Protein structure summary… Lecture 3 Proteins and Disease
Recap… • • • • • •
Proteins are polymers of amino acids (polypeptides) Amino acid polymers are due to formation of peptide bonds 20 R groups = 20 aa’s – 4 subgroups Protein structure has 4 levels: Primary structure = aa sequence Secondary structure = alpha helix beta pleated sheet (due to reactions within the polypeptide backbone) • Tertiary structure = hydrophobic bonds Van der waals interactions Ionic bonds Hydrogen bonds Disulphide bridges (due to interactions between Reactive side chains) • Quaternary structure
• X-ray crystallography – Is used to determine a protein’s threedimensional structure How? • X-ray hits a crystallised protein • Diffracts into many different directions, based on chemical make-up of the protein • 3D image of electrons in protein • Can calculate what atoms, chemical bonds and their order are present
X-ray diffraction pattern
Photographic film Diffracted X-rays X-ray X-ray beam source Crystal Nucleic acid Protein
(a) X-ray diffraction pattern
(b) 3D computer model
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Recap… Proteins are encoded by genes Inherited information carried in genes
Controls the pattern or sequence of mRNA
Functional protein
Proteins and Disease "a disease gene is discovered, which leads to the disease-causing protein, which leads to a definition of the molecular basis of the disease, which enables researchers to develop compounds to cure the disease" Frank Gannon Director SFI
Passage of information from gene sequence to protein structure
Proteins and Disease
Proteins and Disease
Disease Gene
Disease Gene
Disease Message
Disease Message
Disease Protein
ONE GENE defect eg. Huntington’s disease, or cystic fibrosis
Cells can compensate
Disease Protein
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Proteins and Disease
Proteins and Disease Compensatory Pathways
Disease Gene
Disease Message
Compensatory Gene
Compensatory Message
Disease Gene
Multiple Disease Genes
Disease Message
The real story Disease Protein
Functional Protein
Proteins and Disease Disease Gene
Multiple Disease Genes
Disease Message
Disease Fingerprint Disease Protein
Disease Protein
Proteins and Disease Disease Fingerprint
Therapeutic Intervention • New drug targets • New drugs • Early treatment
Diagnostics • New tests • Early diagnosis • Predict response to therapy
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Proteins and Disease
Sickle-Cell Disease: A Simple Change in Primary Structure
• Humans are complex • Scientists use simple models to study disease – Yeast – Drosophila (fruit fly) – Caenorhabditis elegans (worm-nematode)
One Gene
• Sickle-cell disease – Inherited blood disorder
– Results from a single amino acid substitution in the Gene protein hemoglobin (glutamic acid- valine) Variants – Hemoglobin carries oxygen in red blood cells – Symptoms: sickle cell crises • Misshapen angular cells clog tiny blood vessels • Impede blood flow • Physical weakness, pain, organ damage and death
Hemoglobin function • All body cells require oxygen for metabolism -oxygen is non-polar and not soluble in the aqueous blood. • Hemoglobin has a group called "heme", which is at the heart of the protein structure.
• Hemoglobin structure and sickle-cell disease Primary structure
Normal hemoglobin Val
His Leu Thr
Pro Glul Glu
1 2 3 4 5 6 7
Secondary and tertiary structures
Sickle-cell hemoglobin
. . . Primary
Val
His
Leu Thr
α β
Function
Molecules do not associate with one another, each carries oxygen.
Red blood cell shape
Normal cells are full of individual hemoglobin molecules, each carrying oxygen
β α
Pro
Val
Glu
structure 1 2 3 4 5 6 7
Secondary β subunit and tertiary structures
Quaternary Hemoglobin A structure
• At the center of the heme group is the iron +2 metal ion. • The oxygen molecule will ultimately bind to this iron ion
Disease Protein
Quaternary structure
...
β subunit
α β
β α
Function
10 µm
10 µm Red blood cell shape
Exposed hydrophobic region
Hemoglobin S Molecules interact with one another to crystallize into a fiber, capacity to carry oxygen is greatly reduced. Fibers of abnormal hemoglobin deform cell into sickle shape.
• Globular structure
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Sickle cell anemia • 1/10 Africans have this trait • Selective advantage to the disease trait in malarial regions • The malarial parasite remains at a lower density in cells with sickle hemoglobin • Trade off -Fewer malarial symptoms vs -sickle cell symptoms
Proteins and Disease Disease Gene
Multiple Disease Genes
Disease Message
Disease Fingerprint Disease Protein
Breast Cancer-mutant ER receptor • Most common malignancy in women • Estrogen receptors are over-expressed in around 70% of breast cancer cases, referred to as "ER-positive". • Constant growth of Breast cells
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Breast Cancer-mutant ER receptor • Tamoxifen -drug used to reduce ER levels • Cancer cells depend on ER and so die – Cell suicide called ‘apoptosis’
Drug
Gene mutations • Proteins are coded for by genes
The order of bases along the length of the DNA= genetic code instructs what protein is to be made
DNA
mRNA
• Amino acid change is due to a gene defect • A single base change in the DNA of a gene can give rise to a single amino acid change (sickle cell anemia)
Each set of three bases, or codon, specifies a particular amino acid. Amino acids are the building blocks of proteins.
Amino acid
Glutamic acid codon = GAG valine codon = GUG
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Gene Mutations causing SNPs - single nucleotide polymorphisms (SNPs) variations in DNA sequence of genes -can cause an amino acid change Disease
Cause
Trait
Retinitis Pigmentosa
Mutation in gene for transducin
blindness
Spina Bifida
Mutation in gene for Methylene Tetra Hydra Folate Reductase (MTHFR)
Neural tube defect
Spina Bifida
This enzyme MTHFR uses a nutrient called folic acid to help form the neural tube. The variant requires more folic acid:
Normal MTHFR Folic acid
Building blocks for neural tubes Variant MTHFR
Protein Folding
Protein folding
• Unique shape confers unique function • What are the key factors determining shape? -primary structure - sequence effects
-secondary structure – bonds in polypeptide backbone -tertiary structure - bonds between side chains
• Is this the whole story? –NO! -we don’t know all the rules
• Chaperone proteins assist protein folding -protect a new protein from the external environment -provide hydrophillic environment for proper folding
Cylindrical in shape Eg. TRiC
Chaperones
Hydrophobic amino acids
Hydrophillic amino acids
Amino Acid Sequence determines the way the protein will fold in a specific environment using • Hydrophobic interactions • Hydrogen bonds • Van Der Waals forces
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Disease due to misfolded proteins
Prions- misfolded proteins • How can a protein which can not replicate itself be infectious?
- Many diseases are diseases of protein conformation.
• Prions are mis-shapen versions of normal brain proteins – once a prion gets into the brain they interact with the normal version of the protein and convert it to the misfolded- prion version
• This way Prions trigger a chain reaction which increase their numbers
- There is no known cure for prion diseases
• These Prions then polymerise and are toxic to normal cells
- Prion proteins build up in the brain, ultimately causing death
Prions Normal
Disease-causing
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Prions
The mechanism:
PrPc (normal)
Disease due to misfolded or aggregated proteins
Normal brain
PrPsc infects
Normal brain
PrPsc interacts with PrPc
Normal brain
PrPc turned into PrPsc Causing polymerisation Neuronal death occurs
Symtoms begin and accelerate
Aggregates- misfolded proteins Alzheimer’s Disease • Amyloid-related disease-Amyloids are insoluble fibrous protein aggregates • Accumulation of abnormally folded proteins in the brain called β-amyloid plaques • Death of neurons
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Alzheimer’s Disease
(A) Senile plaques (SPs) and neuron loss in entorhinal cortex. SPs show dense cores and radially oriented dystrophic neurites. (B) A typical neurofibrillary tangle in CA3. Bielschowsky silver stain. (C) Amyloid beta protein immunohistochemistry demonstrates frequent plaques in posterior cingulate cortex, accompanied by cerebral amyloid angiopathy (inset). Hematoxylin counterstain. (D) Immunohistochemical stains for hyperphosphorylated tau show aggregation in NFTs and cortical dystrophic neurites
In summary… • A single amino acid change in the primary structure of a protein can cause disease eg. Sickle cell disease • Amino acid changes occur due to SNPs in the DNA sequence of a gene • Chaperones assist protein folding • Many diseases are due to protein mis-folding eg. CJD • Protein structure can be determined by X-ray crystallography
