PROTEINS Nicky Mulder Acknowledgements: Anna Kramvis for lecture material (adapted here)

PROTEINS

Nicky Mulder

Acknowledgements: Anna Kramvis for lecture material (adapted here)

Central dogma of molecular biology

http://www.cem.msu.edu/~reusch/VirtualText/nucacids.htmhttp://www.cem.msu.edu/~reusch/VirtualText/nucacids.htm

Protein building blocks

Proteins are made up of amino acids 20 possible amino acids Each specified/encoded by a triplet of

bases Messenger RNA transcripts translated into

proteins

The Genetic Code

Each amino acid is specified by a triplet of 3 bases (codons)

Codons were elucidated a decade after the discovery of the DNA structure in 1953

If we have the 4 bases A,C,G,T we have 4 x4 x4 = 64 possible codons

Actually 61 codons + 3 stop codons

The Genetic Code

Codon usage varies

Open reading frame

String of in-frame combinations/triplets of bases that specify an amino acid

Starts with ATG (Meth) or Val Ends with stop codon One base insertion or deletion –out of

frame/frameshift

Translating sequences

6 possible reading frames, 3 in each direction

AGTCGGCTGACTGCGTTTACGAATGCGATTACTCCCTT

AAGGGAGTAATCGCATTCGTAAACGCAGTCAGCCGACT

Reverse complement

+1

-1

Translating sequences

6 possible reading frames, 3 in each direction

AGTCGGCTGACTGCGTTTACGAATGCGATTACTCCCTT

AAGGGAGTAATCGCATTCGTAAACGCAGTCAGCCGACT

+2

-2

Translating sequences

6 possible reading frames, 3 in each direction

AGTCGGCTGACTGCGTTTACGAATGCGATTACTCCCTT

AAGGGAGTAATCGCATTCGTAAACGCAGTCAGCCGACT

-3

+3

Getting the final protein

Six-frame translation Find longest ORF with initiation site, start

codon and ending with stop codon

Transcription and translation ATGCGGTGCAACGTGCATCCTAAA

UACGCCACGUUGCACGUAGGAUUU

W G P Y T A K L

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Ribosomes

Protein synthesizers Different subunits for interacting with

mRNA and tRNAs

Copyright-Anna Kramvis 15

Translation process

Amino acid structure

The chemistry of R groups distinguishes amino acids and their properties

Valine Leucine Serine Tyrosine ProlineValine Leucine Serine Tyrosine Proline

Polypeptide chain

Each protein has a unique sequence of amino acids joined into a polypeptide chain

Protein primary structure

Proteins made up amino acids joined by peptide bonds between carboxyl group of one and amino group of the next

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Peptide backbone

Primary structure, disulphide bonds

Secondary structure Held together by interactions (H-bonds)

between peptide backbones

Tertiary structure

Tertiary structure is controlled by the interactions between non-adjacent amino acid R groups

Quaternary Structure

More than one protein chain, e.g. hemoglobin

Possible bonds in proteins

Hydrogen bonds: weak electrostatic attractions between electronegative atom (O or N).

Van der Waals forces: can be attractive or repulsive, depends on distance

Electrostatic interactions or ionic bonds: weak bonds that form between charged groups in aqueous environments

Hydrophobic effects: arise because hydrogen bonded structure of water forces hydrophobic groups into the internal parts of the protein.

Other structures

Summary of protein structures

The function of a protein depends on sequence of

amino acids and requires a precise folding of its

polypeptide chain

Properties of Amino Acids

http://www.jalview.org/help/html/misc/properties.gifhttp://www.jalview.org/help/html/misc/properties.gif

29Copyright-Anna Kramvis

Name     R-Group PropertiesGlycine G Gly HydrophobicAlanine A Ala HydrophobicValine V Val HydrophobicLeucine L Leu HydrophobicIsoleucine I Ile Hydrophobic, two chiral carbonsProline P Pro Cyclic, not terribly hydrophobicPhenylalanine F Phe Hydrophobic, bulkyTyrosine Y Tyr Less hydrophobic (than Phe), bulkyTryptophan W Trp Hydrophobic, bulky (indole ring)Cysteine C Cys Hydrophobic, highly reactive (S-S link)Methionine M Met Hydrophobic (start a.a.)Serine S Ser Hydrophilic, reactiveThreonine T Thr Hydrophilic, reactive, two chiral carbonsLysine K Lys Highly hydrophilic, positively chargedArginine R Arg Highly hydrophilic, positively chargedHistidine H His Highly hydrophilic, positive or neutralAspartate D Asp Highly hydrophilic, negatively chargedGlutamate E Glu Highly hydrophilic, negatively chargedAsparagine N Asn UnchargedGlutamine Q Gln Uncharged

Some protein functions

Information from a protein sequence

MDITIQHPWFKRALGSLYPSRLFDQFFGEGLFEYDLLPFLSSTISPYYRQSLFR

• amino acid composition• molecular weight

MDQHPWFKRAITIVLLGLLPFLSLYPSRLFDQFCGEGLFEYDSSTISCYRQSLFRTVLESG

Single amino acid physical properties

D,E -acidic

C,D,E,H,K,N,Q,R,S,T –polar, active sites, metal binding

V,L,I,M –hydrophobic, membrane

C –disulphide-rich, disulphide bonds

Information from a protein sequence

MDQHPWFKRAITIVLLGLLPFLSLYCPSRLFDQFCGEGLFEYDSSTISYRQSLFRTNVLES

Functionally important regions

Active site/metal bindingHydrophobic region

disulphide bondGlycosylation site

• Transmembrane regions• Signal sequences• Localisation signals (subcellular location)• Targeting sequences• Modification sites

Information from a protein sequence

FAMILY

DOMAIN

MOTIF

SITE

RESIDUE

GKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYN

Physical amino acid properties

Conserved domains

Properties of regions

Conserved sequence

Information from a protein sequence

Protein abundance

Not all genes are expressed all the time, amount of protein is affected by: gene expression -transcriptional regulationPost-transcriptional regulationTranslational regulationPost-translational regulation

Transcription regulation Regulators –enhancers and repressors,

can be cis- or trans-acting Bind to specific sites Sigma factors, anti-sigma factors DNA unwinding DNA methylation Signalling pathways

Post-transcriptional regulation

mRNA half-life Antisense RNA RNA splicing siRNAs

Translational regulation

Ribosomes Translation factors tRNA availability

Post-translational regulation

Transport to appropriate place Protein folding (chaperones) Post-translational modification:

PhosphorylationAcetylationSugars added….

http://jp.senescence.info/thoughts/dna_life.jpghttp://jp.senescence.info/thoughts/dna_life.jpg

Summary of main building blocks of biological systems

Translation exercise

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1. Translate this mRNA using the genetic code table

5’AUGUUUUUGUCGUACUGGUGUCUACCUCAUCAACGUAUUACGAAUAAG3’

Write out the translation using the one letter and three letter conventions.

2. Give the characteristics of each amino acid in the polypeptide chain.

3. How long is the original RNA sequence and how long is the protein sequence?

Additional questions Here is a gene sequence:

5’ AGCAATGCATGCATCGTTATGG 3’

Identify the initiation codon What reading frame is it in? Would translation be affected if the first C was changed

to G, if so, what effect? Would translation be affected if the second last C was

changed to T, if so, what effect?