Lecture 8 Flashcards
T or F, secondary structure can sometimes be predicted by analyzing primary structure of given protein
T; certain amino acids are prone to be involved in one type or the other
What are the alpha helix formers ?
Leucine and methionine
What are the alpha helix terminators
Proline; due to the fact that there is no hydrogen available for bonding and structure resits rotation of right-handed helix –> kinks cause too much strain
What was the Anfinsen Folding Experiment
- ) Native protein (folded)
- ) Treated with beta-ME (reduces –S) and urea (which breaks Hydrogen bonds)
- ) Protein becomes denatured
- ) He noticed when dentaureing agens are removed the native protein returns
– when urea is removed –> natural protein is returned; when denatured protein is oxidized it returns to its natural state
- overalln experiment reveal that the amino acid sequence was theoretically, sufficient to direct folding
What are the 3 factors that the stability of the folded structure of globular proteins depends on?
- The unfavorable conformational entropy change, which favors the unfolded state
- The favorable enthalpy contribution arising from intramolecular noncovalent interactions
- The favorable entropy change of the solvent arising from the burying of hydrophobic groups within the molecule –> think of how water is more ordered when protein is unfolded
Thus, factor 1 works against folding, whereas factors 2 and 3 favor folding. The overall consequence is that the folded structure corresponds to a free energy minimum for the polypeptide under physiological conditions. This is why the protein folds spontaneously.
– all of which leads to a negative change in free energy
What are intramolecular noncovalent interactions?
- charge - charge, hydrogen bonds, Van der Waals, hydrophobic effect
T or F, it is hypothesized that small sections of proteins fold as they are translated
T, newly made protein is folded in 1 minute
T or F, protein folding is mediated by Chaperones
- T, they are proteins that help in correct folding
T or F, disulfide bonds are weak covalent bonds that aren’t usually in intracellular proteins
F, these are covalent bonds that are very stable, but aren’t usually in intracellular proteins as this is a reducing environment
T or F, there are two general classes of HSP: HSP60 and HSP70 are the most common
False, there are three general classes
What is the function of a HSP (Heat Shock Protein)
- they isolate proteins and bind to their hydrophobic groups
T or F, HSP with ATP has a low affinity for unfolded proteins and is found throughout the cell
- False, it has a high affinity for unfolded proteins
How does HSP60 work ?
- GroEL binds to unfolded proteins via hydrophobic residues (because hydrophobic groups are exposed)
- ATP and GroES causes a conformation change that hides hydrophobic residues and forces the protein now into a hydrophilic chamber causing hydrophobic residues on the protein to hide and induces folding –> this is where the chaperone aspect happens
–> ES is the cap and when its added to GroEL ring it changes conformation
- After ATP hydrolysis releases GroES and the folded protein
- Found mostly in mitochondria in eukaryotes, though can be in other locations
What is the role of cofactors typically? What is another name for them?
- stabilizes active state of protein; these are often needed for active enzymes
– also called prosthetic groups
What aside from cofactors stabilizes proteins?
- disulfide bonds –> once protein is completely folded that’s when they form
– ions
T or F, you can predict which amino acids are interacting before 3-D structure
False, you can’t predict which ones are interacting until you see the 3-D structure
What is the general description of Quaternary structure?
- most proteins are made up of more than one subunit
– subunits of polypeptide held together by patches of noncovalent bonds
What is the difference between homodimers and heterodimers ?
- Homodimer: two identical subunits
- Heterodimer: two different subunits
What is an example of a protein with quaternary structure?
- Hemoglobin
- consists of two alpha globin and two beta globin subunits for a total of four subunits
What are typical characteristics to purify proteins by?
- Solubility
- Size
- Charge
- Binding affinity
How do we extract protein?
- in order to release cell’s content we must first break (lyse) the cell membrane –> called homogenization
1. sonication –> breake cells with high frequency sound
- detergent lysis –> use a mild detergent to make holes in plasma membrane
- force –> force cells through small hole using high pressure
- mortar/pestle –> shear cells between a close-fitting rotating plunger and the thick walls of a glass vessel
How does differential centrifugation work?
- it is a way to separate cellular components by mass
- You fractionate the cell into components to see which is the richest in source of protein.
- The homogenate is formed and then an increasing centrifugal force is applied.
– at each step supernatant is removed from pellet that is formed and then subjected to further centrifugation
What is seen at different G’s in supernatant vs pellet?
- At 500g, the pellet will be mainly nuclear fraction
- at 10,000g the pell will be mitochondrial fraction
- at 100,000g the pellet will be microsomal fraction
- and the final supernatant will be the cytoplasm filled with soluble proteins
How does zonal centrifugation work ?
– centrifugation separates matters according to density (m/v)
– often provides a better resolution of separation of particles than differential
