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.
Heterolytic Proteins
- predicting these are much easier than homolytic proteins
- protein with more than 1 side chain that are different but held together by same non covalent interactions
- complementary surfaces determine specific interactions
Homolytic Proteins
- two copies that are exactly the same.
