Lecture 5, protein-protein interactions

Question 1

Affinity

Answer 1

The affinity of a particular molecular interaction refers to it’s strenght.
Can be expressed in Kd -> the lower the higher affinity
Can be expressed in delta G -> the more negative the more stable the complex between a ligand and a protein

Question 2

Specificity

Answer 2

Refers to the preference of a molecule to bind one particular target relative to all others.
Interactions between biological macromolecules usually have hign affinity

Question 3

Fibroblast growth factor and its receptor

Answer 3

FGF binding displaces an autoinhibition domain in the receptor, which blocks dimerization in the absence of FGF. The kinase domains that phosphorylate each other, which activates the receptor.

Question 4

Specificity factor

Answer 4

The ratio of the concentration of ligand bound to the target receptor to the total concentration of ligand bound to all other receptors.
The larger the factor the more specific the binding

Question 5

Ligand concentration and specificity

Answer 5

• When ligand is present at concentrations greater than Kd target, but less than Kd other, most of the ligand will be associated with the target.
• If ligand concentration equals or exceeds Kd other, then both receptors are occupied with ligand, thus lowering specificity.

Question 6

Charge interactions and specificity

Answer 6

Polar or charged residues at an interface will only contribute favorably to the free energy of binding if there are complementary functional groups at specific positions on the opposite surface.

Question 7

Lac repressor

Answer 7

In absence of lactose repressor binds tightly to operator
When lactose is present it binds the repressor, reducing it’s affinity for the operator

Question 8

Two classes of protein-protein interactions

Answer 8

1. The surfaces of two folded protein domains make extensive contact with each other. The residues that interact across this interface may come from several different structural elements within the two proteins.
2. Mediated by specialized peptide recognition domains (bijv. SH2 and SH3). These domains recognize short peptide segements in other proteins

Question 9

SH2 domain

Answer 9

Phosphorylated tyrosine bind to the SH2 domain in an extended conformation. The affinity for phosphorylated tyrosine in not very high, but when tyrosine is not phosphorylated it can’t even be detected.
Sensitivity is depended on target sequence

Question 10

Increased specificity of combinations of domains

Answer 10

If the interactions between peptide binding domains and their targets are not very specific, combinations of these domains are used in a single protein so that the specificity of the final interaction depends on the specificaties of two or more of the component domains beind satisfied simultaneously.
2 domains at specific distance -> only protein with this specific distance between motifs will be bound.

Question 11

Hydrophobic interactions at protein-protein interface

Answer 11

Residues at the interfaces between proteins interacts trough the packing of hydrophobic residues aigainst each other.
There is an increase in hydrophobic sidechains at the interface (not to many, or it would stick to anything hydrophobic and no longer be soluable) this increases affinity.

Question 12

Buried surface area

Answer 12

Protein-protein interfaces are characterized by their buried surface area. This refers to the surface area on the interacting proteins that is accessible to water before the complex is formed, but inaccessible in the complex

Question 13

Interfacial water molecules

Answer 13

Water molecules at the interfaces between proteins.
precisely located on the protein surface, because they form hydrogen bonds with residues on the protein surface.
No entropy gain but they do increase specificity of interaction.

Question 14

Growth hormone and its receptor

Answer 14

Growth hormone triggers dimerization of receptor.
Tyrosine kinases that are bound to receptor phosphorlyate the receptor.
This recruits signaling proteins (STAT’s) which are phosphorylated and then dimerized

Question 15

Protein and DNA/RNA binding

Answer 15

Proteins that bind to DNA or RNA typically do so with surfaces that are enhanched in positive charge.
Protein surfaces in contact with RNA or DNA are enriched in arganine and lysine, and depleted of aspartic acid and glutamic acid. The arganine and lyside sidechans, as well as other polar sidechains make hydrogen bonds to the phosphate group. (makes up 60% of hydrogen bonds between protein and DNA)

Question 16

Double-stranded RNA-binding domain (dsRB)

Answer 16

Many proteins that bind dsRNA contain a module known as dsRB, it makes contact with both the major and the minor groove.
To make contact with both major and minor, the RNA double helix has to be long enough to present two adjacent faces of the minor groove to the protein.

Question 17

direct readout / base readout

Answer 17

Recognition of DNA by a protein in which a specific pattern of hydrogen bond donors, acceptors and nonpolar groups on the protein is matched to a complementary pattern displayed on the DNA

Question 18

Indirect readout / shape readout

Answer 18

When a protein recognizes the ability of a DNA sequence to undergo a particular conformational change

Question 19

Which amino acid is inserted in minor groove and why?

Answer 19

Arginine sidechains are most frequently inesrted into regions of narrowed minor groove. The sequence specificity is not as high for this as it is for most examples of direct readout, because there are multiple A-T and T-A combinations that give rise to a narrowed minor groove.

Question 20

Palindromic sequence

Answer 20

A binding site in DNA that contains an inverted repeat (two copies of a sequence motif, with one running backwards).
Dimeric proteins can bind to such sequences with two fold symmetry

Question 21

Proteins binding DNA as dimers

Answer 21

Many DNA binding proteins occur as dimers and recognize palindromic sequences. Each monomer recognizes the same features in each half of the palindromic sequence, allowing both higher affinity and sequence specificity to be achieved.

Question 22

Zinc fingers

Answer 22

A class of small structural modules that are stabilized by the coordination of zinc by protein sidechains.
The helix of each finger is inserted in the major groove and sequence specific contacts are made to each succesive base. The spacer between modules is quite short, so there are no bases that are skipped, with the three modules reading out the sequence of nine succesive base pairs. The linker between these molecules is flexible and allows them to adapt to the DNA structure.

Question 23

Cooperative DNA binding

Answer 23

When the binding of one protein to DNA increases the affinity of another protein for DNA.

Question 24

Interferon-ß enhancher

Answer 24

The production of interferon-ß is triggered by the binding of multiple proteins to control regions in the DNA known as enhancer elements.
The complex of these proteins with DNA is known as an enhanceosome, and its formation is the first stap in the initiation of transcription

Question 25

KH domain

Answer 25

A class of RNA-binding molecules, that bind with RNA within a cleft, making contact with both the bases and the phophate backbone. Each KH domain interacts specifically with four nucleotides.
Recognition of the RNA by the KH domain relies on hydrophobic bonds between RNA bases and both the backbone and sidechains of the protein.

Question 26

RNA recognition motifs (RRM)

Answer 26

Small domains that recognize RNA trough stacking interactions.
Most RRM interact with RNA across a surface formed by a 4-stranded ß-sheet. Two conseved aromatic sidechains stack with the bases of the nucleic acids.

Question 27

allostery

Answer 27

An allosteric protein or RNA is one in which the activity of the protein or RNA is modulated by interactions that occur at a distance from the active site

Question 28

Graded respons / linear respons

Answer 28

An output that depends on the input function in a hyperbolic fashion, as in a simple binding equilibrium.

Question 29

Ultrasensitivity

Answer 29

An ultrasensitive system is one in which the response to an input is sharper than expected from a simple binding equilibrium.

Question 30

Cooperativity

Answer 30

When the binding of a ligand to a protein is ultrasensitive. As more ligand molecules bind to the protein, the saturation of the protein increases more sharply than would be expected for a normal binding event, as if the molecules cooperate with each other.
Can happen with or without allostery

Question 31

Map kinase pathway

Answer 31

• MAP kinase kinase kinase (MAPkkk) is first activet trough phosporylation
• MAP kinase kinase (MAPkk) is then phosphorylated by MAPkkk.
• Lastly MAP kinase is phospotylated by MAPkk and it will now phosphorylate a number of cellular proteins

Question 32

Positive and negative cooperativity

Answer 32

When two or more ligands bind to a protein in such a way that they mutually reinforce each of their binding affinities, it is called positive cooperativity.
If a ligand makes it more difficult for each other to bind, the phenomenon is called negative cooperativity

Question 33

Functions of hemoglobin

Answer 33

Hemoglobin strasports oxygen form the tissues to the lungs. Four oxygen atoms are bound per hemoglobin tetramer, at each iron-containing heme group. Hemoglobin also transports CO2.

Question 34

Differences Myoglobin and Hemoglobin

Answer 34

• Myoglobin is found in muscle tissue where it serves as a oxygon reservoir. Hemoglobin moves oxygon from the longs to the tissue.
• Myoglobin is a monomer, with one oxygen binding site, while hemoglobin is a tetramer with four oxygen binding sites.

Question 35

Sigmoid binding curve

Answer 35

When the binding of ligands to a protein is cooperative the shape of the binding vurve is no longer hyperbolic. instead it resembles a S shape and is called a sigmoid.

Question 36

Allostary for dimer proteins

Answer 36

A dimeric protein with positive cooperativity switches between two conformations.
T state: all binding sites are in low-affinity conformation
R state: all binding site are in high-affinity conformation.

Question 37

Hill coefficient

Answer 37

This parameter refelects the steepness of the log-log binding isotherm at the point when the protein is half saturated with ligand. A noncooperative system has a hill coefficient of unity. Systems exhibiting positive and negative cooperativity have Hill coefficients that are greater than and less specific than unity.

Question 38

How does a protein sense it has bound oxygen in one of its binding sites? (hemoglobin)

Answer 38

The change in heme structure that occurs upon binding oxygen are transmitted to other subunits of the tetramer.
The structure of each subunit changes upon oxygen binding -> position of helix F is changed

Question 39

Bohr effect

Answer 39

The reduction of the affinity of hemoglobin for oxygen that occurs at lower pH, This phenomenon is important because respiration acidifies venous blood, and the Bohr effect facilitates the release of oxygen from hemoglobin.