Download - Proteins, Composition and Structure
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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:– H2NCHRCOOH
• Amino Group (primary amine)
• Carboxyl Group
• In α-Amino Acids the amino and carboxyl groups are attached to the same C– H2NCHRCOOH
• Variation in the side chain characterises the different types of α-amino acid
What are Proteins?
• α-Amino Acid structure
• Amino Group
• Carboxyl group
• Side Chain
What are Proteins?
• different types of α-Amino Acid make up the building blocks of all proteins
• Grouped into 4 main ‘classes’
– Hydrophobic
– Charged
– Polar
– Special
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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?
• α-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:
RArginine (Arg)
GGlycine (Gly)
DAspartic 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 hardtissues such as– Cartilage, ligaments, tendons,
– Bone and teeth.
• Fibrous structural protein
–Collagen fibrils composed of a triple helix of 3 α-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 prolineand 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 don’t 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 α-helices and β-sheets, resulting from ‘internal’ interactions like ‘backbone’ H-bonding.
– Important examples being the α-helical and
β-sheet units.
α- Helix
β- 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 don’t 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 mechanicalfunctions: – From proteins in the
cell cytoskeleton suchas 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 integrinsand 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 adhesionoccurs
• 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–water–protein 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 18oC 1 hour at 37
oC
Ad
so
rbed
Fib
ron
ec
tin
( µµ µµg
.Sam
ple
-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]
(mg
/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]
(mg
/L)
Day 0 Day 3 Day 7
So What Controls it all???