Lecture 8 Flashcards
Factors determining 2 structure:
- primary structure can sometimes predict secondary structures
- certain amino acids are prone to specific reactions or none (proline)
Amino acids are alpha helix formers:
leucine and methionine
Amino acids are alpha helix terminators:
proline, where no hydrogens are available or bonding and structure resists rotation of the right-handed helix
Anfinsen Folding Experiment:
- An RNAse is the native protein (folded)
- The RNAse is treated with BME (reduces -S-) and urea (breaks H-bonds)
- Denatured protein
- Removed denaturing agents
- Native protein returns
Anfinsen Folding Experiment:
Removing urea and then oxidizing:
native molecule returns
Anfinsen Folding Experiment:
Oxidizing and then removing urea:
molecules with randomly formed disulfide bonds
Conclusion of the Anfinsen Follding Experiment:
Demonstrated that amino acid sequence was sufficient to direct folding
Denaturing of proteins:
changing temperature, pH, or adding solvents like alcohol.urea
Favorability of protein folding:
thermodynamically unfavorable, entropy decreases, but enthalpy increases because of favorable interactions in water
Protein folding depends on 3 factors:
- unfavorable confirmation change, which favors the unfolded state
- favorable enthalpy contribution arising from intramolecular noncovalent interactions
- favorable entropy change of the solvent arising from the burying of hydrophobic groups
2, 3 > 1 = spontaneous
Conclusions of protein folding:
- not a random process as there are too many different possibilities
- small sections fold as they are translated
- mediated by Chaperones
- stabilized by disulfide bonds, but they aren’t usually in intracellular proteins as this is a reducing environment
Chaperones:
proteins that help in correct folding
Examples of chaperones:
heat shock proteins (HSP 60 and HSP70)
Function of Hsp 70:
- has a high affinity for unfolded proteins
- ATP is hydrolyzed, causing a conformation change and enclosing polypeptide in unfolded state (inhibits incorrect interactions)
- ATP is dephosphorylated and releases polypeptide
Function of Hsp 60 (Chaperonin):
- Groel binds to unfolded proteins via hydrophobic residues
- ATP and Groes causes a conformation change that hides hydrophobic residues and forces the protein into hydrophilic chamber and induce folding
- ATP hydrolysis releases Groes and the folded protein
Properties that cause protein stability:
disulfide bonds, ions, and cofactors (prosthetic groups)
Examples of prosthetic groups:
“zinc finger” and heme group
Result of improper protein folding:
disease
Quatenary structure:
subunits held together by patches of noncovalent bonds to form the whole protein complex
Homodimer:
two identical subunits
Heterodimer:
two different subunits
Example of a heterodimer:
hemoglobin consists of two alpha globin and two beta globin subunits for a total of four subunits
Protein isolation:
a protein must be separated from the other cellular components before its function and structure can be further studied
Homogenization:
to lyse the cell membrane
Methods of homogenization:
- sonification: break cells with high-frequency sound
- detergent lysis: use a mild detergent to make holes in the plasma membrane
- force: force cells through a small hole using high pressure
- mortar/pestle: shear cells between a close-fitting rotation plunger and the thick walls of a glass vessel
Production of homogenization:
homogenate/ cell extract
Differential centrifugation:
separates cellular components by mass to determine the richest source of the protein
1. after forming a homogenate, the mixture is centrifuged in a step-by-step fashion of indcreasing centrifugal force
2. At each step, the supernatant is removed form the pellet that is formed, and then subjected to further centrifugation
Zonal centrifugation:
- separates matters according to density
- often provides a better resolution of separation of particles than differential
1. sucrose is centrifuged to create a gradient
2. sample is layered on top of gradient
3. particles settle accoridng to mass
4. colect fractions and do assay
Fractioning by Precipitation:
adding a small amount of salt helps a protein become soluble, but adding a lot can make it precipitate out of solution
Protein purification by Column Chromatography:
solid and liquid phases are collected out the bottom of the tube and measured at absorbance 280 nm
Size Exclusion Chromatography:
separates macromolecules by size and/or shape differences
* larger molecules migrate faster
* smaller molecules migrate through channels within beads
Ion-exchange Chromatography:
separates mainly by charge differences between proteins
1. protein binds the column based on opposite charge
2. column is washed to remove other proteins
3. release by eluting the protein with a pH of the buffer
Affinity Chromatography:
separates molecules according to their affinity to bind to specific ligands (DNA-DBP, Enzymes-Substrates)
1. beads can be covalently linked to the ligant that the target protein will bind
2. results is highly specific purifiation of a single protein
Example of Affinity Chromatography:
- glucose-binding protein attaches to glucose residues (G) on beads
- glucose-binding proteins are released on addition of glucose
