Protein Purification & Secondary Structures

What are 3 types of chromatography?

• Gel filtration

• Ion exchange

• Affinity

How does gel electrophoresis work? (non-denaturring)

Non-denaturing

1. make a gel by polymerizing acrylamide with crosslinker in buffer

2. load protein samples

3. apply voltage (protein separation based on size and charge)

4. detect proteins via stain

Where do they smallest proteins come off in gel filtration? in gel elctrophoresis??

• G.F → very last fraction

• G.E. → very end of lane

What is isoelectric focusing?

when proteins are separated by the pI

What are the 2 separation techniques used in 2-D electrophoresis?

1. Isoelectric focusing for charge

2. SDS-Page (denaturing) for MW

What do different peaks in a mass spectrography represent?

mass-to-charge ratio

what are 3 diseases resulting from improper folding?

Misfolding

• Marfan syndrome

• Amyotrophic lateral sclerosis

• Scurvy

Toxic folds

• Acrapie

• Alzheimer’s disease

• cataracts

rotations around which bonds do the conformation angles phi & psi represents?

phi → N-C alpha

psi → C-C alpha

which backbone bond is unable to freely rotate? why?

peptide bond (C-N bond) is unable to freely rotate because of its partial double-bond character, which locks it into a planar configuration.

what effect does the chirality around C alpha have on the appearance of a Ramachandran plot?

influences the steric constraints on the φ and ψ angles

How does Gel Filtration chromatography work?

1. Protein is applied to either column of gel, beads, or resin

2. buffer is passed through column, & fractions are collected

3. separated by molecular weight

   1. MW determined by comparison to standards

In gel filtration chromatography, why do the larger molecules come out first?

They elute first because they are too big for the column pores

How does ion exchange chromatography work?

• based on attraction between charged groups in protien and column medium (gel or beads)

• selective absorption and elution by pH or salt concentration

What are the types of ion exchange chromatography?

• anion exchanger

  • protein supplies anion

  • - charge

• cation exchanger

  • protein supplies cation

  • + charge

pI

The isoelectric point; the pH at which the molecule carries no net electrical charge.

How does affinity chromatography work?

1. based on selective binding of ligand

2. ligand linked to insoluble col. medium

3. pass protein solution over col.

4. Only the binding protein is absorbed

5. elute with high conc. of unbound ligand

How does gel electrophoresis work? (denaturing)

Denaturing

1. treat protein samples with heat & reducing agent

2. SDS is added to gel & sample

   1. reduced polypeptides with bound SDS form (-) charged bands

3. Polypeptides based on size (larger is at top)

Which bonds rotates on amino acids? What limits rotations?

• N-C Alpha = phi

• C-C Alpha = psi

• rotational freedom is limited by size of the R group

what is a ramachandran plot?

tells the reader what which combinations of psi and phi are favorable

What is the secondary structure of proteins a result of?

Hydrogen bonding between carbonyl oxygens and amide hydrogen of peptide bonds

What are the two fundamental types of secondary structure?

• alpha helix

• beta pleated sheet

What is important about the alpha-helix core structure?

• peptide carbonyl O is H bonded to the peptide N-H, 4 amino acids away

• all H bonds lie parallel to helix axis

• all carbonyl groups point in one direction

• all N-H groups point in opposite direction

• most common helix is right handed

• helices have macro dipole because peptide planes spiral around macro diople

why is Pro called a helix breaker?

no N-H groups because it can destabilize the helices

Why are B pleated sheets roughly perpendicular?

R groups are not exactly in the plane

What are the 2 types of of beta pleated sheets?

• parallel: beta strands run in the same direction

• anti-parallel beta sheets: beta strands run in opposite directions

What should you know about beta turns?

• the Carbonyl O that is H-bonded to amide that is 3 residues away makes a tight turn

• proline and glycine are really the only ones who can handle this tight angle

How does the body solve these folding errors?

Chaperones provide a space where proteins can fold slower

How does the body degrade bad protiens?

Ubiquitin on lysine tags molecules which are sent to a protease chamber.