Lectures 4 - 7 Flashcards
Ways to modify protein structure
- Chemical reagents
- Unnatural amino acids
- In vitro/in vivo synthesis
Use of chemical reagents to modify protein structure
Add a reagent you expect to react with a chemical group in the protein structure - it is non-selective and gives mixed products
Use of unnatural amino acids to modify protein structure
- Allows a specific function to be added
2. Can expand genetic code through promiscuous translation, suppressor codons and orthologous tRNA/AATS pairs
SupE mutation in E.coli
tRNA recognises UAG as a coding codon (pairs with GUC), resulting in a read-through of the STOP codon.
Requirements to incorporate unnatural aa in vivo
Unnatural aa
Cognate tRNA (that binds to aa)
Cognate aminoacyl-tRNA synthetase
Problems with incorporating unnatural aa in vivo
Costly, low yield
Requires special aa to be acylated - in vitro this can be carried out via chemical charging but in vivo must use modified tRNAs/AATS
Requires orthologous tRNA/AATS pairs e.g. don’t want special tRNA to be charged with normal aa
Requires special codons - use unused/low frequency ones
How to engineer tRNA so it is only recognised by cognate AATS
- Make a mutant tRNA ortholog library
- Select tRNAs that are recognised and acylated by cognate AATS
- Kill/remove tRNAs acylated by native AATSs
How to engineer AATS to specifically recognise unnatural amino acid and acylate cognate tRNA
- Produce a library of AATS mutated in the amino acid binding site
- Live or die selection - kill all AATS that incorporate natural aa
In vivo synthesis of unnatural aa
It is more efficient to use host machinery:
1. Start with endogenous precursor
2. Use biosynthetic enzymes
3. Synthesised aa incorporated via orthologous tRNA/AATS pair
Example - p-amino Phe
Homologs
Descended from common ancestor, similar sequence
Paralogs
Proteins related via a gene duplication event, present in the same organism but different function
Orthologs
Same gene and the same function, but different organsisms.
Conformation of most peptide bonds
Trans
Broadest + most restricted Ramachandran plot
Boadest = Gly, most restricted = pro
Alpha helix capping
Capped at N terminus via H bond
Capped at C terminus due to Gly having an LH conformation
Alpha helix forming ability
Dependent on delta Gu
Ala = most likely to form alpha helix
Pro + gly = least likely
Why is Proline often found in 1st turn
Results in a 20 degree bend
Where are amphipathic helices found?
Protein surface
Polyproline helices
LH, no internal H bonds
Antiparallel beta-sheets
One side is exposed the other buried, straight H bonds,, >2 strands
Parallel beta-sheets
H bond at angle - less stable, >5 strands
Globular Proteins
Compact structure, diverse range, hydrophobic core
Motif
Ordered arrangement of secondary structure e.g. Helix-turn-helix (DNA binding protein)
Why does leucine to alanine mutation in globular proteins have a large impact?
Leaves a large cavity - non-optimal core packing
Types of protein cavity mutants
- Steric mutants - shape of cavity changes, not volume
- Extreme volume mutants - increase in volume of cavity
- Polar mutants - polar residue in cavity
Is protein surface normally polar or nonpolar?
Polar
Effect of mutation creating many hydrophobic residues on protein surface?
Reduced solubility, protein aggregation
Benefits of domains
Many protein folding faster, more efficent and can add function
Subunit
1 polypeptide chain stably folded by itself
Non-proteinogenic amino acids
GABA - neurotransmitter
Histamine - immunological response
Thyroxine - hormone (metabolism regulation)
Non-standard aa found in proteins
Hydroxyproline
Lysine methylated by S-adenosylmethionine to create a less reactive version of the aa
Enzyme on/off switch
Ser/Thr phosphorylation via O-glycosylation
Non-specific protein modification
Random hydrolysis of arginine side chain can produce citrulline - cause of autoimmune disease.
Measure of protein ageing
Glycation of protein amino groups
In-vitro chemical modification
via site-directed mutagenesis e.g. amino group acetylation
In vivo fluorescence labelling
Fusion via GFP gene
In vitro fluorescence labelling
Use labels at different wavelengths
Radioactive labelling evaluation [14C, 3H, 35S]
+ only require a few atoms, doesn’t affect properties much
- Safety concerns
Crosslinking reagents
Homobifunctional - both ends of the molecules react with the same functional group
Heterobifunctional - different reactive groups at each end of the molecule - allows for more control
Literature database
Pubmed
Compound database
Pubmed compounds
Sequence database
Genbank
Functional protein information databank
UniprotKB
3D Structure databank
CSD (small molecules), PDB (proteins)
Protein Classes
- Fold - same topology, no evidence for relation
- Superfamily - same fold, related function, probably related
- Family - structure and function very similar, almost defo related
What does sequence alignment show?
Indels, gene duplication events, sequence similarity, synonymous and non-synonymous mutations
Why is it easier to determine homology from protein sequence?
Each letter gives more information, protein evolves slower e.g. may be synonymous mutation - not shown in DNA sequence as clearly
What % sequence required for homology?
> 30%
Types of alignment
- Global alignment - search through an entire sequence looking for similarities
- Local alignment - only searches through single domains
- Pairwise alignment - only compares 2 sequences
- Multiple alignment - more than 2 sequences
Multiple sequence alignment
Used to generate phylogenetic trees
Used to find characteristic protein family pattern
Generated through tree method/divide and conquer method
Types of phylogenetic tree
- Cladogram - shows geneology, branch lengths arbitory
- Additive tree - branch = measure of time
- Ultrametric tree - always rooted and contains timeline
E-value
no. expected hits by chance. Lower number = more relevant result
Sliding window
Approach used to analyse amino acid properties - properties within a window are averaged, and then the window moves along sequence to build up full picture
Protein function assigned based on…
- Genome comparison
- Sequence similarity - more likely in orthologs than paralogs
- Structural similarity - specific fold linked to function
