Lectures 4 - 7 Flashcards by Rachel Stoker

Ways to modify protein structure

Chemical reagents
Unnatural amino acids
In vitro/in vivo synthesis

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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

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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

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SupE mutation in E.coli

tRNA recognises UAG as a coding codon (pairs with GUC), resulting in a read-through of the STOP codon.

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Requirements to incorporate unnatural aa in vivo

Unnatural aa
Cognate tRNA (that binds to aa)
Cognate aminoacyl-tRNA synthetase

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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

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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

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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

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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

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Homologs

Descended from common ancestor, similar sequence

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Paralogs

Proteins related via a gene duplication event, present in the same organism but different function

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Orthologs

Same gene and the same function, but different organsisms.

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Conformation of most peptide bonds

Trans

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Broadest + most restricted Ramachandran plot

Boadest = Gly, most restricted = pro

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Alpha helix capping

Capped at N terminus via H bond

Capped at C terminus due to Gly having an LH conformation

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Alpha helix forming ability

Dependent on delta Gu
Ala = most likely to form alpha helix
Pro + gly = least likely

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Why is Proline often found in 1st turn

Results in a 20 degree bend

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Where are amphipathic helices found?

Protein surface

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Polyproline helices

LH, no internal H bonds

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Antiparallel beta-sheets

One side is exposed the other buried, straight H bonds,, >2 strands

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Parallel beta-sheets

H bond at angle - less stable, >5 strands

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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

1. Steric mutants - shape of cavity changes, not volume 2. Extreme volume mutants - increase in volume of cavity 3. 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

1. Fold - same topology, no evidence for relation 2. Superfamily - same fold, related function, probably related 3. 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

1. Global alignment - search through an entire sequence looking for similarities 2. Local alignment - only searches through single domains 3. Pairwise alignment - only compares 2 sequences 4. 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

1. Cladogram - shows geneology, branch lengths arbitory 2. Additive tree - branch = measure of time 3. 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...

1. Genome comparison 2. Sequence similarity - more likely in orthologs than paralogs 3. Structural similarity - specific fold linked to function