Transcript
Protein Structure
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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
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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-MWhat is the correct sequence?
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Secondary structure
• Simple folding– α-helix– β-pleated sheet– Collagen helix
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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
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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
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Beta sheet
• Parallel or antiparallel sheet
• Backbone H-bonding between C=O 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
R
R
R
R
R
R
R
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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
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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• Beer’s 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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