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BCMB 3100: Test 1
genome |
heritable information |
replication |
process of copying the genome
catalyzed by DNA polymerase |
transcription |
DNA is transformed into RNA |
plasma membrane |
lipid bilayer |
fibrous proteins |
water insoluble
provide mechanical support "tough" |
globular proteins |
compact/ spherical
hydrophobic interrior
hydrophillic exterrior |
allosteric enzymes |
regulate biochemicals through metabolic pathways |
vitalism theory |
1800's- animate world obeys different laws than inanimate world |
Friedrich Wohler |
1828-showed urea could be synthesized from inorganic materials |
Edward/ Hans Buchner |
1897-death of vitalism
fermented sucrose into alcohol w/ yeast extract |
Emil Fischer |
1890- lock and key model
-4 substarte enzyme |
Max perutz |
hemoglobin 3D structure |
John kendrew |
myoglobin 3D structure |
watson/ crick |
determined 3D structure of DNA |
biochemical generalizations |
life requires life
reactions require catalyst
info of life is transmitted through genome |
proteins |
constructed of amino acids linked by peptide bond- forms unbranched polymer
fold into 3D structure
act as catalyst- enzyme |
nucleic acid |
information molecules of cell
-store/ transfer info
constructed of nucleotides
-5-carbon sugar, deoxy/ribose w/ base and phosphate
ex: DNA/ RNA |
DNA |
deoxyribose
nucleotides linked by phosphodiester links
double stranded |
RNA |
ribose
single stranded
mRNA= template for protein synthesis |
lipids |
storage for of fuel/ barrier
much smaller than proteins/ nucleic acids
part hydrophilic/ hydrophobic= allowed to form barriers |
carbs |
fuel- glucose stored as glycogen
linked in chains- branched
|
Eukaryote |
membrane bound compartments:
- nucleus/ ER/ golgi/ mitochondria/ lysosomes/ peroxisomes/ chloroplasts
cell= inside is chemically differnt than envio |
nucleus |
info center of cell
double bounde- contains pores |
mitochondria |
2 membranes
fuel molecules undergo combustion into co2/ H2o - generating ATP
produces 90% of cells energy |
ER |
smooth= processes exergonous chemicals
rough= ribosomes attached- synthesize proteins that will secrete from the cell
-transported into lumen during translation- then folds into proper shape w/ help from chaperones
-leave through transport vesicles |
golgi complex |
recieves TV from rough ER
differnt set of carbs attach
sorts proteins |
endosome |
endocytosis |
lysosome |
digestive enzymes |
H2O |
solvent of life
polar
bound covalently
H-bond= partial + H binds w/ partial - O
- common weak bond
non-polar/ hydrophobic cannot dissolve in water |
weak interactions |
ionic bonds= opposite charged- 2.8A-- 40-200 kj/mol
-maximized in uncharged envio
-weakend by H2O= competes for attraction/ solvation around shell
h-bonds= arise when H is cov bounded to EN atom- 2.6-3.1A--2-20kj/mol
-weaker than cov
stronger in absence of water
van der waals-depend on transient asymmetry in electrical charge
-partial charges-- 3-4A apart-- weaker then h-bonds/ ionic
Hydrophobic interactions= 3-10k j/mol- interaction of non-polar mols |
hydrophobic effect |
non-polar molecules cluster together in H2O
-b/c water binds strongly to itself
- clusters- not b/c of high affinity for each other but because when they dissociate they release h2O molecules
--entropy driven
--spontaneous- no energy required
membrane formation/ protein folding= powered by hydrophobic effect |
amphipathic |
both hydrophilic/ hydrophobic |
Kw |
1.0 x 10^-14 at 25 deg c |
pH= -log10(H+) |
... |
Ka |
acid= proton donor/ base acceptor
Ka= [H][A]/[HA]
pKa= log(1/Ka)
- pH at which the acid is 1/2 dissociated |
henderson hasselbach |
pH= pKa + log(A/HA) |
Amino Acids |
contain: carbon linked to amino group/ carboxylic acid group/ H+/ R-group
neutral pH= exist as zwitterions (dipolar)= NH3+/ COO-
low pH= NH3/ COOH
high pH(9)= NH2/ COO
|
4 AA classification groups |
1. hydrophobic aa w/ non polar r-groups
- only H/ C side chains
-Gly/ Ala/ Leu/ Ile/ Met/ Pro
2. polar aa w/ neutral r groups
-r group contains EN atom- hydrophillic
-Asp/ Glutamine
3. positive aa w/ r groups at pH=7.4
-highly hydrophilic
-Arg/ Lys/ His
4. negative aa= have acidic side chains
-Asp/ Glu |
essential aa |
must be obtained from diet
-His/ Ile/ Leu/ Cys/ Met/ Tyr/ Val/ Phy/ Thr |
hydropathy |
relative hydrophobicity of each aa
- larger it is- more it prefers a hydrophobic environment
--- effects folding |
proteomics |
study of large sets of proteins |
primary structure |
3D structure depends on primary backbone
linear polymer- alpha amino group linked to alpha C
- has directionality
crosslinked peptide chains contain disulfide bonds
-formed through oxidation of cysteine residues |
Sanger |
determined aa sequence of insulin |
secondary structure |
residue by residue conformation backbone of a polymer
a-helix= coiled structure- stabalized by H-bonds between Carbonyl O--- N amnio group
-- right handed
--- rise= .15nm pitch= .54 3.6 residue per turn
B-sheets= 2+ peptide chains (b-strands) not coiled
-- stabalized by H bonds between strands ( carbonyl O--- H amide)
---avg= 6 aa per strand |
conformation |
arrangement of sub groups that are free to change positions without breaking any bonds |
configuration |
arrangement of atoms that cannot be changed without breaking bonds |
turns/ loops |
allow chain to change direction
loops- on protein surface/ interact w/ other proteins/ envio
turns= loops containing less than or equal to 5 residues |
tertiary structure |
3D conformation in native folded state |
motifs |
recurring protein structures |
domains |
independently folded/ compact distinct structural units in protein
- connected by flexible segment |
Quaternary structure |
3D structure of multi-subunit
- multiple chains can assemble in single protein |
dimer |
2 identical subunits |
denature |
unfolding of native conformation
due to chaotropic agents= denature proteins
- ex: urea
- dont cleave bonds- disrupt 2, 3, 4 structures
|
reducing agents |
B-mercaptoethanol
dithiothreital |
christian Anifinsen |
showed aa determined 3D structure |
cooperativity folding |
formation of one part of structure leads to formation of remaining part |
current theory of protein folding: |
not random
cooperative/ sequential
dependent on primary structure
some require help folding= chaperones/ enzymes
rapid < 1s
most have single native 3D shape |
chaperones |
proteins that bind newly synthesized polypeptides/ assist folding
- increase folding rate
- prevent incorrect folding
- most are heat shock proteins
- requires ATP |
collagen |
fibrous protein w/ triple helix
most abundant vertebrate protein
- bone/ tendons/ cartilage/ skin/ vessels/ glue
3 left hand helices
- 3 aa per turn pitch= .94nm rise= 3.1
- high % of Pro/ Gly/ Hyp
- Hyp allows more interchain H-bonding/ stabalization of helix
- Pro/ Hyp prevent a-helices and make collagen rigid
--- strengthened by intra/ inter covalent crosslinks between Aly-Lys |
scruvy |
vitamin c deficiency
weakness in blood vessels/ skin |
cofactor |
small non protein required for enzyme activity |
prosthetic |
metal ion/ non amino acid tightly bound to protein- essential for activity |
holoprotein |
complete= protein w/ all its cofactors |
apoprotein |
w/ out cofactors |
myoglobin |
bond O2- stores/ transports O2 in muscle
prosthetic group= heme= Fe-protoporphyin IX
globular protein w/ 8 a-helices
all polar residues on surface |
hemoglobin |
allosteric protein= changes shape when bound to a particular molecule
transports O2 in blood
prosthetic group= heme= Fe-protoporphyrin IX |
conservative substitutions |
do not affect conformation
- aa in same family
--- ex: Val-- Ile |
nonconservative subs |
effect conformation
ex: Glu-- Val |
bohr effect |
increase of CO2 in blood-results in lower pH- decreases hemoglobins affinity for O2
-p50
- graph shifts right |
globular proteins |
compact shape- many reversible turns w/ a-helix/ b-structure
- 2-16 aa in loops |
4 protein domains |
1. all a-helices/ loops
2. all B sheets and non-repetitive structures that link b-strands
3. mixed a/ B= supersecondary structures
--a-helix/ b-strands alternate
4. a+ B= clusters of a helices/ b sheets in seperate regions of chain |
7 amino acid groups |
1. aliphatic= Gly/ Ala/ Val/ Leu/ Ile/ Pro
2. aromatic= Phe/ Tyr/ Trp
3. sulfur-containing= Met/ His
4. alcohols= Ser/ Thr
5. Basic= His/ Lys/ Arg
6. acidic= Asp/ Glu
7. amides= Asn/ Gln |
gel filtration chromatography |
proteins pass over column filled w/ hydrated porous beads filled w/ carbs
- large molecules exit first |
ion exchange chromatography |
separates proteins over column filled w/ charged polymer beads
-opposite charges attract
- bound proteins elute w/ salt
- non charged elute first |
affinity chromatography |
proteins pass through column of veads containing covalently bound high affinity group for desired protein |
hydrophobic-interaction chromatography (HIC)/ Reverse phase chroma (RPC) |
based on hydrophobic interactions on surface of protein |
polyacrylimide gel electrophoreisis (PAGE) |
chemically inert
polymerized acrylamide matrix of controlled pore size
- allows seperation of proteins based on size/ mass |
SDS-PAGE |
sodium dodecyl sulfate
an ionic detergent used for PAGE
must also add B-mercaptoethanol or dithiothreitol to reduce disulfide bonds
smaller proteins migrate faster
|
pI (isoelectric point) |
pH at which net protein charge= 0 |
isoelectric focusing |
electrophoreisis of proteins w/o SDS in a pH gradient |
assay |
measure of enzyme activity
disappearance of substrate or appearance of product |
gradient centrifugation |
separates extremely small molecules |
western plot |
permits detection of proteins separated by gel electrophoreisis (imunoblotting)
separated in SDS-PAGE gel
transferred to polymer-- then stained w/ fluorescent antibody |
How to determine primary structure |
1st hydrolyze into aa using heat and strong acid
individual aa can be separated by ion exchange chromatography
-visualized w/ flourorescamine
sequenced determined using edman degradation (limited to 50 aa)
- removes 1 residue at a time from the amino end of the peptide
phenyl isothiocyanate reacts w/ terminal amnio group- breaks peptide off- shortned by 1 aa |
Cyanogen Bromide |
CnBr
splits on C=O side of Met |
enzymatic cleavage |
trypsin= cleaves on C=O side of Arg/ Lys
chymotrypsin= cleaves on C=O of bulky hydrophibic and aromatic aa
dissulfide bonds must be removed
-- reduction-- alkylation |
nascant protein |
direct polypeptide product of translation |
6 major groups of enzymes |
1. oxidorecductases= catalyze oxi-redox reactions
-transfer electrons
2. transferases= transfer functional groups
- prominant in aa synthesis/ degradation
3. hydrolazses= cleaves molecules by adding H2O
-Ex: trypsin
4. lyases= adds atoms/ func. groups to a double bond/ reduces to form double bond
5. isomerases= move func. groups in molecule
6. ligases= joins 2 molecules by using ATP hydrolysis
-ex: DNA ligase |
haloenzyme |
complete= w/ cofactors- catalytic ready |
apoenzyme |
w/ out cofactors |