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1
Science of
Biocompatibility
Proteins and protein
adsorption
MAT6312/MTRM312
Dr Karin Hing & Dr Helena Azevedo
Proteins
What are they and what are they made
from?
What do they do?
Why are they important in
Biocompatibility?
Proteins
What are they and what are they made
from?
What do they do?
Why are they important in
Biocompatibility?
What are Proteins?
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What are Proteins?
Organic Polymers known as polypeptides
Made up of linear chains of amino acids
Amino Acids
Specific organic molecules that contain an
amino and a carboxyl group
What are Proteins made from?
Amino Acids have general formula:
H
2
NCHRCOOH
Amino Group (primary amine)
Carboxyl Group
In -Amino Acids the amino and carboxyl
groups are attached to the same C
H
2
NCHRCOOH
Variation in the side chain characterises the
different types of -amino acid
What are Proteins?
-Amino Acid structure
Amino Group
Carboxyl group
Side Chain
What are Proteins?
different types of -Amino Acid make
up the building blocks of all proteins
Grouped into 4 main classes
Hydrophobic
Charged
Polar
Special
21
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What are Proteins?
Hydrophobic
What are Proteins?
Charged
What are Proteins?
Polar
What are Proteins?
Special
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What are Proteins?
-Amino acids
Joined together by peptide bonds
between carboxyl and amino groups
Into specific polymer chains
(or Polypeptides)
To make up the protein
primary structure
What are Proteins?
Eg: RGD sequence critical in cell
adhesion:
R
Arginine (Arg)
G
Glycine (Gly)
D
Aspartic Acid (Asp)
What are Proteins?
Organic Polymers Chains are folded in a
particular way to give the protein a
(secondary/tertiary) 3D structure or
conformation which is in part dependant
on the primary structure
What are Proteins?
With 21 different building blocks vast array
of different proteins possible with widely
varying functions.
Where functionality or activity dependant on
both their primary and secondary structures
Proteins can also work together to achieve
a particular function, and they often
associate to form stable complexes, such
as collagen.
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Collagen
25% of the total protein content in
mammals.
Good tensile strength and toughness.
Main component of both soft and hard
tissues such as
Cartilage, ligaments, tendons,
Bone and teeth.
Fibrous structural protein
Collagen fibrils composed of a triple
helix of 3 -amino acid sequences or
polypeptides
Collagen
Different types of collagen arise from:
Choice of amino acid groups in each chain
Combination of different chains
eg: Glycine-X-Y, where X is usually proline
and Y is often hydroxylysine or hydroxyproline
Currently 28 different types described
Collagen
The sequence of amino acids in a protein
is defined by the sequence of a gene,
which is encoded in the genetic code.
What are Proteins?
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Shortly after or even during synthesis, the
residues in a protein are often chemically
modified by post-translational
modification, which alters protein:
Physical and Chemical properties,
Folding and/or Stability,
Activity and/or Function.
What are Proteins? What are Proteins?
Pro-collagen secreted by cells which then
assembles to form collagen
What are Proteins?
Hierarchical structure of collagen in bone
What are Proteins?
Protein modification in situ by action of
enzymes or other molecules can alter
secondary structure and change protein
activity..
Genes dont have total control!
Proteomics
Epigenomics
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What are Proteins?
Intake of some proteins necessary via
diet, since animals cannot synthesize all
the amino acids.
Through the process of digestion, animals
break down ingested protein
into free amino acids that are
then used in metabolism.
Protein Structure
Made up of linear chains of amino acids
But fold into complex 3D structures as a result
of hydrophobicity, charge interactions and H
bonding
Albumin 66.5 kDa Fibronectin, 440 kDa
Four Levels of Protein Structure Four Levels of Protein Structure
Proteins are all highly ordered molecules
Containing a substantial number of
intramolecular hydrogen bonds as well as
the peptide bonds.
Most H-bonds are formed between amide and
carbonyl groups of the backbone.
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Four Levels of Protein Structure
Primary structure is the sequence of
amino acids in the peptide.
Secondary structure describes repetitive
structural units such as -helices and -
sheets, resulting from internal interactions
like backbone H-bonding.
Important examples being the -helical and
-sheet units.
- Helix
- Sheet Four Levels of Protein Structure
Tertiary structure describes the three-
dimensional arrangement of a single
polypeptide or sub-unit.
Quaternary structure describes the
combination of several independent
tertiary structures in proteins or complexes
with more than one polypeptide chain, i.e.
a multi-subunit complex.
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Tertiary Structure
A single BMP-2
polypeptide will
fold into a 3D
structure
characteristic of a
single sub-unit
Quaternary Structure
Two BMP-2 poly
peptides form a stable
dimer in a butterfly
conformation
composed of two
sub-units
Quaternary structure
This Dimer then interacts with the extracellular
domains (ECDs) of the cell surface membrane
receptors to direct cell differentiation/proliferation
Proteins
Different species have varying levels of
internal stability:
Soft proteins
have a low internal stability
Hard proteins
have a high internal stability
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Proteins
What are they and what are they made
from?
What do they do?
Why are they important in
Biocompatibility?
What do Proteins do?
Proteins are essential parts of organisms
(along with polysaccharides and nucleic
acids) and participate in virtually every
process both intra- and extra- cellularly.
Some have been likened to the bodies
nano-machinary
What are Proteins?
Protein modification in situ by action of
enzymes or other molecules can alter 3D
secondary/tertiary structure and change
protein activity..
Genes dont have total control!
Epigenomics
Proteomics
Control in Biology
Genetics
Constant (relatively) chromasonal based
genetic make up of an individual (DNA) which
encodes mRNA to synthesize protein
Epigenomics
Control and regulation of gene expression
Proteomics
Control and regulation of protein synthesis
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Gene Expression
Epigenomics
mRNA Translation
mRNA Translation required for protein synthesis
A
complicated
business
What do Proteins do?
Some proteins catalyze biochemical
reactions vital to metabolism (enzymes).
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What do Proteins do?
Some have bulk structural or mechanical
functions:
From proteins in the
cell cytoskeleton such
as actin and tubulin,
which form a system
of scaffolding that
maintains cell shape
To the collagens which are present in all
mammalian connective tissue such as bone,
cartilage and ligament
What do Proteins do?
Some have biochemical functions and
participate directly in physiological
processes:
Facilitate interactions
Direct specific responses
Modulate host response
Regulate local molecule transport
Eg: Cytokines, Transcription factors,
Growth factors, enzymes..
What do Proteins do?
Some have interfacial functions and
participate directly in cellular
communication and attachment
processes:
Facilitate cell-cell communication
Direct cell attachment
Regulate cell response to a substrate
Eg: membrane proteins such as integrins
and caderins, extra-cellular adsorption
molecules, matrix proteins..
Proteins
What are they and what are they made
from?
What do they do?
Why are they important in
Biocompatibility?
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Proteins and Materials
Within mS of materials introduction into the
body or body fluids protein adhesion
occurs
The nature of the bio-layer formed plays a
significant role in all subsequent
communication between
the host and the material
Proteins and Materials
Communication occurs through
Protein/surface exchange and modification
Bio-layer/cell interactions





























(i)
(ii)
(iii)
(ii)

(iii)
Materials and Proteins
Protein modification in situ by action of
enzymes or other molecules can alter
secondary structure and change protein
activity..
Similarly interaction with materials can
also alter protein structure and activity or
concentrate specific molecules above a
critical dose
Proteins
What are they?
What are they made from?
What do they do?
Why are they important in
Biocompatibility?
What modulates Protein-Material
Interactions?
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Proteins and Materials
Bio-layer/cell interactions modulated
through specific cell receptors called
integrins
Cells use integrins to interact with host
tissues both directly (eg with collagen) and
through the presence of specialised
adhesion proteins (eg with fibronectin)
this is a normal physiological process
Proteins and Materials
Integrins play a role in cell signalling
Proteins and Materials
Specific receptor and integrin activation
induces particular cell signalling pathways
each of which results in a specific
cell response.
Eg: Fn specific integrin activation of
osteoblast differentiation
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Proteins and Materials

Bioceramic Surface
Soluble
Ionic
Species
Protein-ion
Complex
Protein
Adhesion
Signalling
Cascade
Cell
Phenotype &
metabolism Cell
Migration
Protein/GF
synthesis
Cell
Adhesion
Local pH
Modulation
Cell
Recruitment
Molecule
expression
Division
Cell
Death
Varied Cell responses
Proteins and Materials
For the Medical and Dental Materials
scientist or engineer, need to know:
What materials properties effect protein
adhesion? (Materials response)
What protein adhesion is (or is not) required
for the implant or device to function
appropriately? (Host response)
Proteins and Materials
Adsorption may be promoted or opposed by a number
of enthalpic and entropic changes within the surface
waterprotein system.
1. (Partial) dehydration of protein and sorbent surfaces
2. Redistribution of charged groups at the interface
3. Conformational changes in the protein molecule
The relative significance of each process depends on
the nature of the protein, sorbent, and solvent
Material variables that impact on
host response
Chemical
Bulk material composition
Crystallinity and crystallography
Water content, hydrophobicity/hydrophilicity
Surface chemistry/chemical gradients/surface molecular mobility
Surface energy
Surface charge
Physical
Micro- (or nano) structure
Macro-, micro- and nano- topography
Macro-, micro- and nano- porosity
Mechanical
Elastic constants
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Materials Variables that Modulate
Protein adsorption
Chemical
Crystallinity and crystallography
Water content, hydrophobicity/hydrophilicity
Surface chemistry/chemical gradients/surface molecular mobility
Surface energy
Surface charge
Physical
Micro- (or nano) structure
Macro-, micro- and nano- topography
Macro-, micro- and nano- porosity
Proteins and Materials
Proteins are multifaceted charged molecules
that are predominantly hydrophobic
Protein adsorption/desorption
occurs in an aqueous environment (double layer)
occurs under competitive conditions
is dynamic
Nature of the Solvent also critical
Defining the Biological environment
Blood Composition (Lentner 1981/Kokubo1990):
Cell Volume: 38.5% Serum Volume: 61.5%
Serum Proteins: 65-80 g/L
Serum Ion Concentration (mM)
Sodium
Potassium
Calcium
Magnesium
142
5
2.5
1.5
Chlorine
Bicarbonate
Phosphate
Sulphate
103
27
1
0.5
Environmental Sensitivity
Temperature
Protein Composition
0
0.5
1
1.5
2
10% 10% + Fn 10% 10% + Fn
1 hour at 18
o
C 1 hour at 37
o
C
A
d
s
o
r
b
e
d

F
i
b
r
o
n
e
c
t
i
n

(

g
.
S
a
m
p
l
e
-
1
)
HA SA
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Environmental Sensitivity
Solute
Composition
Variation in ion release:
(a) without (b) with serum proteins
A B C D
Day 0 29 29 29 29
Day 3 2.03 4.46 2.66 0.56
Day 7 0.83 0.86 0.66 1.13
0
5
10
15
20
25
30
[
P
]

(
m
g
/
L
)
Day 0 Day 3 Day 7
A B C D
Day 0 33.1 33.1 33.1 33.1
Day 3 29.43 20.66 13 4.26
Day 7 21.63 20.1 13.96 5.4
0
5
10
15
20
25
30
35
[
P
]

(
m
g
/
L
)
Day 0 Day 3 Day 7
So What Controls it all???