class 12+13

in the presence/absance of DNA…

the AAs are in different conformations

• proteins change their shape

conformational changes

• ordered changes in shape that happen every time

• doesn’t count when disordered proteins are flopping around

quarternary structure

• multiple polypeptide chains’ subunits interact with each other

• only when there are multiple polypep. chains

how to denature a protein (5)

1. detergents→ disrupt hydrophobic interactions by messing with charge

2. organic solvents→ disrupt hydrophobic interactions

3. acids or bases→ deprotonate/protanate side chains→ messing with ionic bonds

4. heat→ beak all noncovalent bonds by adding KE

5. reducing agents→ reduce disulfide bonds

if we remove denaturing agents→

the protein may lead to self-assembly to native conformation

if the protein refolds after removing denaturation agents, what does it tell us about the protein? 2

• 1° structure has enough info for proper 3° structure

• proteins are moving to lowest free energy→ everything is moving to the happiest state

steps of protein refolding

1. local 2° structure fold first → ionic interactions

2. move near other 2° structures until they are happy→ form a 3° structure

free-energy funnel

start with a lot of unfolded states→ high free entropy

→ then groups settle more into organized structures

→ lower free energy even though less structural disorder

molecular chaperones

prevent nonselective interactions during protein folding

• keep protein unfolded until they only bind with desired peptides and itself

• also helps move newborn proteins across membranes (keep unfolded until through hole in membrane)

Hsp 70 family

• help keep denatured proteins unfolded and stable in excess heat until the heat is gone and it can refold

• transport proteins to chaperonins

• transport proteins through membranes

chaperonins

allow large new proteins to assemble without interference

GroEL+GroES

essential for proper folding in E. coli

• 2 rings of 7 subunit cup looking things

• helps unfolding proteins fold (brought by chaperones)

• GroES sits on top and forces proteins to fold

how does GroEL work? (4)

1. starts with 2 subunits on top of each other and in the cup are all hydrophobic molecules

2. protein goes in and the hydrophobic parts interact with the walls

3. GreES goes on the top like a lid and through atp hydrolysis → forced hydrophobic inside of cup to flip and be charged

4. protein freaks out and tries to get hydrophobic parts away from walls→ forces it to fold

how are protein-protein structures held together?

by noncovalent bonds

• between domains/motifs

yeast 2 hybrid

the transcription factor from yeat and split it in half→ take DNA binding domain → attach to a protein→ take domain that activates transcription→ put on another protein→ if they interact→ start of transcription

homologs

members of the same family

paralogs

homologs from same organism that had a gene mutation→ multiple mutations over time→ dif function

orthologs

homologs with the same function → emerged from divergence over speciation

• ex. my DNA polymerase vs. Honey’s DNA polymerase

analogs

dif genes (not from same family)

• have developed through evolution→ same function

isoforms

dif forms of same protein in dif. locations or in dif. stages of life → paralogs

polyacrylamide gel electrophoresis (PAGE)

migration of proteins in electric field based on molecular size, shape, and charge density

how does SDS-PAGE work?

denature proteins by boiling with SDS detergent→ removes shape and charge as variables

• so we can determine only based on size

loading dye

• tells us how fast things are moving through the gel because we know how fast it’s moving

protein staining

allows us to visualize all the different proteins in a gel

• SDS-PAGE gel

what proteins show up in a gel electrophoresis?

the big concentrations of a type of proteins the cell is making

how does protein staining work?

1. stain SDS-PAGE gel

2. soak gel in staining solution with acetic acid+ alc+ dye → pour out solution

3. soak in detain solution (same as stain solution w/out dye)→ dye stops binding to gel

4. see lines of proteins

how to figure out which line is the desired protein?

western blot

protein hybridization

specific antibodies that bind to a protein

western blot

1. transfer lines for gel electrophoresis onto another sheet of paper

• by alternating the direction of current

2. wash specific protein of interest binding antibodies onto paper

3. everything other than protein washes off

4. 2nd antibody to bind to 1st antibody→ 2nd antibody cases protein to light up

two-dimensional gel electrophoresis

used when you wanna analyze all the proteins in a cell and see what happens

pI two-dimensional gel electrophoresis

in a tube of gel with varying pHs

• cell applied at top and current applied

• pulls proteins to where their isoelectric point is → balance pull on pos and negative side of gel

put tube on top of SDS-PAGE→ proteins separated based on weight → can see effects before and after smth done to change cell

steps to purify a protein

1. break open cell to release protein into solution → crude extract → remove junk in solution so there is just cytoplasm left → use centrifuge

2. fractionate→ add salt to try and find concentration that separates our protein from others

3. dialysis

4. repeat fractionation and dialysis until we get a pretty pure concentration of protein

how are proteins separated?

based on:

1. size

2. charge

3. binding properties

4. protein solubility

how is protein purification measured?

increase in specific activity

chromatography

separating things in a mixture of dissolved components

• I did this in lab→ mixture+poured on top of beads in a fractional column → beads have a (pos or neg) property→ dif thing move through matrix → separate

in chromatography, if a molecule has high affinity for the matrix…

it moves slower through it

high performance lipid chromatography (HPLC)

use pumps to move immobile phase to the mobile phase through pumps → greatly improves resolution

dialysis

put solution of protein and a ton of salt into dialysis bag (with holes bigger than salt)

2. but bag into a thing of solution not containing salt → causing salt to diffuse through bag and leave protein behind

fractionation

separating protein into a lot of dif tubes and mixing it with dif concentrations of thing to find the most concentrated form of the protein

selective precipitation

add salt until proteins fall out of solution

at low ion strength

proteins tend to stay in solution

at high ion strength

proteins tend to fall out of solution (solubility decreases)

crude extract

release of proteins into solution