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Protein Structure and Analysis
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Importance Protein Structure Initiative
NIH; $600 million, 10 years Food
Cheese: Chymosin (cow stomach) know engineered
Enzymes: detergent Bioremidiation Etc….
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Protein Structure Polypeptides
long, linear polymers 20 amino acids (monomers) joined by peptide bonds
Many functions Enzymes, structural components
(collagen),insulin HgB, albumin (egg whites), actin/myosin, antibodies….
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Protein Structure
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Protein Structure
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Protein Structure
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Levels of Protein Structure Primary structure: sequence of amino
acids
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Levels of Protein Structure Secondary structure:
α-helix or β-pleated sheet hydrogen bonds between amino acids
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Levels of Protein Structure Tertiary structure:
Overall shape of polypeptide chain chemical interactions of side chains
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Quaternary Structure 2 or more polypeptide chains
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Denature proteins Change the shape of the protein – change
is activity Primary level - mutations Heat or a change in pH
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Sir Archibald Garrod (1909) Inborn errors of metabolism – disease
caused by the inability to produce specific enzymes
Ex. Alkaptonuria: urine appears black – contains the chemical alkapton (turns black when exposed to air)
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Beadle and Tatum (1941) One gene – one enzyme Bread mold
Wild type: grow on minimal agar – synthesize all needed materials
Mutant: cannot grow on minimal agar – cannot synthesize needed nutrients
Mutant + minimal agar + 1nutrient at a time = pinpoint defective enzyme
One gene : one enzyme (polypeptide)
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RNA Structure RNA nucleotides
ribose (sugar) Deoxyribose in DNA
bases (uracil, adenine, guanine, or cytosine) Thymine in DNA
Phosphate group Single stranded
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Types of RNA mRNA
copy of the DNA message Created during transcription Every 3 bases is called a codon
TAC CGT GGC TATAUG GCA CCG AUA
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Ribosomes
Composed of ribosomal RNA (rRNA) and proteins
large ribosomal subunit and small ribosomal subunit
Eukaryotic and prokaryotic
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RibosomeStructure
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tRNA (Transfer) “transfer” amino acids to ribosome mRNA codon specifies which tRNA
(transport a specific amino acid) tRNA has a complimentary anticodon
tRNA UAC CGC GGC UAUmRNA AUG GCA CCG AUA
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Genetic Code mRNA codons
3 nucleotides (AAU, UAA…) specify a sequence of amino acids Nirenberg and Matthaei – poly-U
(phenylalanine) 64 codons (43)
61 code for amino acids 3 codons are stop signals
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Genetic Code Is redundant
some amino acids have more than one codon
Is virtually universal suggesting all organisms have a common
ancestor few minor exceptions to standard code
found in all organisms
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Genetic Code - wobble hypothesis
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DNA to Protein Information encoded in DNA
codes sequences of amino acids in proteins
2-step process:1. Transcription2. Translation
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Transcription Synthesize messenger RNA (mRNA)
from DNA Occurs in the nucleus RNA Polymerase
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Translation Synthesizes polypeptide chain
Requires mRNA, tRNA and ribosomes
Codon sequence of 3 mRNA nucleotide bases specifies one amino acid or a start or stop signal
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Transcription – level 2 RNA polymerases (RNA synthesis)
Attaches to the promoter region of the gene
Carries out synthesis in 5′ → 3′ direction; attaches to a free 3’ end
Uses a nucleoside triphosphate base
DNA ATT TCA GATRNA UAA AGU CUA
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Translation: Initiation Initiation factors bind to small ribosomal
subunit; mRNA displays initiation codon (AUG) tRNA anticodon (UAC) attaches – carries
f-methionine Lg. ribosomal subunit completes
ribosome
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Translation: Elongation Proceeds 5’ to 3’ A tRNA with a complimentary anticodon
enters the A-site and binds to the mRNA codon
Peptide bond forms between the two amino acids
tRNA that was occupying the P-site, shifts to the E-site, tRNA in A-site shifts to the P-site and a new tRNA moves into the unoccupied A-site – repeats….
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Translation: Termination Stop codon occupies the A-site (UAG,
UAA, UGA) No matching tRNA anticodon Stops translation
Ribosome sub-units separate
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mRNA Editing Primary transcript contains
Exons – expressed Introns – not expressed, removed We have 20,000+ genes and produce
100,000+ proteins – alternate splicing 231,667 exons
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Alternate Editing | 1 | I | 2 | I | 3 | I | 4 | I |
1,2,3,4 or 1,3,4, or 1,2,4, or 123, or 2,3,4
1 gene and 5 different proteins Titan gene 178 exons
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Modifications to mRNA 5’ cap: modified guanine nucleotide
Protects mRNA from hydrolytic enzymes “Attach here” signal for ribosome
3’ end: poly-A tail Protection from hydrolytic enzymes
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Proteins in Biology Cytoskeleton(support),
metabolism(enzymes, hormones), immunity (antibodies), skeletal (collagen, ligaments, tendons, muscle…), communication(chemical messengers)
Fibrous proteins: keratin (skin,nail,fur,hair), myosin (muscle, collagen
Globular: signaling, antibodies, enzymes
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Proteins in Biotechnology Food industry Textiles: size (stiffen) fabrics, spider silk Biofuels, bioremidiation Detergents Insulin growth hormones….
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Protein Analysis Quantification
Colormetric analysis Beer’s law: the quantity of light absorbed
by a substance dissolved in a nonabsorbing solvent is directly proportional to the concentration of the substance: the darker the color the greater the concentration
Measured with a spectrophotometer Generate a standard curve; interpolate data
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Protein Analysis Colormetric analysis
Bradford Assay 1976 M.Bradford Coomassie Blue G-250
Reacts with R-Group of certain amino acids and turns from reddish-brown to blue
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Labs
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Bradford Assay Quantify proteins Coomassie Blue: interacts with R-groups
of specific Beer’s Law: absorbance of a specific
wavelength of light by a solute is directly proportional to the concentration of the solute Correlation between the darkness of the
blue color and the amount of protein
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Coomassie Amino Acid Interactions Pg 6 Lab: binds to proteins in 3 ways Arginine: electrostatic binding of sulfate
groups Electron stacking: interaction between
aromatic groups of the dye and AA’s Hydrophobic interaction with polar AA’s
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SDS-PAGE Quantify DNA #bps; linear bps ~ the
same size (purine:pyrimidine) Proteins: variable sizes and MW’s of
AA’s (89-204 kD); AA composition varies from protein to protein
Dalton: mass of 1 H atom; 1.66 x 10-24
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Polyacrylamide gels: smaller pores/tighter matrix Separate smaller fragments of DNA and
proteins Two phases:
upper stacking gel (4%) – stacks up the different size proteins so they run uniformley
Lower resolving (20%)
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Laemmli Buffer Tris: correct pH SDS
Dissolve cell membrane – release proteins Coats protein uniform (-) charge; separate by
size not charge (AA’s can be -/+) Bromophenol blue: running dye DTT (dithiothreitol): bad odor: reducing agent;
breaks disulfide linkages (cysteine) protein completely unfolds
Heat: denatures 3 and 4 structure
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SDS-PAGE Gel TGS Buffer
TRIS; pH SDS; keep protein denatured Glycine: ions electrophoresis
Precision Plus Protein Kaleidoscope prestained standard Prestained proteins known molecular wgts – see
gel running Actin/Myosin Standard: positive control/reference
protein Coomassie Stain: blue
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Western Blot W. Neal Burnette (1981)
Pun Southern blot: Edwin Southern Transfer protein to nitrocellulose gel
Protein negative (SDS) pulled from gel towards the + electrode
Gel is fragile Protein is embedded in gel matrix – difficult
to reach Immunodetection remove protein from membrane
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Blocker: 5% non-fat milk protein Covers areas of gel not occupied by proteins –
prevents non-specific binding of antibodies Antibodies
Primary: attaches to target protein Secondary: attaches to primary catalyzes
oxidation of the colormetric substrate: ahs HPR (horseradish peroxidase) attached to it
Colormetric substrate: 4-chloro-1-napthol (4CN)
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Chromatography Used to purify molecules by separating
individual components from complex mixtures Two Phases (of chromo)
Mobile phase: solvent and the molecules to be separated
Stationary phase: medium through which the mobile phase travels; paper, resin (glass beads)
Molecules separate because they travel at different rates
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Chromatography Types Size Exclusion (SEC): porous beads
packed into a column Lg. molecules pass around the beads; sm.
Molecules go through the beads and move through column at a slower rate
Affinity: antibodies are place in a column: mobile form added the protein of interest sticks the antibody while the others pass through
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Chromatography Types Ion Exchange: glass beads in column
have a charge (+ or -); the bead charge is the opposite of the protein of interest
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Enzymes biological catalyst
increases speed of a chemical reaction without being consumed
Complex globular proteins Lower activation energy (EA)
Energy needed to start a reaction Very selective
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Lock and Key HypothesisInduced FitE + S E-S Complex E + P
Substrate binds to enzyme’s active site forming enzyme–substrate complex changes shapes of enzyme and substrate induced fit helps break and form bonds
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Factors that Affect enzyme activity Substrate concentration Enzyme concentration pH
Changes the electrical charge, affects hydrogen bonds – affect tertiary/quartenary structure
Temperature 2X increase/10 degree C increase Drops quickly after 40 C Change enzyme shape
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Temperature and pH
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Enzyme and Substrate Concentration
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Feedback Inhibition and Metabolic Pathways End product inhibits earlier reaction in metabolic pathway
Prevents cells from wasting chemical resources
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Allosteric Enzymes Allosteric – “other site” bind to allosteric sites (noncatalytic sites) changes shape of active site
(confirmation) modifies the enzymes activity
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