Protein Structure

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


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


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-MWhat is the correct sequence?


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 N-H 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-P-

G-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


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


Protein Structure

Documents

native structure

d structure

final structure aa

actual structure

normal structure

study structure

myoglobin structure

primary sequence of