Protein Structure and Analysis

Importance Protein Structure Initiative

NIH; $600 million, 10 years Food

Cheese: Chymosin (cow stomach) know engineered

Enzymes: detergent Bioremidiation Etc….

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….

Protein Structure

Protein Structure

Protein Structure

Levels of Protein Structure Primary structure: sequence of amino

acids

Levels of Protein Structure Secondary structure:

α-helix or β-pleated sheet hydrogen bonds between amino acids

Levels of Protein Structure Tertiary structure:

Overall shape of polypeptide chain chemical interactions of side chains

Quaternary Structure 2 or more polypeptide chains

Denature proteins Change the shape of the protein – change

is activity Primary level - mutations Heat or a change in pH

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)

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)

RNA Structure RNA nucleotides

ribose (sugar) Deoxyribose in DNA

bases (uracil, adenine, guanine, or cytosine) Thymine in DNA

Phosphate group Single stranded

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

Ribosomes

Composed of ribosomal RNA (rRNA) and proteins

large ribosomal subunit and small ribosomal subunit

Eukaryotic and prokaryotic

RibosomeStructure

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

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

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

Genetic Code - wobble hypothesis

DNA to Protein Information encoded in DNA

codes sequences of amino acids in proteins

2-step process:1. Transcription2. Translation

Transcription Synthesize messenger RNA (mRNA)

from DNA Occurs in the nucleus RNA Polymerase

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

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

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

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….

Translation: Termination Stop codon occupies the A-site (UAG,

UAA, UGA) No matching tRNA anticodon Stops translation

Ribosome sub-units separate

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

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

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

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

Proteins in Biotechnology Food industry Textiles: size (stiffen) fabrics, spider silk Biofuels, bioremidiation Detergents Insulin growth hormones….

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

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

Labs

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

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

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

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%)

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

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

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

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)

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

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

Chromatography Types Ion Exchange: glass beads in column

have a charge (+ or -); the bead charge is the opposite of the protein of interest

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

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

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

Temperature and pH

Enzyme and Substrate Concentration

Feedback Inhibition and Metabolic Pathways End product inhibits earlier reaction in metabolic pathway

Prevents cells from wasting chemical resources

Allosteric Enzymes Allosteric – “other site” bind to allosteric sites (noncatalytic sites) changes shape of active site

(confirmation) modifies the enzymes activity