Download Protein Structure

Protein Structure
(Campbell text chapters 4 and 5)
(shape is everything)
Why study structure?
• Structure helps us understand function
• Many disorders are due to aberrant protein
structure
– Sickle cell anemia
– Can aid in design of therapeutics
• Disruption of structure causes disruption of
function
– denaturation
Native Structure
• The normal structure
found in an organism
• Functional structure
– Human Insulin
Prosthetic Groups
Non-amino acid
molecule that is
necessary for the
protein to function
• Ex: heme in
hemoglobin
• Mostly an organic
molecule
– Often contain metal
ions
Stabilization of Protein Structure
• H-bonds
– Backbone
– Side chain
• Disulfides
• Electrostatic
• Nonpolar forces
Driving of protein folding
• What provides the energy?
– Stuff gets moved pretty far
Requires energy
Driven by entropy increase of solvent
Levels of Protein Structure
• Primary
• Secondary
– Supersecondary
• Tertiary
• quaternary
Primary Structure
• Amino acid sequence
– Read amino to carboxyl
– Below is the sequence for insulin
malwmrllpl lallalwgpd paaafvnqhl cgshlvealy
lvcgergffy tpktrreaed lqvgqvelgg gpgagslqpl
alegslqkrg iveqcctsic slyqlenycn
– Can get sequences from the link below
http://www.ncbi.nlm.nih.gov/
Determination of primary structure
• Biochemical
– Enzymatic digestion
– Sequential Edman degradation
• Molecular biological
– Determine nucleotide sequence
– Deduce amino acid sequence
Determination of primary structure
• Determine amino acid content
– Boil protein in 6 M HCl
• Protein cleavage
– Use enzymes that cut at different places
• Chymotrypsin: cuts at C-end of aromatic aa
• Trypsin: cuts at C-end of + charged aa
• Cyanogen bromide: cuts after M
– Determine sequences of peptides
– Line up
Primary Sequence determination
example
• Trypsin Digests:
N-T-W-M, D-T-W-M-I-K, G-Y-M-Q-F-V-L-G-M-S-R
• CNBr digests:
Q-F-V-L-G-M, D-T-W-M, S-R-N-T-W-M, I-K-G-Y-M
What is the correct sequence?
Answer
Secondary structure
• Simple folding
– α-helix
– β-pleated sheet
– Collagen helix
α-helix
• Right handed helix
• 3.6 aa/ turn
• Backbone H-bonding
between carboxyl oxygen
and amino nitrogen 4 aa
away
– C=O on aa 1 bonds to N-H
on 5
– C=O on aa 2 bonds to N-H
on 6 etc
• Proline doesn’t fit into
helix
α-helix
• R groups perpendicular to
helix
• To right is view down
helix axis
• AA close in primary
sequence of protein not
necessarily close in final
structure
• AA far away in primary
often close in final
structure
Beta sheet
• Parallel or antiparallel
sheet
• Backbone H-bonding
between C=O and NH on adjacent strands
• Sheet is pleated
Beta Sheet side view
• R- groups below and
above plane of sheet
• R-groups on adjacent
aa very far apart
R
R
R
R
R
R
R
Collagen helix
• Triple helix
• Helices wrapped
around each other.
• Sequence is G-X-PG-X-P etc
• Found in tendons and
ligaments
• Very strong
• H-bonding between
strands
Supersecondary structure
• Structural motifs
• Repeating patterns of secondary
structures
– Beta barrels
– Alpha-beta-alpha-beta
– Examples in next slides
Helix- turn- helix
Beta sandwiches
4 alpha bundle
Beta Barrel
Alpha beta horseshoe
Tertiary Structure
• Final 3-D structure of the protein
• Folded native form
• aa close together in primary sequence
often far apart in tertiary
• aa far apart in primary may be close in
tertiary
• NCBI HomePage
Myoglobin structure
Quaternary Structure
• Subunits
• Made from more than one polypeptide
chain
• Held together by weak interactions
Hemoglobin
Protein Structure Determination
• X-ray crystallography
– X-rays diffract off of molecule
– Pattern is Fourier transform of actual structure
– 2 angstrom resolution
– Have to make crystal
• NMR
– Similar to MRI technology
– Can do in solution
– Resolution not as good
Protein purification
• Start with source
– Cells from animal or plant
– Molecular bio
• Grind up
• Centrifuge
Protein purification
• Chromatography
– Ion exchange
– Size exclusion
– Affinity chromatography
• Use of antibodies
Protein Purification
• Assays
– Functional
• Activity
– Define
– Protein amount
• Colorimetric
• Beer’s law
• ELISA
– Determination of specific activity
• Activity / mg protein
SDS PAGE
• Sodium Dodecyl
Sulfate
PolyAcrylamide Gel
Electrophoresis
• Separates by size
• Distance travelled is
function of log(MW)
• Native v denaturing
Answer to sequence
D-T-W-M-I-K-G-Y-M-Q-F-V-L-G-M-S-R-NT-W-M
• Back