Biochemistry Flashcards
how far apart do h bonds hold water molecules apart
3A
deltaG > 0
endergonic reaction, not spontaneous
deltaG=0
equilibirium
deltaG< 0
exergonic reaction, spontaneous
what happens if you change a protein’s solvent
denatures - hydrpphobic effect
when can any weak acid or base act as a buffer
when ph is almost pka
why can amino acids act as buffers at different phs
2 different ionisable groups, 2 different pkas
zwitterion
no overall charge but is ionised
ala
alanine
a
alanine
arg
arginine
r
arginine
asn
asparagine
n
asparagine
asp
aspartate
d
aspartate
cys
cysteine
c
cysteine
gln
glutamine
q
glutamine
glu
glutamate
e
glutamate
gly
glycine
g
glycine
his
histidine
h
histidine
ile
isoleucine
i
isoleucine
leu
leucine
l
leucine
lys
lysine
k
lysine
met
methionine
m
methionine
phe
phenylalanine
f
phenylalanine
pro
proline
p
proline
ser
serine
s
serine
thr
threonine
t
threonine
trp
tryptophan
w
tryptophan
tyr
tyrosine
y
tyrosine
val
valine
v
valine
CORN law
l isomers
COOH, R, H, NH2
resonance
partial double bond properties due to sharing electrons between N and O so no rotation around peptide bond
what do phi and psi angles describve
shape of proteind
ramachandran plot
shows angles around individual alpha carbons
each dot is a pair of phi and psi
shows steric limitations placed on amino acid residues in proteins
why is trans conformation preferred
8kj/mol more stable than cis
why is trans more stable than cis
steric hindrance
why is proline sometimes cis
less steric hindrance
what physicochemical interactions determine 3D shape of proteins
salt bridges
h bonds
vdw interactions
hydrophobic interactions
covalent bonds
disulphide bonds
charged amino acids
asp
glu
his
lys
arg
polar amino acids
asn
ser
the
aliphatic amino acids
ala
ile
leu
met
val
aromatic amino acids
phe
trp
tyr
what can form between oppositely charged side chains
salt bridgeswh
which amino acids are ionised at physiological ph
asp and glu
what interactions do polar amino acids form
h bonds
what interactions do aliphatic a.a form
van der waals
when is the attraction of a.a to non-polar atoms maximised
1A apart
why are atoms held apart
energetically favourable to be at that distance
what do hydrophobic interactions do to bipolar a.a residues
bury non-polar side in core and leave polar outside
why do aromatic a.a not appear inside of protein folds
too big
why is proline found on extremities of proteins
disrupts backbone H bonding in alpha and beta
soluble proteins
in cytosol/ plasma rather than membrane
properties of alpha heliux
right handed/ clockwise
no free space inside
side chains point outwards
alpha helix favoured residues
met, ala. glu, lys
alpha helix unfavored residues
pro, gly, asp
h bonding in alpha heliux
every carbonyl o forms h bond with amide h 4 residues along
pitch
how far it goes up every time it goes round
coiled coils
stripes of amino acids wrap around each other on different helices
3 10 helix
every 3 residues
pi helix
every 5 residues
beta sheets properties
h bonds between adjacent backbones
antiparallel favoured as h bonds perfectly perpendicular
beta barrel
entirely made of beta strands joined by h bonds
example of beta barrels
retinol binding protein
green fluorescent protein
loops and turns
most variable and biologically active parts of proteins
reverse turn beta hairpin
links beta strands
greek key motif
fold beta hairpin over 2 beta strands
effect of proline on secondary structures
forms kinks in alpha helices
do not form beta sheets
non-allosteric interaction
protein binds one ligand
P+L<>PL
allosteric interaction
protein binds multiple ligands
changes affinity
P+LA+LB<>PLA+LB<>PLALB
Hb structure
2 alpha chains, 2 beta chains
HbF structure
2 alpha chains, 2 gamma chains
alpha chain
141 a.a
beta chain
146 a.a
prosthetic group
non a.a group
Mb structure
one chain - 154 a.a
one haeme group
similar 2 and 3 structure to hb
haeme group
porphorin ring sitting in hydrophobic cavity
distal and proximal his
what stabilises the distal his in haeme
o2 forms h bonds with it
P+L<>PL
what do the forward and reverse reactions represent
ka
kd
P+L<>PL
what is the velocity of the forwards reaction (vf) dependent on
ka
[P] and [L]
P+L<>PL
what is the velocity of the reverse reaction (vr) dependent on
kd
[PL]
mass action at equilibrium
[P][L]ka = [PL]kd
caclulaction for
dissociation constant
kd/ka = Kd = [P][L]/[PL]
fractional occupation equation
Y = [L]/Kd+[L]
low [L]
low binding site occupation
[L] = Kd
half of binding sites are occupied
[L] much greater than Kd
most binding sites are occupied
when does Kd differ
if protein has multiple possible ligands with different affinities
Kd equation for myoglobin
[deoxyMb]*[O2]/[oxyMb]
what is Kd substituted for with gases
p50
p50 equation for myoglobine
[deoxyMb]*p50/[oxyMb]
p50
partial pressure to fill 50% of binding sites
fractional occupation equation in terms of o2
Y=pO2/P50+pO2
rank body systems in descending order in terms of po2
lungs
resting tissues
exercising tissues
Tense form
deoxyhaemoglobin
what affinity for o2 does the tense for have
low
relaxed form
oxyhaemoglobin
what affinity does the relaxed form have
high
what does the sigmoidal curve for haemoglobin show
positive binding cooperativity
binding of one o2 increases affinity for other sites
hill coefficient = 1
no cooperative binding
hill coefficient >1
positive cooperativity
does the t or r state have a higher p50
t state
how does equilibrium shift as more o2 bind
favours the r state
2,3-bpg
allosteric effector
binds to T state more tightly than R state
increases p50
the Bohr effect - H+
H+ as allosteric inhibitor
binds preferably to T state
the Bohr effect - co2
co2 binds directly to N terminal groups of Hb forming carbamate
reduces affinity for O2
importance of purifying proteins
prevent interference with experiments
remove proteins with related activity
impure proteins resistant to forming crystals in x-ray crystallography
advantages of using prokaryotes to produce proteins
very easy to manipulate genome
easy to grow in large cultures
high yield
disadvantages of using prokaryotes to produce proteins
different post translational modifications to mammals
poor folding of complex proteins
advantages of using unicellular eukaryotes to produce proteins
easy to manipulate genome
easy to grow in large quantities
high yield
mammalian like post translational modifications possible
disadvantages of using unicellular eukaryotes to produce proteins
moderate ability to produce more complex proteins
advantages of using cultured mammalian cells to produce proteins
full range of post translational modifications
can fold complex proteins
disadvantages of using cultured mammalian cells to produce proteins
difficult to genetically manipulate
hard to grow in large quantities
poor yield
very expensive growth medium
assay method
add sample of enzyme to substrate
mix and follow absorbance in spectrophotometer
liquid phase of column chromatography
solution containing protein mixture
stationary phase of column chromatography
porous solid matric
column chromatography: gel filtration
separates based on size of protein
column chromatography: ion exchange
charge of protein
column chromatography: hydrophobic interaction
separates based on hydrophobicity
column chromatography: affinity chromatography
separates based on protein interactions
enzyme-substrate
antibody-antigen
how does gel filtration chromatography work
carb polymer beads
small molecules enter aqueous spaces within beads
large molecules cannot enter beads
how does ion exchange chromatography work
medium has a permanent charge
protein has many charged amino acids
isoelectric point
pH at which protein has no overall charge but is ionised
how does charge change as pH becomes more basic
overall charge decreases depending on numbermof ionisable side chains
how do you alter what proteins bind to the column
change pH
how to eliminate proteins from ion exchange column
change pH
increase buffer’s salt concentration
stepwise or gadient
does stepwise or gradient have a higher resolution
gradient
SDS-PAGE protein purification
based on size
gel gives proteins negative charge
voltage passes through gel - makes positive
heavier proteins don’t move as dark as as lighter proteins
examples of co-enzymes/cofactors
metal ions, NADH, haem
where are hydrophobic amino acids found in enzymes
inside
similarities of enzymes to chemical catalysts
catalyse reaction without changing
reaches equilibrium faster but does not shift equilibrium
differences of enzymes to chemical catalysts
high substrate specificity
high catalytic power
require less intense conditions
Kcat
number of molecules a single enzyme can bind and convert to product every second when substrate is not a limiting factor
how is enzyme activity determined
assay measuring
rate of product formation
rate of loss of substrate
rate of production of cofactor
equation to calculate enzyme activity
d[P]/dt = -d[S]/dt
why must initial rate be calculated
substrate used up in reaction reduces rate
enzyme activity unit
amount of enzyme which transforms 1 micromole of substrate per minute at 25 degrees
specific activity
number of enzyme units per mg of protein - purity
Vmax
maximum rate
Km
Michaelis constant
measure of affinity for a substrate
line weaver-burk plot
reciprocal of m-m plot
y=mx+c of line weaver burk
1/V = Km/Vmax * 1/[S] + 1/Vmax
irreversible enzyme inhibit
covalent bonding to active site
competitive reversible enzyme inhibitors
bind to active site
overcome at high [S]
Vmax same but Km increases
non-competitive enzyme inhibitors
bind to allosteric site
Vmax reduces, Km same
how does lineweaver burk plot change with inhibitors
gradient becomes steeper
why can’t we perform acid-base catalysis experimentally
not possible to have 2 pHs at once
how does RNase perform hydrolysis
His 12 acts as base
His 119 acts as acid in first step
reverse in second step
substrate steered into active site by oppositely charged residues
what contributes to the catalytic activity of RNase
enhanced reactivity of side chains
orienting of substrate wrt catalytic groups of enzyme
why is L-lactate dehydrogenase stereospecific
Arg 109 angles carbon down to ensure L-isomer produced
chymotrypsin
c-terminal side of bulky hydrophobic and aromatic amino acids
trypsin
c-terminal of K or R
elastase
c-terminal side of small amino acid
mechanism of identifying enzyme’s catalytic triad
nucleophilic attack - his acts as base
acyl-enzyme intermediate - His acts as acid
substitution of tetrahedral intermediate
general base catalysis
specificity pocket
orients peptide bond for cleavage by catalytic triad
zymogen
inactive protease which must be cleaved to become active
how are serine proteases regulated biologicallty
zymogens secreted from pancreas to duodenum
trypsin activated by enteropeptide in duodenum
trypsin inactivated by pancreatic trypsin inhibitor
assumptions of the fluid mosaic model
proteins at low concentrations
constant thickness
lipids are all the same
truth of fluid mosaic model
many protein complexes
many different types of lipids
bilayer constantly changes shape to match protein
all membranes are different
amphipathic
hydrophobic and hydrophilic regions on the same molecule
properties of cholesterol
polar OH group so slightly amphipathic
controls membrane fluidity and packing
why is the membrane asymmetric
each monolayer has a different lipid composition
many cytosolic proteins bind to specific lipid head groups
lipid movement
lateral diffusion
hydrocarbon chains are flexible and dynamic
lipids rotate freely around their vertical axis
properties of rigid gel phase
occurs at low temperatures
restricts lateral diffusion
transition temperature is lower with shorter chain hydrocarbons
evidence for lateral diffusion
different lipids labelled with fluorescent markers
after 40 minutes they had integrated
conclusion: free diffusion of cell surface proteins with hybrid membrane
properties of integral membrane proteins
embedded in bilayer
bound to membrane by hydrophobic forces
can only b e separated from membrane using disrupting agents
single or multipass
insoluble in aqueous buffers
properties of peripheral/ extrinsic proteins
bound to surface by h bonds or salt bridges
easily dissociate from membrane under mild conditions
can be anchored to lipids
what is unique about membrane proteind
either wholly helix or wholly sheet
physical chemistry of membrane proteind
many hydrophobic amino acid
present hydrophobic surface to acyl chains
difficult to study outside of membrane environment
properties of alpha helices in the membrane
thermodynamically stable
all h bonds are satisfied
hydrophobic residues face the acyl chains
properties of beta sheets in the membrane
alternate polar and hydrophobic aa
hydrophobic residues face bilayer core, polar residues face interior
intra-chain h bonds between strands
transporter
down or against conc gradient
channel
only allow diffusion down conc gradient
na+/k+ ATPase
actively exports 3Na+ and imports 2K+ using 1 ATP
inward sodium gradient
negative delta G
free energy used to drive transporters
prokaryotic membranes
electron transport chain generates proton gradient
drives ATP synthase
genome
all genetic information of an organism
gene
basic unit of inheritance
difference between ribose and deoxyribose
ribose has OH on the 2’ carbon
purines
double ring structure
A&G
pyrimidines
single ring structutre
C, U & T
why is RNA unstable
2’ OH acts as nucleophile to break phsophodiester link
char gaff’s rule
pyrimidine:purine = 1:1
%C=%G, %A=%T
why do bases stack
hydrophobic interaction
denaturation
double strand to single strand
annealing
single strands to double strands
minor groove properties
1.2nm
narrow and deep
major groove properties
2.2nm
wide and shallow
persistence length
length of DNA along which a thermally excited bend of 1 radian occurs
why is short DNA stiff
electrostatic repulsion of phosphates pushes against bending
energetically favourable that bases are stacked nicely
protein-DNA interactions
proteins bind and recognise specific DNA sequences
recognise dna damage
bind DNA non-spefifically]
dNTP
deoxyribo nucleotide triphosophate
DNA polymerase 3
main replicating enzyme
9 subunits
250-1000 nucleotides per second
3’-5’ exonuclease activity
proofreading
DNA polymerase requirements
dNTPs as precursors
can only add dNTP to 3’ end of nucleic acid
3’ primer
magnesium ion
initiating DNA replication
initiator binds to origin
easily melted A&T nucleotides separated
DNA helices binds to break more H bonds
ssDNA binding proteins
prevents reannealing of sDNA during replication
DNA polymerase removes them as it goes along
properties of primase
synthesises RNA primers
binds to 3’ hydroxyl
no proofreading
no specific initiation sequence
frequency of priming is different in leading and lagging strands
how often does replication restart in the lagging strand
~1000 bases
direction of DNA replication
5’ to 3’
purpose of mg ion in polymerase catalytic site
stabilise the phosphate
activate OH to make it a better nucleophile
requirements for polymerase catalytic site
asp residues
mg ion
triphosphate
nuclease
cleaves phosphodiester bonds
exonuclease
ends of nucleic acid
endonuclease
within nucleic acid
prokaryotic connecting Okazaki fragments
polymerase 1 removes primers and replaces with DNA
3’-5’ exonuclease rpoofreading
DNA ligase catalyses phosphate linkage
terminating replication
tus binds to terminating sequence
physical block to replication fork
direct repair
specific base damage
removed directly by enzyme
mismatch repair
incorrect bases
base excision repair
range of damaged bases
nucleotide excision repair
wide range of bulky DNA damage
RNA secondary structures
helices
hairpins
bulges
RNA base interactions
AU, GU, AUA triple
prokaryotic RNA polymerase requirements
all dNTPs
3’ hydroxyl to attach dNTPs
starts at A or G
promoter DNA and sigma unit to initiate
bacterial promoters properties
where RNA polymerase starts transcription
define which strand is copied
requires sigma factors
different strengths
promoter strength
stronger promoters produce more proteins
promoter unwinding
positions new nucleotide into the active site of RNA polymerase
defines where transcription starts
rho-dependent transcription termination
occurs at specific sequences
binds to rut sites in transcription
pulls RNA out of RNA polymerase so RNAP falls off
rho-independent trasnscription termination
occurs at specific sequences
transcription inhibitors
rifampicin
binds to beta subunit of bacterial RNAP
prevents initiation but not elongation
blocks path of elongation at 2-3nt length
transcription inhibitors
actinomycin D
intercalates into DNA
prevents initiation and elongation
can also interfere with replication
which amino acids to pyrimidines generally code for
hydrophobic
which amino acids to purines generally code for
hydrophillic
silent or synonymous mutations
same Amino acids
missense or non synonymous
changes 1 amino acid
nonsense or stop
truncated protein
frameshift
scrambled protein structure
properties of tRNA
small, 74-93 nucleotides long
folds from clover to L shape
wobble position
1st and 2nd codons bind to tRNA normally but 3rd is less constrained
activation of amino acids
catalysed by amino acyl tRNA transferases
aa added to ATP releasing 2Pi
aa added to tRNA forming amino acyl tRNA
releases AMP
why must amino acids be activated
peptide bond formation between free amino acids is unfavourable
can ribosomes check aa
no
amino acid recognition
synthetases are highly specific
correct aa has highest affinity for active site of synthetase
tRNA recognition
synthetases must recognise correct tRNA
structurally and chemically complimentary
aa proofreading
after initial attachment, aa forced into editorial site
only incorrect aa fit
once in, hydrolysed from tRNA
peptide tRNA
bound to mRNA and had polypeptide chain attached
amino acyl tRNA
free with next amino acid in chain attached
prokaryotic 50S component
Mr 1.6 million
rRNA and proteins
prokaryotic 30S component
Mr 900k
rRNA and proteins
initiation of translation in prokaryotes
5’-3’ at AUG or GUG codon
30S component binds to ribosome binding site and places AUG in active site
IF 1,2 and 3 + GTP allow 50S component to bind
forms 70S initiation complex
elongation of translation in prokaryotes
amino acyl tRNA binds to empty A site
proofread and EF-Tu dissociates
peptide transferase reaction
large subunit translocation
small subunit translocatiom
explain proofreading during elongation of prokaryotic translation
GTP hydrolysed
incorrect tRNA dissociates
what escorts tRNA to the A site
EF-Tu
describe the peptide transferase reaction
bond to peptidyl tRNA broken
chain transferred to amino acyl tRNA
synthesis starts at N terminus and new amino acid is added to C terminus
catalysed by ribozyme
ribozyme
23S rRNA
explain large subunit translocation
large subunit moves forward
stabilised by EF-G
GTP binds to A site
energetically unfavourabke
explain small subunit translocation
GDP released
small subunit moves to next codon
energetically favourable
termination in prokaryotic translation
release factor binds to stop codon in A site
C terminus hydrolysed and protein released
why does the release factor cause ribosome to detach
ribosome changes conformation so subunits detach and mRNA is released
why are prokaryotic transcription and translation coupled
dna and ran are both in the cytosol
antibiotics properties
stall initiation
prevent elongation
induce miscoding
basal transcriptional activity
genes are always on unless controlled
strength of promoter dictates gene expression
genetic switches
repressors or activators
proteins that bind to specific dna sequence controlled by binding of a ligand
repressors
bind to operators
cause RNA to detach
operator
overlaps with RNAP binding site
activators
bind to sites which do not overlap with RNAP binding site
helps RNAP bind to promoter
purpose of the lac operon
low glucose but still need to synthesise ATP so uses lactose
the lac repressor
RNAP binds in the presence of lactose
releases lac repressor from operator
the CAP activator
CAP detects cAMP
CAP binds to operon, cAMP binds to CAP
RNAP binds
when are high levels of cAMP present
low levels of glucose
lac operon when glucose is present and lactose is not
expression of lac operon repressed
lac operon when glucose is not present but lactose is
lac operon expressed
no glucose or lactose in lac operon
expression repressed
glucose and lactose present in lac operon
expressed
lamda repressor process
lamda repressor binds to OR1
at high concentrations more lambda repressor binds to OR2
at higher concentrations binds to OR3 SO RNAP cannot bind
cro repressor blocked
why does lama repression fail
DNA damage so proteolysis of lambda repressor
cro repressor promoter no longer blocked
cro repressor
RNAP binds to cro promoter producing cro repressor
lamda bacteriophage genes expressed
lytic state reachedq
lamda repressor high, cro low
lysogenic state
lamda repressor low, cro high
lytic state
size of DNA double helix
2nm
size of beads on a string form
11nm
size of chromatin fibre of nucleosomes
30nm
why can we not be sure of 30nm fibre
never actually been observed in the cell
may only exist due to extracellular environment
size of chromatin fibre folded into loops
700nm
size of mitotic chromosome
1400nm
structure of histone
2 copies of 8 proteins
chromatin remodelling enzymes
use energy from hydrolysis of ATP
ISW1 enzyme
slides nucleosome along DNA
SW1/ SNF enzyme
removes nucleosomes from DNA
histone acetyltransferase function
relaxes chromatin via acettylation
histone deacetylase function
condenses chromatin
differences of eukaryotic DNA replication to prokaryotic genes
during S phase
slower DNAP
multiuple origins of replication as multiple chromosomes
end replication problem solved by telomerase
differences of eukaryotic transcription to prokaryotic transcriptiuon
RNAPs require accesory factors for each stage in the cycle
promoters are more complex
RNAPs transcribe through chromatin
why does pre-mRNA require processing
unstable
cannot leave nucleus
cannot bind to ribosome
why can only RNAP2 transcripts be processed
1&3 do not produce translatable mRNA
5’ capping process
M7G linked through onverted 5’-5’ triphosphate bridge to initiating nucleotide of a nascent script
why is 5’ capping important
prevent RNA degradation by exonucleases
allows transport from nucleus to cytoplasm
initiates translation
recruits splicing factors
mRNA splicing
introns removed by spliceosome complex
why is splicing useful
different protein combinations can be produced by combining different exon sequences
polyadenylation process
ployA tails added to mRNA at the end of transcription by poly-A polymerasew
why is polyadenylation useful
protects RNA from degradation
transport of RNA from nucleus to cytoplasm
assists action of ribosome
termination
preparing DNA for recombination
add phenol and centrifuge
aqueous layer is DNA and RNA, phenol layer is protein
add ethanol
DNA precipitate left
source of restriction endonucleases
bacteria
restriction endonuclease recognition sites
specific 4-8bp palindromic sequences
activity of restriction endonucleases
cut dsDNA
leaves 5’ sticky ends or blunt ends
digestion frequency
how often RE cuts
4^n
4 = number of bases
n = length of recognition sequences
DNA ligation
covalent bonding of fragments to each other
complementary base pairing forms H bonds
process of DNA ligation
T4 DNA ligase joins nucleotides
uses 2ATP
produces 2 AMP and 2 Pi
both gaps closed
how to produce a viral vector
replace lamda DNA with foreign DNA
produce viral assay
how to produce bacterial plasmid vector
many unique RE sites to insert foreign DNA
cloning eukaryotic genes using reverse transcriptase
synthesises cDNA using oligo(dT) primer which binds to poly-A
RNA/DNA hybrid forms
RNAse H digests RNA
ssDNA forms hairpin which primes cDNA
DNAP added to form DNA
S1 nuclease opens hairpin
in-vitro DNA synthesis in a tube
ssDNA primer + 4 dNTPs + ssDNA template + Taq DNA polymerase
heat to 95 degrees to break H bonds
Taq polymerase properties
heat resistant but prone to error
no proofreading
PCR for gene cloning
not dependent on RE sites
greater specificity
PCR for viral screening
highly sensitive
shows virus before symptoms appear
PCR for forensics
DNA fingerprinting
familial linkage
sanger DNA sequencing
dideoxyribonucleotides
determine order of bases
screen mutations of variants
validates PCR
Sanger’s method
template + primer + dNTPs + 35-S-dCTP + Taq DNAP
tubes have different dNTPs
each reaction terminates at a different base
add products to respective lane of gel
electrical field
read manually 5’-3’
improvements to sanger’s method
flourescent dNTPs over radioactive
performed in a single tube
