MOB lectures
Biochemistry
1. Introduction and regulatory strategies
The chemistry in all living systems is the same: reversible interactions of
biomolecules are mediate by three kinds of noncovalent bonds
- Electrostatic interactions (1.5 kcal/mol) (+-)
- Hydrogen bonds (1-3 kcal/mol) (keeps DNA
together)
- Van der Waals interactions (0.5-1 kcal/mol)
(distance is important)
- Hydrophobic interactions (reduced
interaction with water)
Allosteric regulations → interaction between
molecules.
Hydrogen bonds important
- Cell system is water based
- Reversible
- Every amino acid has a free O2, N, H. → H-bond can be made with every aa
Knowledge about free energy is necessary to understand
interaction between molecules. The free energy difference
of the products minus that of the substrates of a reaction
determine the direction of the reaction. (ΔG).
Free energy of the reaction → depends on the
concentration of reactants. Hydrogen bonds can change
equilibrium of reactions.
Allosteric regulation → switching proteins on/off.
- Often by binding of a molecule
- Change conformation = inhibiton or activation
Allostery – definitions
-Allosteric enzyme: an enzyme that can be in two states,
active and inactive. In the active state the enzyme binds its
substrate at its binding site and carries out its reaction.
In the inactive state the enzyme is unable to bind its
substrate. This is due to binding of a modulator molecule
elsewhere that alters the shape of the binding site, making
it inactive.
This way different signals can be perceived and integrated by the same protein
-Allosteric inhibitors: inhibitors act as ‘modulators’ in enzyme execution as they can attach
themselves to an enzyme that will alter the binding site for the enzyme, rendering it unusable and
therefore rendering the enzyme inactive.
-Allosteric protein: a protein that has underwent a fundamental structural change after reacting in
the presence of another molecule. This will alter its ability to react with that particular type of
molecule in the future.
-Allosteric transition: the transitional stage of a changing structure in a protein.
,Covalent modification of proteins can affect:
- Cellular location
- Interactions with other proteins (or others)
- Degradation
- Activity
Allosteric = more dynamic
Modifications are:
Phosphorylation can only happen to serine, threonine or tyrosine residue
Acetylation only to lysine
Myristoylation and farnesylation allow the protein to sit in membrane. → long chains of carbons and
here and there a hydro group = non-polar. → when post-translationally added to protein =
dramatically change localization of protein.
Myristoylation = addition to cysteine
Farnesylation = added to cysteine
Ubiquitin (protein) tags proteins for destruction
- Proteins can be tagged with single or multiple ubiquitin polypeptides
- Four linked ubiquitin molecules is the primary signal for degradation of the linked protein by
proteasomes.
Some post translational modifications are reversible, not all.
Most common covalent modification: about 30% of the cellular proteins are phosphorylated. Kinases
put the terminal phosphate group of ATP on the protein. Phosphatases remove the phosphate group.
The reaction is reversible, but takes energy and time. Although phosphatases remove the phosphate
group, the energy is lost. → it takes 2 regulatory steps : addition and removal. Not like an allosteric
effect. Phosphorylation is very often a regulatory step.
Phosphorylation is a highly effective means of regulating the activity of target proteins
1. 2 strong negative charges are added → empowers molecule to have more electrostatic
interactions
2. Directional new hydrogen bonds → more H-bonds, more interactive
3. Due to large change in free energy equilibria can change 10.000 fold
4. Very fast kinetics
5. Large rate of amplification possible → 1 kinase phosphorylates many other kinases and other
molecules
6. ATP as common phosphoryl group is linked to energy status → allows to connect signaling to
energy status of the cell more ATP = less energy demand = phosphorylate certain compounds
7. Reversible
→Carried out by enzymes: protein kinases. Kinases need to be under control, otherwise there is a
problem → massive effects on signaling pathway if something is phosphorylated that shouldn’t be
phosphorylated. Kinases regulate themselves. → allosteric regulation or covalent regulation systems.
,Ways protein kinases are regulated:
→each can direct regulate protein kinases, mostly allosteric regulation of a covalent system
Protein kinases have intrinsic specificity. For example protein kinase: cAMP activated protein kinase
A. protein kinase A is involved in a lot of different processes, controlled by second messenger cAMP.
protein kinase A phosphorylates Ser or Thr residues within a consensus sequence:
Arg-Arg-X-Ser-Z Arg-Arg-X-Thr-Z.
X= small, Z= large hydrophobic
→pkA : probably hydrophobicity on one site, Arg = positively charged and polar.
pkA consist of 2 subunits.
1. Catalytic subunit (blue)
2. Regulatory subunit (yellow) → does nothing but inhibiting catalytic subunit. Does this by
binding with a pseudo substrate (almost identical to substrate sequence)
Usually bound by a pseudo substrate with the sequence: Arg-Arg-Gly-Ala-Ile. With the addition of 4
cAMP’s it becomes active. Substrate sequence is Arg-Arg-Gly-Ser-Ile.
Pseudo substrate → ala cannot be phosphorylated, but is very identical = inhibition. cAMP binds to
regulatory subunit = conformational change = regulatory subunit lets go of catalytic subunit = pkA
active.
➔ Double allosteric regulation (regulatory and cAMP)
Both arginines interact with negatively charged glutamine of the enzyme. Serine is normally exactly
sticking towards the phosphate that needs to be transferred.
Pseudo will not be recognized as non-substrate because it only lacks the OH group that needs to be
phosphorylated normally.
2.Signal Transduction Pathways
The principles of signal transduction → a molecular circuit. Whether the signal is
perceived from the outside or inside will need a different path. Often signals are
received at the membrane. Amplification is usually happening to make sure that a signal
leads to something in the cell. → 2nd messengers used for amplification.
How a signal is perceived depends on environment and cell.
- Membrane receptors transfer information from the environment to the cells interior
, - Second messengers relay information from the receptor-ligand complex
- Protein phosphorylation is a common means of information transfer and amplification
- Signal termination process
- Evolution of signal transducing proteins
Every ligand has its own receptor. → specific response. ➔ specific ligand, specific receptor. What
happens downstream can be very diverse.
Seven-transmembrane-helix receptors or GPCRs (G-protein coupled cell surface
receptors)
→easy to spot in genomes → 7 very hydrophobic areas
→ligand bound in the center. The 7 helixes form a barrel. Changing an amino acid on the
inside will not change the conformation but will change the specificity for the ligand binding.
Different amino acids in the center = binding different ligand.
There are hundreds and hundreds of these receptors in the human genome. → versatile
receptor
→Specific receptor for adrenaline
Biological functions mediated by 7TM receptors
- Smell
- Taste
- Vision
- Neurotransmission
- Hormone secretion
- Chemotaxis
- Control of blood pressure
- Cell growth and differentiation
- Development
When a molecule binds to the receptor, it will change the order a bit = change the alpha helixes
relative to one another → something binding on the outside will change the conformation relative to
each other on the inside. → transfer information from the outside to the inside of the cell.
Convey info across the membrane if you have 1 transmembrane protein: nearly impossible to convey
conformational change across the membrane if you have 1 transmembrane protein. → relative to
what? Membrane is fluid, constantly changing.
Single TM receptors are very abundant → solution = dimerization. Bind ligand and dimerize =
conformational change relative to something else.
All G-proteins belong to the same family and get
activated by 7 TM receptors and dramatically change
conformation when activated by 7 TM receptors.
Galpha = alpha subunit of G-protein. How they all
operate is very similar. What response it will give is
specific. What they activated by depends on the specific
receptor.
G-proteins cycle between GDP- and GTP-bound forms.
All G-proteins are heterotrimeric complexes → 3 subunits. Alpha subunit actually is the G-protein. →
G comes from GTPase = hydrolyses GTP. It can do that in active site in alpha subunit. Active site
contains a P-loop. GTP and GDP can both bind, only GTP is hydrolyzed.
Beta and gamma subunit inhibit the alpha subunit (catalytic subunit).
Biochemistry
1. Introduction and regulatory strategies
The chemistry in all living systems is the same: reversible interactions of
biomolecules are mediate by three kinds of noncovalent bonds
- Electrostatic interactions (1.5 kcal/mol) (+-)
- Hydrogen bonds (1-3 kcal/mol) (keeps DNA
together)
- Van der Waals interactions (0.5-1 kcal/mol)
(distance is important)
- Hydrophobic interactions (reduced
interaction with water)
Allosteric regulations → interaction between
molecules.
Hydrogen bonds important
- Cell system is water based
- Reversible
- Every amino acid has a free O2, N, H. → H-bond can be made with every aa
Knowledge about free energy is necessary to understand
interaction between molecules. The free energy difference
of the products minus that of the substrates of a reaction
determine the direction of the reaction. (ΔG).
Free energy of the reaction → depends on the
concentration of reactants. Hydrogen bonds can change
equilibrium of reactions.
Allosteric regulation → switching proteins on/off.
- Often by binding of a molecule
- Change conformation = inhibiton or activation
Allostery – definitions
-Allosteric enzyme: an enzyme that can be in two states,
active and inactive. In the active state the enzyme binds its
substrate at its binding site and carries out its reaction.
In the inactive state the enzyme is unable to bind its
substrate. This is due to binding of a modulator molecule
elsewhere that alters the shape of the binding site, making
it inactive.
This way different signals can be perceived and integrated by the same protein
-Allosteric inhibitors: inhibitors act as ‘modulators’ in enzyme execution as they can attach
themselves to an enzyme that will alter the binding site for the enzyme, rendering it unusable and
therefore rendering the enzyme inactive.
-Allosteric protein: a protein that has underwent a fundamental structural change after reacting in
the presence of another molecule. This will alter its ability to react with that particular type of
molecule in the future.
-Allosteric transition: the transitional stage of a changing structure in a protein.
,Covalent modification of proteins can affect:
- Cellular location
- Interactions with other proteins (or others)
- Degradation
- Activity
Allosteric = more dynamic
Modifications are:
Phosphorylation can only happen to serine, threonine or tyrosine residue
Acetylation only to lysine
Myristoylation and farnesylation allow the protein to sit in membrane. → long chains of carbons and
here and there a hydro group = non-polar. → when post-translationally added to protein =
dramatically change localization of protein.
Myristoylation = addition to cysteine
Farnesylation = added to cysteine
Ubiquitin (protein) tags proteins for destruction
- Proteins can be tagged with single or multiple ubiquitin polypeptides
- Four linked ubiquitin molecules is the primary signal for degradation of the linked protein by
proteasomes.
Some post translational modifications are reversible, not all.
Most common covalent modification: about 30% of the cellular proteins are phosphorylated. Kinases
put the terminal phosphate group of ATP on the protein. Phosphatases remove the phosphate group.
The reaction is reversible, but takes energy and time. Although phosphatases remove the phosphate
group, the energy is lost. → it takes 2 regulatory steps : addition and removal. Not like an allosteric
effect. Phosphorylation is very often a regulatory step.
Phosphorylation is a highly effective means of regulating the activity of target proteins
1. 2 strong negative charges are added → empowers molecule to have more electrostatic
interactions
2. Directional new hydrogen bonds → more H-bonds, more interactive
3. Due to large change in free energy equilibria can change 10.000 fold
4. Very fast kinetics
5. Large rate of amplification possible → 1 kinase phosphorylates many other kinases and other
molecules
6. ATP as common phosphoryl group is linked to energy status → allows to connect signaling to
energy status of the cell more ATP = less energy demand = phosphorylate certain compounds
7. Reversible
→Carried out by enzymes: protein kinases. Kinases need to be under control, otherwise there is a
problem → massive effects on signaling pathway if something is phosphorylated that shouldn’t be
phosphorylated. Kinases regulate themselves. → allosteric regulation or covalent regulation systems.
,Ways protein kinases are regulated:
→each can direct regulate protein kinases, mostly allosteric regulation of a covalent system
Protein kinases have intrinsic specificity. For example protein kinase: cAMP activated protein kinase
A. protein kinase A is involved in a lot of different processes, controlled by second messenger cAMP.
protein kinase A phosphorylates Ser or Thr residues within a consensus sequence:
Arg-Arg-X-Ser-Z Arg-Arg-X-Thr-Z.
X= small, Z= large hydrophobic
→pkA : probably hydrophobicity on one site, Arg = positively charged and polar.
pkA consist of 2 subunits.
1. Catalytic subunit (blue)
2. Regulatory subunit (yellow) → does nothing but inhibiting catalytic subunit. Does this by
binding with a pseudo substrate (almost identical to substrate sequence)
Usually bound by a pseudo substrate with the sequence: Arg-Arg-Gly-Ala-Ile. With the addition of 4
cAMP’s it becomes active. Substrate sequence is Arg-Arg-Gly-Ser-Ile.
Pseudo substrate → ala cannot be phosphorylated, but is very identical = inhibition. cAMP binds to
regulatory subunit = conformational change = regulatory subunit lets go of catalytic subunit = pkA
active.
➔ Double allosteric regulation (regulatory and cAMP)
Both arginines interact with negatively charged glutamine of the enzyme. Serine is normally exactly
sticking towards the phosphate that needs to be transferred.
Pseudo will not be recognized as non-substrate because it only lacks the OH group that needs to be
phosphorylated normally.
2.Signal Transduction Pathways
The principles of signal transduction → a molecular circuit. Whether the signal is
perceived from the outside or inside will need a different path. Often signals are
received at the membrane. Amplification is usually happening to make sure that a signal
leads to something in the cell. → 2nd messengers used for amplification.
How a signal is perceived depends on environment and cell.
- Membrane receptors transfer information from the environment to the cells interior
, - Second messengers relay information from the receptor-ligand complex
- Protein phosphorylation is a common means of information transfer and amplification
- Signal termination process
- Evolution of signal transducing proteins
Every ligand has its own receptor. → specific response. ➔ specific ligand, specific receptor. What
happens downstream can be very diverse.
Seven-transmembrane-helix receptors or GPCRs (G-protein coupled cell surface
receptors)
→easy to spot in genomes → 7 very hydrophobic areas
→ligand bound in the center. The 7 helixes form a barrel. Changing an amino acid on the
inside will not change the conformation but will change the specificity for the ligand binding.
Different amino acids in the center = binding different ligand.
There are hundreds and hundreds of these receptors in the human genome. → versatile
receptor
→Specific receptor for adrenaline
Biological functions mediated by 7TM receptors
- Smell
- Taste
- Vision
- Neurotransmission
- Hormone secretion
- Chemotaxis
- Control of blood pressure
- Cell growth and differentiation
- Development
When a molecule binds to the receptor, it will change the order a bit = change the alpha helixes
relative to one another → something binding on the outside will change the conformation relative to
each other on the inside. → transfer information from the outside to the inside of the cell.
Convey info across the membrane if you have 1 transmembrane protein: nearly impossible to convey
conformational change across the membrane if you have 1 transmembrane protein. → relative to
what? Membrane is fluid, constantly changing.
Single TM receptors are very abundant → solution = dimerization. Bind ligand and dimerize =
conformational change relative to something else.
All G-proteins belong to the same family and get
activated by 7 TM receptors and dramatically change
conformation when activated by 7 TM receptors.
Galpha = alpha subunit of G-protein. How they all
operate is very similar. What response it will give is
specific. What they activated by depends on the specific
receptor.
G-proteins cycle between GDP- and GTP-bound forms.
All G-proteins are heterotrimeric complexes → 3 subunits. Alpha subunit actually is the G-protein. →
G comes from GTPase = hydrolyses GTP. It can do that in active site in alpha subunit. Active site
contains a P-loop. GTP and GDP can both bind, only GTP is hydrolyzed.
Beta and gamma subunit inhibit the alpha subunit (catalytic subunit).
