Lecture 10: Protein Structure & Function Part 2

Question 1

Post-translational modifications

Answer 1

• Protein structure and function can be changed by these
• Covalent modification of amino acid side chains changes their chemical properties
• Proteolytic cleavage removes amino acids from the original translated sequence
• A given protein may have multiple potential modification sites, not all of which may be used at the same time
• Allows the protein to be responsive to different environmental conditions

Question 2

Phosphorylation

Answer 2

• Addition of a negatively charged phosphate group to the R-group of serine, threonine, tyrosine
• Bacterial cells can phosphorylate histidine residues (usually positive or neutral depending on conditions)
• E.g. Serine (R = -CH2-OH) to Phosphoserine (R= -CH2-O-PO3-2)

Question 3

Protein kinases

Answer 3

• Phosphate comes from ATP, forming the phosphorylated amino acid residue + ADP
• This reaction catalyzed by these

Question 4

Protein phosphatases

Answer 4

• Phosphorylation is reversible; phosphate removal is catalyzed by these
• Reversible just means that the phosphate group can be removed, but not placed back onto ADP; this is energetically unfavorable

Question 5

Effects of phosphorylation on protein structure & function

Answer 5

• Many changes in protein structure and activity are driven by phosphorylation because:
• Each phosphate group adds 2 negative charges to the protein
• Changing the charges can drive major structural changes (attractive or repulsive forces between amino acids), activity changes (ability to interact with ligands or acid-base chemistry), or changes in protein solubility (interacts between salts and charged amino acids)
• The added phosphate group may create a new recognition site that allows other proteins to bind to the phosphorylated protein (“SH2 domain” is a phosphotyrosine-binding motif)

Question 6

Ubiquitin

Answer 6

• Added to the lysine of proteins
• Small cytosolic protein (76 amino acids)
• Covalently attached to proteins (reversible)
• Serves as a tag that can either: mark proteins for degradation or direct proteins to specific locations in the cell

Question 7

Effects of the addition of ubiquitin to proteins

Answer 7

• Adding a single ubiquitin to a lysine usually directs the protein to a part of the cell
• There can be multiple lysines on a protein
• Chains of ubiquitins usually mark a protein for degradation
• Ubiquitin is the only covalent modification that is an actual protein

Question 8

Ligands

Answer 8

• All proteins bind to other molecules - those molecules are called these of the protein in question
• A protein’s physical interaction with other molecules determines its biological properties
• Because the binding of these is generally achieved by noncovalent bonds, it’s reversible and usually doesn’t require enzyme catalysis
• Unlike the binding of these, some covalent modifications are reversible and some are not, for example glycosylation is irreversible

Question 9

The strength of ligand binding

Answer 9

• In aqueous solution, molecules are in constant motion, bumping into one another
• Bonds are still strong even though there aren’t covalent bonds
• Protein binding must be “strong” enough to withstand the jolting of molecular motions
• Ligand binding sites are 3D
• The amino acids that contribute to binding a ligand are often quite far apart on a protein’s primary sequence but come together when the protein folds
• Hence the important of correct folding for protein function

Question 10

How is ligand binding strength achieved?

Answer 10

• 3D complementarity of binding

- The formation of several noncovalent bonds (the more noncovalent bonds, the stronger the binding)

Question 11

How strong is ligand binding?

Answer 11

• Consider a reversible reaction in which a protein binds its ligand:
  A + B AB
  A = protein, B = ligand, and AB = protein-ligand complex
• k ON (—>) and k OFF ( Ka = kON/ kOFF
• > Kd = 1/Ka
• Lower dissociation rates = lower Kd values = stronger binding
• > Vice versa for Ka

Question 12

Nucleotide triphosphates and protein function

Answer 12

• Play a huge role in protein function
• There can be direct phosphorylation by ATP (where the phosphate group is directly adding to the protein) or using nucleotide triphosphates as ligands for proteins (GTP is more frequently used)
• In using the nucleotide triphosphates as ligands, the protein itself is involved in hydrolyzing the nucleotide triphosphate
• In using the nucleotide triphosphates as ligands, the protein is turned on and off by attaching either GDP or GTP to the protein, unlike direct phosphorylation
• Guanosine Triphosphate = Activated energy carrier and also binds to proteins to regulate their activity

Question 13

Turning a protein on or off using GTP as a ligand

Answer 13

• Turning the protein on requires the catalysis of guanosine nucleotide exchange factors (GEFs)
• Turning the protein off requires the catalysis of GTPase activating proteins (GAPs)
• A phosphate group is removed in turning off of the protein, while turning on the protein requires the GDP to be exchanged for GTP
• The protein can hydrolyze the GTP attached to it when turned on, but usually protein will be needed to be turned off before this happens

Question 14

What does having multiple interaction sites mean for proteins?

Answer 14

• Allows proteins to act as molecular integrators
• Some proteins need several modifications in order to be activated, while others only need less to have different degrees of activity
• Example: Cyclin dependent kinases, which participate in cell cycle regulation by driving the cell into the different stages
• Activity is regulated by:
• Phosphorylation at one site in order to activate the kinase
• Dephosphorylation at another site in order to activate the kinase
• Binding to a cyclin is also necessary to activate the kinase