M2C: Protein Structure 2

Front 1

Protein Stability

Back 1

Proteins must be folded to be active. They are usually more stable while folded, but not too much. Therefore, it only takes a few mutations to unfold and render inactive a protein. Common in disease.

Front 2

Unfolded proteins

Back 2

Do not stay in one state, fluctuate between many unstable states. Depends on polar, hydrophobic, etc. Usually not much secondary structure.

Front 3

Factors/forces that determine protein stability

Back 3

Hydrophobic effect, conformational entropy, hydrogen bonding, electrostatic interactions, steric repulsion, trosional strain.

Front 4

Hydrophobic Effect

Back 4

Free energy is lowered if water is not in contact with hydrophobic residues with which it cannot H-bond. For this the free energy of a protein is lowered (desirable) if hydrophobic residues are folded to the inside of the protein with the polar molecules to the outside in contact with the aqueous environment (body).

Front 5

Conformational Entropy

Back 5

Loss in conformational entropy is one of the most important factors in destabilizing proteins. Conformational entropy refers to the number of conformational states (structures) a protein adopts in a given thermodynamic state. Folded proteins have little conformational entropy while unfolded ones can have large numbers of conformations. Nature trends towards entropy and therefore pushes proteins in this direction. More at greater temps (denaturing with heat).

Front 6

Hydrogen Bonding

Back 6

H-bonding and the lack of H-binding have a great effect on protein stability. Polar molecules need to find partners with which to H-bond in the folded state as easily as the unfolded (very easy in aqueous environment).

Front 7

Electrostatic Interactions

Back 7

Charged aa can either stabilize or destabilize proteins depending on same or different charges. Important in DNA binding proteins because DNA is highly negative.

Front 8

Steric Repulsion

Back 8

Atoms do not like to lie on top of each other and in a protein, this will not occur. Folding must occur to avoid steric repulsion. With mutations that change sterics, new folding may need to occur (often less favorable) or unfolding may occur.

Front 9

Torsional Strain

Back 9

Bonds have a preferred angle. Some protein folding forces bonds out of these angles. Glycine is the most flexible aa due to its small (H) side chain, and is often used to avoid torsional strain. Mutations that change Gly to something else are destabilizing for the protein.

Front 10

Mutations and Protein Stability

Back 10

Mutations are usually neutral or deleterious to stability, rarely stabilizing.

Front 11

Mutations to surface vs. inside of protein

Back 11

Mutations to the surface of a protein usually have little effect on the protein unless it directly effects the function of the protein. Mutations to the interior of the protein are often destabilizing.

Front 12

Small to big mutations in proteins

Back 12

Generally bad because can cause torsional strain or steric repulsion.

Front 13

Big to small hydrophobic mutations in proteins

Back 13

Generally bad because less hydrophobic surface area buried in folding.

Front 14

Hydrophobic to polar mutation in proteins

Back 14

Usually very destabilizing because polar molecules will want to interact with polar environment, not be buried in the interior of the protein.

Front 15

Mutations that switch out Glycine

Back 15

Usually destabilizing because causes torsional strain.

Front 16

Mutations that cause switch to a Proline

Back 16

Proline is large and will destabilize the protein if placed in an area with inappropriate torsional angles.

Front 17

Mutations that cause a change in a charged residue that interacts with other charged residues

Back 17

Usually destabilizing because charge interactions can change

Front 18

Mutations that cause a change in a good H-bound polar residue

Back 18

Usually destabilizing unless replacement can make a similarly good bond.

Front 19

Mutations that remove a group that interacts with a metal or co-factor

Back 19

Destabilizing.

Front 20

MSA

Back 20

Multiple Sequence Allignent. A way to try to find a destabilizing mutation. Homologous proteins from similar species are lined up so that they best match. In similar proteins, the regions that are less important in function are more likely to have mutations while areas that are crucial to function will have very few. A mutation in one of these crucial sequences will likely be deleterious.

Front 21

Protein Drugs

Back 21

Proteins are often ineffective drugs in their natural form. For this, proteins are redesigned to increase effectiveness. They must be stable, soluble, remain in the blood stream, not attacked by immune system, and have desired activity.

Front 22

To increase protein solubility in protein drugs...

Back 22

1. Exchange Cys for Ser to avoid sulfide bond aggregation.
2. Replace hydrophobic residues on exterior of protein for polar residues to reduce hydrophobic aggregation.
3. Alter the net charge and iso-electric point (pI). A protein with a net charge (pI does not equal the pH of the solution) will be more soluble.

Front 23

To increase protein stability for protein drugs...

Back 23

If protein is more stable, won't unfold, won't be cleaved, won't aggregate.
1. Remove free Cystines so they don't sulfide bond while in unfolded state.
2. Computational tools used to find useful mutations that will cause more stable folding.
3. Stop protein degradation by proteases by removing protease recognition sites and by shortening loops as they are often protease targets.

Front 24

Pharmokinetics: slower clearance of protein drugs

Back 24

1. Small proteins are more easily removed by kidney filtration. Increase molecular weight by binding protein to another protein, or to a carbohydrate.
2. As protein drug (ligand) is taken into a cell by a cell-surface receptor, it is often degraded. Histidines can be added to protein to cause change in charge upon entrance into the cell (endocytosis) and therefore release from the membrane receptor. Degradation is therefore reduced.
3. Higher pI of Insulin causes precipitation upon injection and therefore slower release in body. Also Insulin sometimes changed to monomeric active form before injection so that reaction is immediate and drug can be taken AT mealtime instead of an hour before.

Front 25

Redesigning Activity

Back 25

Human Growth Hormone dimerizes its receptor upon binding with protein. Has two binding sites and is an activator or inactivator depending on which is bound. HGH has now been created with only one binding site or the other so that function is definite.

Front 26

Immunogenicity

Back 26

Immune system attacks many altered protein drugs. Most protein drugs come from outside sources (non-human) and are attacked. Few have been successful for this reason. One success is Murine.

Front 27

Basic Principles of Protein Folding

Back 27

How a protein folds is coded in its sequence. Needs only water to occur. More common for a protein to fold in random order but always the same way. Always a chance that it will fold in an unexpected way. This is more common with bigger and more complex proteins.

Front 28

Misfolded Protein

Back 28

Proteins can misfold or fold in new and unexpected conformations. This is usually deleterious. A good example is amyloid fibrils found in BSE, Creutzfeldt-Jakob Disease, and Alzheimer's. Structural unit is repeating beta sheets. Do not know if fibrils are cause of the disease, but they form rigid plaques that can block and damage cells. Contribute to disease at least.

Front 29

Random folding and non-specific aggregation of protein

Back 29

Can happen as being created by ribosome. Can't fold until completed. Chaperones are fix for this.

Front 30

Molecular Chaperones

Back 30

Over 20 proteins called chaperones created by body to help other proteins fold correctly.

Front 31

Heat-Shock Protein 70

Back 31

Hsp-70. A chaperonin that keeps proteins unfolded until they are fully released by ribosome.

Front 32

GroEL Family of Chaperonins

Back 32

Cylindrical protein complexes comprise two rings stacked back to back. ATP and GroES are used as GroEL protein grabs unfolded protein, folds it, and releases it.

Front 33

Fibril seeding

Back 33

Many fibrils caused by misfolding of proteins are formed by a nucleation process. Presence of a very small fibril can cause other proteins to aggregate. This is why protein misfolding can be infectious!

Front 34

Cystic Fibrosis

Back 34

Caused by the misfolding of an ion channel protein. For this, there are not enough of these functioning proteins to regulate osmotic balance across the cell membrane.