Transcript Protein Structure and Analysis

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
acids
structure: sequence of amino
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
2 or more
polypeptide
chains

Structure
 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

bases (uracil, adenine, guanine, or
cytosine)
 Thymine


in DNA
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 TAT
AUG GCA CCG AUA
Ribosomes
 Composed
of ribosomal RNA (rRNA)
and proteins
 large ribosomal subunit and small
ribosomal subunit
 Eukaryotic and prokaryotic
Ribosome
Structure
tRNA (Transfer)
 “transfer”
amino acids to ribosome
 mRNA codon specifies which tRNA
(transport a specific amino acid)
 tRNA has a complimentary anticodon
tRNA
mRNA
UAC CGC GGC UAU
AUG GCA CCG AUA
Genetic Code
 mRNA



3 nucleotides (AAU, UAA…)
specify a sequence of amino acids
Nirenberg and Matthaei – poly-U
(phenylalanine)
 64


codons
codons (43)
61 code for amino acids
3 codons are stop signals
Genetic Code
 Is

 Is


redundant
some amino acids have more than one
codon
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. Transcription
2. 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 GAT
RNA 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 Psite 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’


 3’

cap: modified guanine nucleotide
Protects mRNA from hydrolytic enzymes
“Attach here” signal for ribosome
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
matrix

Separate smaller fragments of DNA and
proteins
 Two


gels: smaller pores/tighter
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




Precision Plus Protein Kaleidoscope prestained
standard



TRIS; pH
SDS; keep protein denatured
Glycine: ions electrophoresis
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


Antibodies



Covers areas of gel not occupied by proteins –
prevents non-specific binding of 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 Hypothesis
Induced Fit
E + S
 Substrate



E-S Complex
E + P
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