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 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

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?

• 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 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…


• Some proteins catalyze biochemical reactions vital to metabolism (enzymes).

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• 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


• 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…..


• 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




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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


• 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 modulates Protein-Material Interactions?

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• 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


• Integrins play a role in cell signalling


• 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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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


• 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)


• 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 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???

Documents

Proteins, Composition and Structure