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

Central dogma of molecular biology http://www.cem.msu.edu/~reusch/VirtualText/nucacids.htm 2

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 4

The Genetic Code Codon usage varies 5

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 +1 Reverse complement AAGGGAGTAATCGCATTCGTAAACGCAGTCAGCCGACT -1

Translating sequences 6 possible reading frames, 3 in each direction AGTCGGCTGACTGCGTTTACGAATGCGATTACTCCCTT +2 AAGGGAGTAATCGCATTCGTAAACGCAGTCAGCCGACT -2

Translating sequences 6 possible reading frames, 3 in each direction AGTCGGCTGACTGCGTTTACGAATGCGATTACTCCCTT +3 AAGGGAGTAATCGCATTCGTAAACGCAGTCAGCCGACT -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

http://www. virtualsciencefair. org/2004/mcgo4s0/public_html/t3/RNA http://www.virtualsciencefair.org/2004/mcgo4s0/public_html/t3/RNA.html 12

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Ribosomes Protein synthesizers Different subunits for interacting with mRNA and tRNAs 14

Translation process Copyright-Anna Kramvis 15 15

Amino acid structure The chemistry of R groups distinguishes amino acids and their properties

Polypeptide chain Each protein has a unique sequence of amino acids joined into a polypeptide chain Already in 1905 it was known that proteins consist of amino acids linked by peptide bonds. Chain lengths vary from 5 to 4000 amino acids. There are 20 amino acids and proteins consist of various mixtures of these amino acids. Valine Leucine Serine Tyrosine Proline 17

Protein primary structure Proteins made up amino acids joined by peptide bonds between carboxyl group of one and amino group of the next www.columbia.edu commons.wikimedia.org

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 25

Summary of protein structures

The function of a protein depends on sequence of amino acids and requires a precise folding of its polypeptide chain 27

Properties of Amino Acids http://www.jalview.org/help/html/misc/properties.gif 28

Hydrophobic, two chiral carbons Proline P Pro Name   R-Group Properties Glycine G Gly Hydrophobic Alanine A Ala Valine V Val Leucine L Leu Isoleucine I Ile Hydrophobic, two chiral carbons Proline P Pro Cyclic, not terribly hydrophobic Phenylalanine F Phe Hydrophobic, bulky Tyrosine Y Tyr Less hydrophobic (than Phe), bulky Tryptophan 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, reactive Threonine T Thr Hydrophilic, reactive, two chiral carbons Lysine K Lys Highly hydrophilic, positively charged Arginine R Arg Histidine H His Highly hydrophilic, positive or neutral Aspartate D Asp Highly hydrophilic, negatively charged Glutamate E Glu Asparagine N Asn Uncharged Glutamine Q Gln Copyright-Anna Kramvis 29 29

Some protein functions proteins enzymes bind ions structural membranes connective tissue muscle fibres extracellular hormones antibodies 30

Information from a protein sequence MDITIQHPWFKRALGSLYPSRLFDQFFGEGLFEYDLLPFLSSTISPYYRQSLFR amino acid composition molecular weight

Information from a protein sequence Single amino acid physical properties MDQHPWFKRAITIVLLGLLPFLSLYPSRLFDQFCGEGLFEYDSSTISCYRQSLFRTVLESG 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 Functionally important regions MDQHPWFKRAITIVLLGLLPFLSLYCPSRLFDQFCGEGLFEYDSSTISYRQSLFRTNVLES Active site/metal binding Hydrophobic region Glycosylation site disulphide bond Transmembrane regions Signal sequences Localisation signals (subcellular location) Targeting sequences Modification sites

Information from a protein sequence FAMILY Conserved sequence DOMAIN Conserved domains MOTIF Properties of regions SITE RESIDUE Physical amino acid properties GKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYN

Protein abundance Not all genes are expressed all the time, amount of protein is affected by: gene expression -transcriptional regulation Post-transcriptional regulation Translational regulation Post-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: Phosphorylation Acetylation Sugars added….

Summary of main building blocks of biological systems http://jp.senescence.info/thoughts/dna_life.jpg 40

Translation exercise 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? Copyright-Anna Kramvis 41 41

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?