Protein Structure (Campbell text chapters 4 and 5)

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Protein Structure (Campbell text chapters 4 and
5)

• (shape is everything)

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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

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Native Structure

• The normal structure found in an organism
• Functional structure
• Human Insulin

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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

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Stabilization of Protein Structure

• H-bonds
• Backbone
• Side chain
• Disulfides
• Electrostatic
• Nonpolar forces

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Driving of protein folding

• What provides the energy?
• Stuff gets moved pretty far
• Requires energy
• Driven by entropy increase of solvent

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Levels of Protein Structure

• Primary
• Secondary
• Supersecondary
• Tertiary
• quaternary

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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/

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Determination of primary structure

• Biochemical
• Enzymatic digestion
• Sequential Edman degradation
• Molecular biological
• Determine nucleotide sequence
• Deduce amino acid sequence

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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

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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

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Secondary structure

• Simple folding
• a-helix
• ß-pleated sheet
• Collagen helix

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a-helix

• Right handed helix
• 3.6 aa/ turn
• Backbone H-bonding between carboxyl oxygen and
  amino nitrogen 4 aa away
• CO on aa 1 bonds to N-H on 5
• CO on aa 2 bonds to N-H on 6 etc
• Proline doesnt fit into helix

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a-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

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Beta sheet

• Parallel or antiparallel sheet
• Backbone H-bonding between CO and N-H on
  adjacent strands
• Sheet is pleated

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Beta Sheet side view

• R- groups below and above plane of sheet
• R-groups on adjacent aa very far apart

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Collagen helix

• Triple helix
• Helices wrapped around each other.
• Sequence is G-X-P-G-X-P etc
• Found in tendons and ligaments
• Very strong
• H-bonding between strands

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Supersecondary structure

• Structural motifs
• Repeating patterns of secondary structures
• Beta barrels
• Alpha-beta-alpha-beta
• Examples in next slides

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Helix- turn- helix
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Beta sandwiches
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4 alpha bundle
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Beta Barrel
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Alpha beta horseshoe
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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

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Myoglobin structure
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Quaternary Structure

• Subunits
• Made from more than one polypeptide chain
• Held together by weak interactions

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Hemoglobin
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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

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Protein purification

• Start with source
• Cells from animal or plant
• Molecular bio
• Grind up
• Centrifuge

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Protein purification

• Chromatography
• Ion exchange
• Size exclusion
• Affinity chromatography
• Use of antibodies

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Protein Purification

• Assays
• Functional
• Activity
• Define
• Protein amount
• Colorimetric
• Beers law
• ELISA
• Determination of specific activity
• Activity / mg protein

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SDS PAGE

• Sodium Dodecyl Sulfate PolyAcrylamide Gel
  Electrophoresis
• Separates by size
• Distance travelled is function of log(MW)
• Native v denaturing

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Answer to sequence

• D-T-W-M-I-K-G-Y-M-Q-F-V-L-G-M-S-R-N-T-W-M
• Back