2/3 Structures Flashcards
Anfinsen Ribonuclease Experiments
- Christian Anfinsen studied the reversible denaturation of bovine ribonuclease using urea
- provided proof that
1. primary protein structure had all the necessary info to guide protein folding
2. native (folded) proteins are thermodynamically stable
3. got a nobel prize
Denaturation
- the disruption (or unfolding) of tertiary and secondary protein structure that leads to the loss of protein function
- also refers to the disruption of nucleic acid structure and function
Denaturing agents and treatments
- Heat and Temperature
- disruption of H-bonds, leads to protein unfolding. - Strong Acids/Bases
- protonation/deprotonation of sidegroups - Water soluble organic solvents (alcohol)
- Interfere with hydrophobic interactions - Detergents
- Disrupt hydrophobic interactions - High salt concentration
- interferes with salt bridges, removes water) - Reducing agents
- reduce disulfide bridges - Mechanical Stress
- breaks weak forces holding structure together
Conformational entropy of folding
- conformation entropy works against folding
- high to low entropy=decrease in entropy=negative S
- random coil= high entropy
- folded = low entropy
1. There must be certain features of protein folding that yield large negative values for delta H
2. There must be other ways to increase delta S
Negative delta S makes a ______ contribution to delta G
- positive
- conformational entropy change works against folding bc we want a negative delta G
Why form salt bridges
- charge to charge interactions at neutral pH help to stabilize entropically unfavorable conformations of folded proteins
The folding of a globular protein is thermodynamically favorable therefore, the free energy change for folding must be
- negative
The folding process goes from a random coil to a single folded structure and involves a _____ in randomness and entropy
- decrease
- delta S is negative
Charge-Charge interactions
- between positively and negatively charged side chain groups
- creates an electrostatic attractive force called a salt bridge
- the loss of salt bridges is a partial explanation for acid or base denaturation of proteins
Internal hydrogen bonds
- good hydrogen bond donors or good acceptors
- hydrogen bonds are relatively weak in aqueous solution, but their large number can add a considerable contribution to stability
Van der Waal Interactions
- in proteins the interactions between nonpolar groups can make significant contributions to protein stability because they are densely packed and make a large number of vdW contacts
Change in enthalpy
- dominated by the differences in noncovalent interactions between the unfolded and folded states
- a favorable energy contribution from the sum of intramolecular interactions more than compensates for the unfavorable entropy of folding
Unfolded states
- noncovalent interactions between extended polypeptide and solvent water
Folded state
- fewer interactions with water and many more intramolecular interactions
Hydrophobic affect
- also stabilizes protein folding via increasing entropy
- will result in an increase in entropy when hydrophobic residues are buried within the protein interior
The three main factors influence folding and stability of globular proteins
- Unfaorable conformational entropy change, which favors the unfolded state (high entropy bc many conformations)
- Favorable enthalpy (delta H) contribution arising from intramolecular noncovalent interactions
- favors folding - favorable gain of solvent entropy(delta S) from burying hydrophobic groups (The hydrophobic effect)
- As hydrophobic side chains cluster in the interior they release ordered solvent molecules from clathrate structures
- “favors folding”
Purpose of disulfide bonds between cysteine residues
- to further stabilize the folded three dimensional structure
Cofactors to stabilize tertiary structures
- folded proteins can be further stabilized by binding of metal ions, binding of cofactor or binding of prosthetic group (small molecule that is required for protein to be active.)
Holoprotein
- a protein in complex with an ion, cofactor or prosthetic group
- when the ion or cofactor is bound
- stabilizes the active conformation
Apoprotein
- a protein that requires an ion, cofactor, or prosthetic group but is not complexed with it
- when the ion or cofactor is stripped or absent from the protein
Predicting of secondary structure from amino acid sequence
- based on observed distributions of amino acids in helix vs sheet conformations (Ala=helix, Val=sheet)
- Amphiphilic α helix shows repeating patterns of side chain polarity every 3-4 residues
- Amphiphilic β strand shows repeating patterns of side chain polarity every other residue
Spectroscopic techniques to predict protein secondary structures
- Infrared spectroscopy
- Ultraviolet spectroscopy
- Fluorescence spectroscopy
- Circular dichroism
- Mass spectrometry
- Nuclear magnetic resonance (NMS)
Prediction of tertiary structure from amino acid sequence
- prediction is difficult due to the need to correctly predict interactions between residues that are far apart in the primary structure
- about 60% computations are accurate
Prediction of quaternary structure from amino acid sequence
- has multisubunits
- 2 bands = definite different subunits (heterolytic and homolytic)
- the interactions between the folded polypeptide chains in multi subunit proteins are the same kinds that stabilize tertiary structure like salt bridges, van der waals, hydrogen bonding, and hydrophobic effect.
