Lecture 13 + 14 Flashcards
Explain the molecular interactions that stabilize a folded protein
- non covalent interactions : hydrogen bonds, hydrophobic interactions, ionic interactions, van der Waals forces
- covalent bonds: disulfide bonds
Protein folding: native and non-native states
- native state (folded) -> denatured state (unfolded or misfolded)
- native state is the most stable (lowest energy), delta G less than 0
What can cause denaturation
- raising the temp
- extremes of pH
- chaotropic agents (8M urea or 6M guanidinium)
- detergents that disrupt hydrophobic (sometimes polar) interactions
- most proteins are insoluble when denatured due to newly-exposed hydrophobic regions than cause protein aggregation and precipitation
- some proteins can refold after denaturation under the right conditions
Explain the Anfinson refolding experiment and how this proves that disulfide bonds function to stabilize a protein fold, not to direct the fold
- some denatured proteins will spontaneously refold in vitro, ex. ribonuclease
- information needed for the correct 3D conformation of the protein is inherent in the amino acid sequence of a polypeptide
- not all proteins refold spontaneously in vitro
- some fold during protein synthesis
- some need help (chaperones)
- some cannot fold or are unstable without their binding partners
- reducing agents like beta-ME and DTT reduce disulfide bonds, as the bonds are reduced the reducing agent is oxidized and forms disulfide-bonded dimers
Reduction and denaturation of ribonuclease
- denaturants such as urea and guanidinium chloride disrupt weak, non-covalent interactions that stabilize folded proteins
- if the protein has disulfide bonds reducing agents (reductants) such as beta-ME and DTT reduce the covalent disulfide bonds
Explain the Levinthal folding paradox
- proteins cannot possibly sample the almost infinite number of possible conformations in getting to their final folded structure, that instead they fold along an energy landscape, with folding intermediates
Explain co-translational protein folding
- proteins fold as they are synthesized, first into secondary structures that associate into domains, which associate to form the native protein
- also occurs for in vitro folding but in that case the entire protein is available for folding
A folding funnel - Energy landscape
- folding occurs along “energy surfaces”
- limited number of secondary structure elements: helices, sheets, and turns
- limited conformational space can be explored during folding
- state A and B are unfolded (non-native) states (many different conformations)
energy landscape - descent towards lowest energy state (native conformation) - state A and B go through energy minima - folding intermediates - on their way to the native folded structure (N)
3 proposed models of protein folding pathways
hydrophobic collapse model - assembly of secondary structures
framework model - assembly of tertiary structures
nucleation model - hierarchical assembly
Describe the thermodynamics of protein folding
- delta G for folding is negative
- conformation entropy works against folding
- entropy contribution from the hydrophobic effect works in favor of folding
- enthalpy contribution works in favor of folding
What is the role of chaperones
- help in protein folding in vivo
- clamp type bacterial chaperon Hsp70
- ATP hydrolysis
- bacterial chaperonin GroEL-GroES
GroEL-GroES protein-folding cycle
- GroES and ATP bind to the GroEL ring, trapping an unfolded protein within the folding chamber
- conformational change releases GroES, and folded protein exits the lower chamber
- ATP hydrolysis causes a conformational change in the upper chamber that facilitates protein folding and resets the lower chamber for another round
- a new unfolded protein enters the lower chamber and the cycle continues
What is the difference between protein domains, globular and fibrous proteins
domains - compact locally folded and stable regions
- associated with a particular function
- 40 to 200-250 aa
- fewer than 40 = difficult to fold stably
- more than 300 difficult to fold correctly
- single domain made up of a single stretch of primary sequence
- interior consists of non-polar, both polar and non-polar residues on the outside
fibrous proteins - highly elongated molecules whose shapes are dominated by a single structure
globular proteins - have compact roughly spherical shapes, ex. RNase A, chymotrypsin, myoglobin
- examples of globular proteins -> predominantly alpha-helix, predominantly beta-sheet, mixed alpha-helix, beta-sheet
Examples of quaternary structures
- Cro protein of bacteriophage
homodimer - identical subunits - hemoglobin -> heterodimer
bacterial pili - hair like filaments on bacterial surfaces - made up of thousands of subunits of the pilin protein
- pilus filaments have multiple diverse functions in bacterial pathogenesis: motility adhesion, DNA uptake
What are the various post-translational modifications that occur for some proteins
- addition of hydroxyl groups to prolines by the enzyme prolyl hydroxylase -> hydroxyprolin - results in abnormal collagen fibers and tissue abnormalities
- addition of second carboxylate group to the carbon of glutamate -> carboxyglutamate
- addition of carbohydrates, fatty acids
- phosphorylation of serine, threonine or tyrosine hydroxyl groups - reversible regulation
- acetyl groups are often added to the amino terminus of proteins to make them more resistant to degradation
- acetylation of lysines on histones to remove their positive charges