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34 Cards in this Set

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  • Back
Protein Stability
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.
Unfolded proteins
Do not stay in one state, fluctuate between many unstable states. Depends on polar, hydrophobic, etc. Usually not much secondary structure.
Factors/forces that determine protein stability
Hydrophobic effect, conformational entropy, hydrogen bonding, electrostatic interactions, steric repulsion, trosional strain.
Hydrophobic Effect
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).
Conformational Entropy
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).
Hydrogen Bonding
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).
Electrostatic Interactions
Charged aa can either stabilize or destabilize proteins depending on same or different charges. Important in DNA binding proteins because DNA is highly negative.
Steric Repulsion
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.
Torsional Strain
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.
Mutations and Protein Stability
Mutations are usually neutral or deleterious to stability, rarely stabilizing.
Mutations to surface vs. inside of protein
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.
Small to big mutations in proteins
Generally bad because can cause torsional strain or steric repulsion.
Big to small hydrophobic mutations in proteins
Generally bad because less hydrophobic surface area buried in folding.
Hydrophobic to polar mutation in proteins
Usually very destabilizing because polar molecules will want to interact with polar environment, not be buried in the interior of the protein.
Mutations that switch out Glycine
Usually destabilizing because causes torsional strain.
Mutations that cause switch to a Proline
Proline is large and will destabilize the protein if placed in an area with inappropriate torsional angles.
Mutations that cause a change in a charged residue that interacts with other charged residues
Usually destabilizing because charge interactions can change
Mutations that cause a change in a good H-bound polar residue
Usually destabilizing unless replacement can make a similarly good bond.
Mutations that remove a group that interacts with a metal or co-factor
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.
Protein Drugs
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.
To increase protein solubility in protein drugs...
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.
To increase protein stability for protein drugs...
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.
Pharmokinetics: slower clearance of protein drugs
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.
Redesigning Activity
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.
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.
Basic Principles of Protein Folding
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.
Misfolded Protein
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.
Random folding and non-specific aggregation of protein
Can happen as being created by ribosome. Can't fold until completed. Chaperones are fix for this.
Molecular Chaperones
Over 20 proteins called chaperones created by body to help other proteins fold correctly.
Heat-Shock Protein 70
Hsp-70. A chaperonin that keeps proteins unfolded until they are fully released by ribosome.
GroEL Family of Chaperonins
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.
Fibril seeding
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!
Cystic Fibrosis
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.