Helical Peptides, Amyloid Inhibitors and Prediction of Protein Function Andrew Doig

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Helical Peptides, Amyloid Inhibitors and
Prediction of Protein FunctionAndrew Doig
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1. The a-Helix
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Surveying the PDBExample Side Chains at the
N-Terminus of the Helix

• Gln, Glu, Asp, Asn, Ser, Thr and His are favoured
  at N1 and N2
• Hydrogen bond to NH groups at the N-terminus

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Experiments on Helical PeptidesExample
Hydrophobic-Polar Interactions (Ile-Lys)

• Peptide Sequence Helix Content
• Ac-AKAIAAAKAIAAAKAKAGY-CONH2 51
• Ac-AKIAAAAKIAAAAKAKAGY-CONH2 37
• Ile-Lys energy is -0.25 kcalmol-1

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Cooperativity in Side Chain Interactions
Arg c1 trans

• Arg-Phe -0.08 kcalmol-1
• Phe-Met -0.35 kcalmol-1
• Arg-Phe-Met -1.39 kcalmol-1
• Side chain interactions are much stronger when
  present together
• Fixing side chain into one conformation costs
  energy
• But in triplet, only pay this once

Phe c1 trans
Met c1 gauche
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Phosphorylated Helical Peptides

• Helix Content () ?G phosphorylation
• Site Sequence OH OPO3H- OPO32- OPO3H- OPO32-
• N-cap SAAAAQRAAAARAGY-NH2 31 24 47 0.3 -0.6
• N1 Ac-SAAAAQRAAAARAGY-NH2 50 51 61 -0.4 -1.6
• N2 Ac-ASAAAAQRAAAARGY-NH2 42 56 68 -1.1 off scale
• N3 Ac-AASAAAAQRAAAARGY-NH2 42 49 54 -0.7 -2.3
• Mid Ac-AAAQRAAAASAAAARGY-NH2 38 25 17 0.6 1.2
• Phosphorylation is very stabilising at N-terminus
  (more than any other amino acid when 2-)
• Destabilising in helix interior
• Phosphorylation may affect protein function by
  inducing or breaking helical structure

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2. Inhibitors of Amyloid Formation
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b-Sheets Aggregate
Designed b-sheet peptides usually aggregate At
least 20 peptides or proteins aggregate to form
toxic amyloid Probable cause of Alzheimers
disease, BSE, type II diabetes, Parkinsons etc.
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Inhibition of Amyloid Formation
Association of free peptides inhibited
Association of N-methylated peptide
b-sheet
Association of N-methylated peptide
Association of free peptides inhibited
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N-Methylation to Prevent b-Sheet Assembly
Natural peptides
Both edges free to associate with other b-strands
Backbone methyl groups block one edge of peptide
strand
Methylated peptides
This edge blocked
This edge free to hydrogen bond as normal
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N-Methylated Inhibitors of b-Amyloid (Ab)

• Aggregation of 40/42 amino acid peptide Ab is
  probable cause of Alzheimers disease
• Hundreds of N-methylated peptide fragments
  investigated as potential inhibitors
• Compared to other known derivatised peptide
  inhibitors
• Work done in collaboration with start-up company
  - Senexis

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Inhibition of 100mM Ab(1-40) aggregation
Thioflavin T assay
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Electron Microscopy
Ab(1-40) Ab(1-40) NMe1
Ab(1-40) NMe3 Wild type fibrils
Altered morphology Few fibrils
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Inhibition of 100mM Ab(1-42) toxicity by selected
Meptides at 11 stoichiometry
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3. Prediction of Protein Function from Structure
? Non-Enzyme
? Enzyme
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Orphan Genes and Structures

• ? 200 genomes (? 25 eukaryotes, 150 bacteria, 20
  archaea)
• ? 50 unknown function
• PDB (July 6th 2004)
• 26000 structures
• 408 unknown function

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Function from Structure

• Aim - assigning function for orphan structures
  (no similar sequence or fold)
• Two Classes - Enzyme/Non-Enzyme
• Support Vector Machines machine learning

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Structural Features

• Amino acid composition
• Amino acid composition of surface
• Cofactors (NAD, ATP, FAD)
• Metals (Fe, Mg, Cu, Ca)
• Disulphides
• Size of largest cleft
• Secondary structure
• Surface fractal dimension

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Accuracy

• 52 features 77
• 36 features 80
• Easier to predict an enzyme (90) than a
  non-enzyme (69)
• Extended to predicting EC number and to predict
  functional class from sequence for orphan genes

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