protein structure and post translational modification

post translational modification

alternation to amino acids after protein synthesis

where can post translational modification occur

n terminus
c terminus
side chain

how does post translational modification occur (what bonding)

covalent bonds

where does phosphorylation occur

the cytosol

where does glycosylation occur

the golgi and ER

phosphorylation

addition of phosphate group to a polar group

what enzyme does phosphorylation

kinase

dephosphorylation

removal of a phosphate group

what enzyme does dephosphorylation

phosphatase

which amino acids commonly undergo phosphorylation

HYTS

what happens during phosphorylation

a charge is introduced to polar uncharged amino acids that will alter interactions with other nearby amino acids

glycosylation

addition of a carbohydrate (glycan aka sugars)

what spots can glycosylation occur

n terminus
side chain

which amino acids can glycosylation occur at the n terminus

NR

which amino acids can glycosylation occur at the side chain

STY

what are some benefits of glycosylation

can help with adhesion (cell/cell binding), increasing antigen binding affinity, and drug optimization for monoclonal or polyclonal antibodies

somatic hypermutation

mutates own binding site for better antigen binding affinity via glycosylation. sugars add to proteins at the cell surface to serve as receptors for signaling molecules

what is the cancer immunotherapy agent that sounds like our professor

herceptin

what does herception target

HER2 receptor: it is typically overexpressed in certain breast cancers

what happens with 2 amino acids that are positively charged and close to each other

TS metallica analogy
the pKas of the amino acids will shift and it will not have the expected structure or stability

consequences of 2 similar charged groups

1. electrostatic repulsion between the two amine groups results in an unstable interaction that pushes them apart

2. to minimize the electrostatic replusion one group is motivated to get rid of a proton to make the interaction more favorable

3. the electrostatic repulsion would lower the pKa so it would be more likely to donate the proton to reach a more stable interaction

4. the pKa can shift down as much as 1 unit to stabilize itself.

5. phosphorylation turns the charge neutral

what happens when you have oppositely charged groups in close proximity to one another

the positive amino acid is stabilized by the negatively charged amino acid

shifts pKa in opposite direction: pKa raises and pH raises

consequences of oppositely charged groups

1. the electrostatic attraction results in a really stable interaction between positive amine group and negative carboxyl group

2. the attraction makes it less likely that the amine group wants to donate a proton so the pKa shifts up as much as 1 unit because it is already in a favorable interaction

3 ways to alter a proteins stability

1. post translational modification

2. pKa shift resulting in some sort of pH shifts

3. Adding ions to solution so the charges are shielded from them

ionic strength

ions shield each charged group from one another which stabilizes like charges and prevents strong ionic interaction from different charges

what can ionic strength do

Ionic strength can shield like charges and separate them out just enough to allow them to collapse. They can also collapse onto separate charges allowing them to separate slightly, allowing some tertiary kink in stability bc you cant separate them

consequences of 2 similar charged groups

1. electrostatic repulsion results in an unstable interaction between the two amine groups

2. to minimize the repulsion one group is motivated to get rid of a proton to make the interaction more favorable

what does ionic shielding do to a protein

Reduces Repulsion and Attraction: Ions cluster around positive and negative charges on the protein surface (forming an ionic atmosphere or electric double layer), which dampens long-range electrostatic attractions and repulsions between different parts of the protein or between separate protein molecules.

what happens when you have anions and cations condense on amine and carboxyl groups

the electrostatic attraction is minimized and the charges are shielded from each other

Like Charges (+/+) example

• Two positively charged residues (e.g., Lysine/Lys and Arginine/Arg) are forced next to each other.

• The Mechanism: Same charges experience strong electrostatic repulsion. To alleviate this unfavorable high-energy state, the system shifts to neutralize one of the charges.

• pKa Shift:

  • For (+/+) residues, one amino acid will give up its proton easier to become neutral. The pKa decreases (becomes more acidic).

Like Charges (-/-) example

• two negatively charged residues (e.g., Aspartate/Asp and Glutamate/Glu) are forced next to each other.

• The Mechanism: Same charges experience strong electrostatic repulsion. To alleviate this unfavorable high-energy state, the system shifts to neutralize one of the charges.

• pKa Shift:

• For (-/-) residues, one amino acid will aggressively hold onto or grab a proton to become neutral. The pKa increases (becomes less acidic/more basic).

Opposite Charges (-/+) example

• The Setup: A negatively charged residue (e.g., Asp) next to a positively charged residue (e.g., Lys).

• The Mechanism: The opposite charges form a favorable salt bridge (ionic bond). This electrostatic attraction strongly stabilizes the charged states of both residues.

• pKa Shift:

  • The negative residue wants to stay negative: its pKa decreases (holds onto the negative charge at lower pH).

  • The positive residue wants to stay positive: its pKa increases (holds onto the proton at higher pH).

Which ions condense where, and why?

• Anions condense directly around positively charged patches (+/+) or basic clusters.

• Cations condense directly around negatively charged patches (-/-) or acidic clusters.

• Why: Electrostatic attraction drives these mobile counterions to accumulate near the surface charges of the protein, forming an ordered "cloud" or double-layer to minimize local electrostatic free energy.

What happens to the protein after ions condense?

1. Electrostatic Screening (Debye Screening): The cloud of counterions effectively "masks" or dampens the long-range electric fields of the amino acids. The effective Debye length decreases.

2. Structural Impact:

   • If local repulsion was destabilizing the protein, increasing ionic strength stabilizes the fold by shielding that repulsion.

   • Conversely, if opposite attractions were holding the protein together, high salt can disrupt them, causing the protein to unfold or lose specific binding affinity.

how can pH and ionic strength stabilize amino acids that have similar or opposite charges

pH and ionic strength stabilize charged amino acids by altering their protonation states and screening electrostatic forces

role of pH

• Protonation and Charge States: pH determines whether amino acid side chains carry a positive, negative, or neutral charge based on the local pKa value

• Opposite Charges (Salt Bridges): At physiological pH, basic amino acids like lysine and arginine are positively charged, while acidic amino acids like aspartate and glutamate are negatively charged. These opposite charges attract each other to form stabilizing electrostatic interactions known as salt bridges inside protein structures.

• Similar Charges (Repulsion vs. Control): Amino acids with similar charges (e.g., two positive groups like Lys and Arg, or two negative groups like Asp and Glu) repel one another. Adjusting the pH near or past their pKa can neutralize one of the groups (e.g., turning a basic side chain neutral), reducing electrostatic repulsion and preventing protein unfolding.

role of Ionic Strength

• Charge Screening: Ionic strength refers to the concentration of ions such as Na+ or Cl- in the surrounding solution.

• Stabilizing Similar Charges: High ionic strength shields (screens) repulsive forces between amino acids with similar charges by clustering counter-ions around them, making close placement energetically more favorable.

• Modulating Opposite Charges: Moderate ionic strength optimizes attractive forces between opposite charges (salt bridges). However, excessively high ionic strength can weaken these salt bridges by competing with the charged side chains for electrostatic interactions.