O
Asp
CH2
Glu
CH
OOC
COO
-
CH2
CH2
OH
O
-O
-
O
-
P O
O
-Glu
Acidic macromolecules
Ser
PSer
Macromolecules and organic matrix-mediated biomineralization
SYSTEM
FRAMEWORK
ACIDIC
Bone and dentine Collagen
Glycoproteins (osteopontin, osteonectin)
Proteoglycans (chondroitin sulfate)
Gla-containing proteins
Osteocalcin
Tooth enamel
Amelogenin
Glycoproteins (enamelins)
Mollusc nacre
-chitin
Silk-like proteins (MSI 60)
N16/N14
Lustrin A
Glycoproteins (nacrein, N66)
Crab cuticle
-chitin
Glycoproteins
Diatom shells
Frustulins
Glycoproteins (HEP200, silaffins)
Silica Sponges
Silicatein
???
Plant silica
Cellulose
Proteins/carbohydrates
Matrix macromolecules in bone
collagen (90 wt%) + non-collagenous proteins and proteoglycans
Biosynthesis of collagen
osteoblast
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
extracellular space
enzymatic removal of short peptides from filament ends

self-assembly of collagen fibrils

formation of cross-links

mature collagen fibrils

biomineralization
H O
H H O
Collagen – type I
N C
N C
C
H O
C
N C C
H2C CH2
CH2
H
H 2C
CH2
CH
OH
1000 amino acids  30 % glycine (Gly) + 20% proline (Pro) + hydroxyproline (Hyp)
[Gly-X-Y]338 triplets often as [Gly-Pro-Hyp]
small Gly  triple superhelix
steric constraints  helical backbone
tropocollagen
coiled-coil
3.3 residues /turn
Pro
Pro
Gly
Pro
Gly
Pro
Tropocollagen interchain interactions: 280 nm
steric, H-bonding (NH-OC, OH)
A
covalent crosslinks involving lysines
B
1.5 nm
C
Tropocollagen – assembly of collagen fibrils
Revised quarter-stagger model: five overlapping zones
Mismatch due to crosslinks near C and N ends.
CN
C N
N
C
N
C
[110]
Collagen
Groove
Direction
Side face
Top face
Hole zones: 40 x 5 nm
End face
[110]
[001]
Collagen
Fibril
Axis
HAP crystals aligned in hole zone/grooves
grooves
Non-collagenous proteins in bone
MACROMOLECULES
MOLECULAR
MASS (x 103 )
Acidic glycoproteins
Osteonectin
Sialoprotein II
Phosphoprotein
Phosphophoryns (dentine)
44 (bovine)
200
40
100 (human)
Proteoglycans (cartilage)
Bone proteoglycans
Cartilage proteoglycans
350
1,000
Gla proteins
Osteocalcin
Matrix Gla protein
COOH
N C C
CH2
CH
OOC
COO
-Glu
O
H
OH
O
H
H
H
OH
Asp/Glu
Asp[Glu]9
Asp/Glu/PSer
[Ser-Asp]n [PSer]8
chondroitin sulfate
chondroitin/keratin sulfate
-carboxyGlu (x3)
-carboxyGlu (x5)
6
15
H H O
COMPOSITION
CH2OSO3O
O
OH
H
H
H
H
H
NHCOC H3
Chondroitin 6-sulfate
CH2OH
O
OH
H
H
H
H
H
OH
H
O
CH2OSO3O
OH
O
H
H
H
Keratan sulfate
NHCOC H3
Tooth enamel proteins
amelogenins
Only 5 % organic macromolecules
180 amino acids (hydrophobic, Pro, Leu ..)
25k monomer  20 nm nanospheres (gel)
spatial control of c axis growth
enamelins
Ameloblast
60k highly acidic (Asp, Glu)
Ca2+
Amelogenin
monomer
HPO42-
sheath around HAP crystals
20nm
nanosphere
Hydroxyapatite
crystal
+ enamelin sheath
c axis
Aragonite
Matrix macromolecules from shell nacre
Antiparallel -pleated sheet
Acidic
macromolecules
MSI 60, N16; Ala, Gly-rich
Silk-fibroin-like
hydrophobic proteins
-CO, -NH hydrogen bonds
-chitin
a
b
c
 - chitin; R = -NHCOMe
nacrein
[Asp-Glu-PSer]
Laminated hybrid structure
Macromolecules from silica biomineralization
Diatoms
Frustulins
HEP200
(HF-extractable)
Silaffins
high Mr (75k) glycoproteins
[Cys-Glu-Gly-Asp-Cys-Asp]
+ [Gly]n
25% Ser/Thr
+ 20% Asp/Glu
Sponge spicules
Silicatein
low Mr (4 to 17k)
polylysine repeats
+
oligo-N-methylpropylamino
H H O
N C C
CH2
CH2
CH2
x3 subunits; 20% Ser/Thr
catalytic (hydrolytic) properties in vitro
CH2
NH2+
[CH2 CH2 CH2 NH+]n
C H3
Organic matrix-mediated nucleation
G
G*N(1)
no organic surface
G*N(2)
r*(2)
r*(1)
The activation energy for nucleation is
lowered by specific interfacial
interactions between functional groups
on the organic matrix and ions in
supersaturated solution.
r
organic matrix
– 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
I
G
II
B
B
B
1
1
1
III
A
no matrix
2
matrix
1
A, B polymorphs
A
A
1
1
2
2
1
A
2
2
2
kinetically favoured
(no matrix)
2
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
Inorganic nucleus
Lattice
geometry
Charge
Polarity
Stereochemistry
Space
symmetry
Topography
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
A
High capacity binding 
+
Low affinity binding 
B
C
high localised supersaturation
migration of surface-bound ions to nucleus
Or, charge matching of preformed nuclei in regions of high spatial charge density
Electrostatic accumulation – nucleation in ferritin
nucleation groove
FeIII
FeII
O2
2FeII + O2 + 4H2O  2FeOOH + H2O2 + 4H+
ferroxidase centre
Surface topography
concave
convex
concave surfaces – high spatial charge density
+
3-D clustering of ions
convex surfaces –
dissipated charge density
planar substrates – localized charge distributions
planar
good nucleation sites
poor nucleation sites
limit on number of nucleation sites
2-D nucleation sites
structural matching
Structural matching – the geometric model
-
-
-
+
+
-
+
+
-
x
+
-
+
-
+
+
-
+
+
+
-
-
-
-
+
+
+
-
-
+
+
y
x
Organic
matrix
-
+
-
Nucleating
crystal
+
-
nacre
Distances between regularly spaced binding sites on the surface of the
organic matrix are commensurate with lattice spacings in particular crystal
faces.
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,
cooperativity
charge balance
stereochemistry in crystal face
Calcite ( ) vs aragonite ( ) (001) faces
Similar lattice geometry but different Ca2+ and
CO32- stereochemistry
Calcite; CN = 6, planar CO32- all coaligned
Aragonite; CN = 9, planar CO32- x2 types
0.496
0.797
0.499
120°
90°
0.499
(001) face; values in nm
Oriented nucleation on soap films
hydrophilic headgroup
surfactant
hydrophobic tail
Langmuir monolayers
supersaturated CaHCO3(aq)
air
Limiting area for single alkyl chain
= 0.2 nm2
Oriented nucleation of calcium carbonate under Langmuir monolayers
CH3(CH2)16COOH
unit cell a axis  to monolayer surface
CH3(CH2)19OSO3H
c axis  to monolayer surface
NO oriented nucleation under CH3(CH2)17OH
Ca2+ binding required !
A
air
0.5 nm
carboxylate monolayer
Ca2+ binding
supersaturated
CaHCO3 (aq)
0.5 nm
Matching of headgroup
distance and Ca2+ spacing
B
in nucleated crystal face
0.5 nm
sulfated monolayer
Ca2+ binding
0.5 nm
Matching of headgroup
and orientation of
CO32- anions in nucleated
crystal face