Lecture 10 - BIO 311 PROTEIN SRUCTURE and DETECTION METHODS...

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BIO 311 PROTEIN SRUCTURE and DETECTION METHODS
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R = side chain – this is variable Amino acids The building blocks of proteins C H R N H H C O OH NH 2 = primary amine group NH 2 = primary amine group N H H COOH = carboxyl group
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Peptide Bond C H R N H H C O OH C H R N H H C O OH amino acid # 1 amino acid # 2 C H R N H H C O C H R N H C O OH a peptide bond! H H 2 O O OH H
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Protein Synthesis Remember, DNA is transcribed to RNA 5’ to 3’ RNA is translated to protein from N- terminus to C-terminus 5’ to 3’ N to C Done on ribosomes with the assistance of tRNA
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Genetic Code
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Enhanced Green Fluorescent Protein What does “enhanced” mean? 1. Codon usage has been switched to correspond to “human” codon-usage by creating 190 silent base changes. 2. The Kozak consensus translation initiation site has been placed in the upstream mRNA sequence. 3. Double amino acid substitutions of a red-shift variant form of GFP are used for brighter fluorescence.
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Amino Acid Codon % usage in humans Anticodon in tRNA Glutamine CAG 73 % CTG Glutamine CAA 27 % UUG Q Human Codon Usage
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Protein Synthesis AAA AAG GCCGGCUGG AAGUUA TTTCGGCCGACCTTCTTCAAT Lys Ala Gly Trp Lys Lys Leu ribosome tRNA Transcription Translation DNA 5’ 3’ N C RNA Protein (polypeptide)
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Levels of structure in protein architecture Primary Secondary Tertiary Quaternary
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Primary Structure Order of amino acid sequence At this point the molecule is called a polypeptide Lys Ala Gly Trp Lys Lys Leu
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Coding region: complement(24. .743) Protein sequence: 239 aa MVSKGEELFT GVVPILVELD GDVNGHKFSV SGEGEGDATY GKLTLKFICT TGKLPVPWPT LVTTL SYG V Q CFSRYPDHMK Q HDFFKSAMP EGYV Q ERTIF FKDDGNYKTR AEVKFEGDTL VNRIELKGID FKEDGNILGH KLEYNYNSHN VYIMADK Q KN GIKVNFKIRH NIEDGSV Q LA DHY QQ NTPIG DGPVLLPDNH YLST Q SALSK DPNEKRDHMV LLEFVTAAGI TLGMDELYK EGFP CAG
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Secondary Structure The spatial arrangement of amino acids 2 types of arrangements α - helix β - sheet
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Bonding Between Amino Acids of a Polypeptide Chain (a) H - bonds (a) H - bonds (b)Disulfide bonds (Cys and Cys) (b)Disulfide bonds (Cys and Cys) (c) Ionic bond (Asp and Lys) (c) Ionic bond (Asp and Lys) (d) Hydrophobic bond (Val and Ile) (d) Hydrophobic bond (Val and Ile)
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Bonding Stabilizes Secondary Structure of the Alpha Helix The NH of an amino acid forms an H- bond with the CO of the amino acid 4 residues earlier
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Bonding Stabilizes Secondary Structure Anti-parallel β - sheet Parallel β - sheet
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Tertiary Structure Globin fold β - barrel The 3 – D structure of the protein
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Bonds Stabilize Tertiary Structure as well Hydrophobic Hydrophobic bonds bonds H-bonds Disulfide bonds Ionic bonds
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Green Fluorescent Protein (GFP) The protein has 239 amino acids The three dimensional structure was determined in 1996 Looks like a barrel with a light bulb – the fluorophore Can be detected by fluorescence of the imbedded fluorophore at amino acids 65, 66 and 67 http://www.rpc.msoe.edu/cbm2/gfp1.htm
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S S Quaternary Structure of an Antibody V H C L V L C L Arrangement of multiple protein molecules in a multi-subunit complex
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2 Different Types of Proteins Based on Structure Fibrous proteins Globular proteins
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Lecture 10 - BIO 311 PROTEIN SRUCTURE and DETECTION METHODS...

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