bis 102

midterm 2

Structes

Primary structure

see if angiogenin destroys RNase (meaning there is enzyme activity)

in the x-ray, some x rays get deflected at different angles (dark sopts are where it is hit) comes up with 3d structure were all atoms are

helps determine second and tertiary struct

sequence a protein

stategy 1. generate small fragments

2. look for overlapping sequeces

Tools that Sanger had

trypsin down K and down R

chumotryspin aromatic a.a down

Sanger N-terminal chemistry

DNP is yellow

we need the yellow guy and run through stnadards to find who he is

thin layer crmo togrsahy

arrow is polar solvent (polyamide plate)

plate si never homegenoues

nonpolar for the secon picture

Overlapping sequences

trypsin cuts right hand side

Secondary structure
reverse turn

one h bond holds reversre turn

many times its a proline not 2 though

cant have big guys or repusion

no di sulifde bonds

α-helix

most are right handded because most are made o l amino acids

rgroups on the outside are pointing down

residue 1h bond with residue 4

(-60,-50

# of h bonds=n-4 (n=residues)

avg length 10 aa residues

What causes an α-helix to form?

-glycine too flexible

roline wrong geo no h on N

-/- +/+ repulsion

W-W F-F steric hinderance

seerine mpefer h bond with h20

α-helix

dots are h bonds, in gray is the planes that are formed by the atoms), r groips are yellow

β-pleated sheea

serveral hydrogen bonds (how many depends on length) weaker if they are not in striaght line, both diagrams can make different mixture toghter

a helix stand by itslrf byt b can have family vin diseal

Ramachandran plot

organe is anti b pleated beta sheet

Myoglobin

this protien is sittign in muscles cells

etc is where o becomes water

mb has a tricky taks: how do yuo carry )2 without becoming oxdized

Mb 17000MW

the functional protein has heme ← protroporphin IX +Fe2+

apoprotein with out function

heme

letters are ring systems, iron i bound to N from cov. bonds, 5 covanletn bonds 4 ring and 1 from behind , amino acid is histdine base form

90-degree heme

all bottom arrows are illed

Side view of heme in Hb subunit (α or β

~13+/- on left (salt bridges)

Where are all of these salt bridges?

5 salt brdiges in the central cavity of the molecule at the end of the channel

Other salt bridges of deoxy Hb

CO2

bottom forms new+/ -

-0 to Nh is carbamate

carbmate+ H

binding co2 is alllosteric not binfin whereo2 binds but inderctly affect O2

Wherre do protones binds

where is ther net bindin of H+ at these sites

1 dramtic changes in pka oxy Hb →← deoxy HB

2. more H+ in tissues

Ph= pka +log b/a

7.6=6.2 +log b/a

hence no salt bridge

1. virtually no + charge on H

2. r is too far away coulomb’s Law

for tissue 7.2=7.7+logb/a

a=1/1.316=76%

What environmental factors affect a pKa

2 which xharge is thermo favored

what will lead to the lower energy

3. up to accpmlish thiw

2. which charge is thermo favored

3. pka down to accomplisjed

Cl-

pka up

dr ani

urea know struct, it forms h bonds

top 1% activity 1/105

→ trace amounts mercapteothanol 10gr 4C

bottom 95% actvitvit

red dots are urea, at time zeor no urea so net flow is out

con.1. all info is in the 1

2. 3 is the ost thermo stbale

3. disuslfide bonds prodive strngth but weak bonds are more important

in viv they are syntg as inactive precursors

proinsulin (took pic)

What if all of the RNase A was not completely denatured?

amino acids→synthesice RNase A

Kinetics of protein folding

How then does protein folding occur?

How then does protein folding occur?

no one path to fnial 3 struct

right is order collpased

Several diseases are associated with improper protein folding:

1. protein disulfuide isomerase

   catalyzes disulfide reaction in the ER

2. proline race mase

trans 1000 cis 1

trans 4 csi 1

← proloene racemarse

3. chaperionins proten complexes that allo phobi interactions a second chance

inerior holds 90000mw

red is incorreclty folded

interior loines with bydrophobic residues

bottom atp hydrolysis

lid off then gren ort

protien does not fild faster

What if a protein cannot be repaired?

ubiquitin proteasome

MM equation

Burk plots identify different types of reversible inhibitors:

Active site participants:

Binding?

all 20 aa res idues

cofactors mg2+,Zn2+, Fe2+, Mn2+

coezemzmes : NAD FMN lipoic acid biotin
Catalysis?

NU attckers C S K H H2O

gen acid cat. (dono pro at actvit site) - 7 inozable group can do this (in prtonated form)

gen bas cat - 7 inozable group can do this argine

cofactos

coenzymes

ph affects

all above

struct of enzymes

substrates (any chargees care about ph)

Chymotrypsin mechanism

How do enzymes speed up chemical reactions?

Bradford assay

1 enzyme Z assy

2. bradford protein assay

1. need to reduce te vol

2. 2 need to remove nucleic acids and cho polymers (if kept in they will bind to everything)

we will do two NH42 si4 cuts cuts refer to precipate

0%-100% saturation ~4.2M at 4*C

the first cut removes nucleic acids and CHO polymers

th second cut reduces the voulume and safty the enzyme

at best givs 2x purfication

how does ammonium sulfate fractionation work

comp for water

1 enzyme Z assy and bradprotein essay calcualte specific activity fold purfication and % yeild

affinity chromatography

1. only technique that can separt biologically active form from denautre from

2. can give 2000x purficatipn

why might it not work

1. lignd presented to the enzyme inthe wrong confoguration

2. enzyme has two substrate and the other substart must bind first

2. phospholipids

phosphodiester 4 amine

phosphatidylcholine PC

phosphatidlyserine PS

terpenoids

SDS fatty acids lyoplipids

facilitated diffusion

steropsefcific

saturable