Structural & chemical biological Flashcards
(113 cards)
1
Q
Why is synthetic chemistry used and how
A
many biochemical mechanisms cant be achieved with only Amino Acids
so uses
Cofactors and Post-translational modifications
2
Q
what challenges can be faced with synthetic modification of proteins
A
- the need for specific biological conditons
-> non extreme pH
-> can denature in organic
solvent - reactions must occur fast
- site-selectivity for one group to avoid modification of unwanted sites
-chemical linkage between small molecule and protein need to be stable
3
Q
how is synthetic chemistry work
native amino acid modification
A
N-terminal modification
-> Edman degradation
Lysine modification
->NHS coupling
-> isothiocyanate coupling
Cysteine modification
-> alpha-halo carbonyl
-> Maleimide coupling
4
Q
what are the advantages and disadvantages of Lysine modification
A
adv - ease of reaction
readily synthesised electrophilic partners for reaction
dis - not site selective
5
Q
what type of modification is maleimide coupling and the advantages and disadvantages
A
cysteine modification
adv - site specific control
dis - can be reversible under physicological conditions which can lead to off site toxicity
6
Q
what is incorporating unnatural functionality
A
manipulating natural biological processes for protein modification
7
Q
methods of incorporating unnatural functionality
A
unnatural amino acid mutagenesis
metabolic engineering
-> using azides
8
Q
what types of functionality might you want to add to a protein
A
fluorophore, toxic warhead, pegylates, UV active tag and biotinalation
9
Q
what direction are peptide chains drawn and written
A
N - terminus to c - terminus
10
Q
What does it mean when peptide chain ends with -NH3
A
Nothing doesnt affect n-terminus direction still matches convention but c terminus is capped with an amide. This however makes it more susceptible to a nucleiophilic.
11
Q
what is a glycoconjugate
A
a carbohydrate bonded to something that isn’t a carbohydrate
12
Q
what is mutarotation?
A
the equilibrated transition of a monosaccharide from acyclic (open chain) to cyclic form. This creates stereoisomers based on the position of the anomeric hydroxyl group in either the equatorial position / beta (pointing down) or axial / alpa (pointed up)
13
Q
what is the most stable conformation of hexose sugars
A
chair conformation specifically 4c1 chair conformation with OH in the equatorial position
14
Q
what is required for mutarotation to occur
A
a free hydroxyl in the anomeric position it ois not possible to form anomeric stereochemistry without OH this means that O-R cannot form stereochemistry.
15
Q
how to draw glycosidic bond structures
A
read left to right with the sight carb being first
alpha or beta being how it bonds - axial or equatorial
the two numbers: first carbon bonding taken from the anomeric position bonds to the second number od the second carb.
16
Q
draw glucose molecule
A
look at notes
17
Q
draw galactose
A
notes
18
Q
draw N-acetylglucosamine
A
notes
19
Q
draw mannose
A
notes
20
Q
how do you achieve effective coupling between amino acids
A
increase reactivity: making better leaving groups
encouraging regio/chemoselectivity: protecting groups
avoid loosing stereochemistry or racemisation
21
Q
how can you minimise racemisation of amino acids
A
using a coupling reagent to activate the c terminus,
while written in the n terminus direction for actual peptide synthesis work backwards in the c-terminus to n-terminus direction to try avoid oxazolone formation.
22
Q
why does racemisation occur in amino acids
A
the formation of resonance either between protection groups and amino acids or amino acids in peptides.
This causes intermolecular cyclisation to form oxazolones which forms a planar center losing stereochemistry
23
Q
what are coupling reagents including common examples and how do they vaguely work
A
activate the OH group on an amino acid to increase reactivity. DCC, HATU, pyBOP. HIghly nucleophilic and they help to form a good leaving group in an amino acid.
24
Q
how to decide which protecting group to use
A
stability : in reaction conditions
orthongonality : is the reactant and protection group compatible
Selectivity: the ability to install at a specific position
25
what are amines/ n-terminus protected as ........ which are
carbamates N - C - O
Boc with base
26
what are carboxylic acids/ c terminus protected as ........ which are
esters c=o-o-r
methyl ester with HCl catalyst
benzyl ester with HCl catalyst
allyl ester with HCl catalyst
tert-butyl ester
27
what are hydroxyl/ some side chains protected as ........ which are
alkyls -o-c-r silyl ethers -o-si-r3
tert-butyl ether base (NaH)
benzyl ether base NaH
silyl ethers
28
side chains that require hydroxyl protection
serine, threonine, tyrosine
29
what is solid supported synthesis of proteins
the construction of peptides on insoluble resin beads
-> peptide is immobolised on resin
->reagents are in solution that is washed away after reaction
->product is cleaved off after complete synthesis
30
how do resins bind to the first amino acid
c-terminus of first amino acid
covalent bonding to functionalised resin
31
how do amino acids bind to different types of resin
merrifeld resin : substitution of chloride to OH side
Wang resin : OH forms ester bond with OH side
Rink amide resin : NH2 forms amide bind at c terminus
all with coupling agents
32
why do you generally use Fmoc instead of Boc
to avoid releasing peptide during deprotonation
33
what coupling reagent do you normally use
DCC
34
What coupling reagent do you use in sold support synthesis
DIC
35
what does merrifield resin require to be cleaved?
HF - highly corrosive & toxic
36
what do other types of resins use for cleavage to avoid the use of HF
TFA, it allows for a positive mesmeric effect
37
advantages and disadvantages of SPPS
Adv:
- no work-up required
- can be automated
Dis:
-as purification isnt possible while bound accumulation of impurities and by-products
-final purification can be challenging due to multiple steps
- limited amount of peptide can eb produced
38
challeneges in glycosidic bond formation
reactivity: OH is a poor leaving group
regioselectivity: many hydroxyl groups make the positions sugars link challenging
stereoselectivity: can form both alpha and beta anomer
39
how to protect the primary hydroxyl group
most reactive species as a hemiacetyl
triphenylmethyl ether: very sterically bulky
non-selective (will bind to all other hydroxyl)
- acetyl esters
- benzoyl esters
40
how to selectively protect the anomeric hydroxyl group
fisher glycosylation - MeOH + H+ HCl catalyst
41
how do you deprotonate non selective hydroxyl acetyl ester groups
h2NNH2 (hydrazine)
42
how to protect 2 neighbouring hydroxyls and then selectively break one linkage
Add PhCH(OMe)2 and acid cause linages at c4 and c6 that coonect together via the OMe sidechains
to cleave c4 NaBH4
to cleave c6 LiAlH4
43
methods of activating the anomeric hydroxyl group in glycosidic bond formation
the instalation of a good leaving group
-> causes the formation of oxocarbenium ion
formation of glycosyl bromide
-> using HBr and a silver salt for activation to remove Br group
trichloroacetimidate
-> NaH (base) deprotonates O- attacks CCl3CN
-> activated by Lewis acid
thioethers
(though these arent particularly reactive in themselves and need further activation
44
what factors decide stereochemistry in glycosylation reactions
the anomeric effect explained by
- dipole minimisation in beta dipoles are aligned causing repulsion
- hyperconjugation : alpha anomer has orbital overlap with axial lone pair and antibonding orbital leading to stabilisation
neighbouring group participation
- bicyclic oxonium ion intermediate sterically hinders attack by acceptor
reactivity of the acceptors
- sometimes more reactive intermediates forgo the oxocarbenium intermediate therefore the anomeric effect doesnt occur and Beta is the major produce
intramolecular tethering
in order to favour 1,2-cis glycosidic bonds
forms a temporary linker between acceptor and donor, holding acceptor in place for preferential attack of the donor from one face of the ring
45
what is click chemistry
the bioorthagonal reactions between azides and alkynes that occurs at fast speeds
1,3-dipolar cycloaddition that is accelerated by a cu catalyst to make a cyclic product
pH 7.4
46
what is the big disadvantage of CuAAC and how is this combatted
Cu(I) is cytotoxic in living cells
so strained alkynes are used in order to achieve this, in strain promoted azide alkyne cycloaddition (SPAAC)
cant use copper so use strained ring
47
how is palladium useful for protein modification
-hydrogenation catalyst by coordinating unsaturated double bonds
-> alkenes to alkanes
-Pd triggered 'decaging' of proteins
->inactivates proteins by protecting with other functional groups
-Pd mediated cross coupling (suzuki-miyaura)
-> forms c-c bonds between organoboron and organic halide in the presence of K2CO3 and ligand
48
why can carbonyl groups be utilised in synthetic protein modification
electrophilic carbonyl groups are biorestrictive meaning they can be exploited to control metabolites and small biological molecules
49
what is the prime condition for carbonyl reaction to form imine and why is this the case
pH 4.5
lower pH - too much amine is protonated and not nucleophilic
higher pH - not enough H+ to protonate the OH groups
50
whats the main drawback for imine formation
reversible reaction in presence of excess water
51
Draw the mechanism with conditions for carbonyl to imine
see notes
52
alternate protucts that can be made using carbonyl chemistry and why they can be good?
htdrazones and oximes
a lot more stable in biological conditions than imines however some hydrolysis still observed
53
what is the wittig reaction when has it been used and what does it do
solution to the issues with mylotarg (a drug for leukemia that had unexpected partial cleavage of linker)
wittig reaction makes alkenes from aldehydes using phosphonium ylide
uses neutral pH
54
what is the protein databank
it is a database containing the atomic models of 3D proteins and nucleic acids made in 1971
data found on there is predominantly x ray crystallograph, but contains NMR and electron cryo-microscopy too.
EM data is on a fast rise currently
55
what's the spread of PDB organism data looking like
primarily human, followed by common mouse, E.coli
56
what are the amino acids typically used in hydrolysis
the charged residues
histidine, lysine, arginine, glutamic acid and aspactic acid
57
is a peptide bond planar
not completely due to tortional strain of ~180 degrees. this has a wiggle room of ~ +- 10 degree
58
what are rotamers
different conformations that the amino acid side chains can form due to tortional strain.
these can change with substrate binding
more rotatable bonds = more rotamers
59
why do beta pleated sheets tend to be antiparallel
more points of contact between strands mean more stablising interactions
60
how do disulphide bonds form
s in one cystein being within 2A of another
61
how do salt bridges form
his, lys or arg
being within 4 A of
Glu acid or asp acid
62
how do pi stacking interactions form
tyr, phe or try
within 4A
of each other either parallel or perp
63
common 3D folds
thioredoxin - twisted beta central alpha outside
Greek Key - Y shaped beta up/down form antibodies
tim barrel - repeated alpha beta
Rossmann fold - repeated beta alpha
EF hand - helix turn helix
jellyroll - all beta moves between start and end
64
how is quaternary structure written out
homo or hetero
number of different 3d struc
-mer
65
what is homology
shared common ancestory
66
what are paralogues
a pair of genes that are ancestorally from the same gene but are now in different places in the genome
67
what are orthologues
genes form different species of the same function that have a shared ancestoral gene
68
what are analogues
genes of similar function that have no shared ancestory
69
how can we infer homology from protein sequence similarity
compare sequences in the FASTA format generally colour coordingates the conservation og genes
70
how do point mutations effect protein evolution
what a single amino acid is exchanged for another it can:
-completely conserve character if its from the same group preventing fold disruption
-cause a massive disruption by being from a different group effecting fold due to differences in polarity, size, charge etc.
71
how can point mutation effect bond forming behaviour via the GroEL example
Glu 461 mutation causes a large conformational rearrangement and much lower thermostability
salt bridge cant form causing denaturation at 60 degrees whereas wild can withstand
72
what is exon shuffling
it is when exons from different genes are brought together in order to form new genes
73
types of post translational modification
phosphorylation
methylation
hydroxylation (adds OH to proline ring)
acetylation (most commonly lys)
oxidation
74
how does the chou-fasman algorithm work
predicts the likely secondary structure of proteins based upon each residues likelihood of adopting conformations, alpha, beta, turn
if two sequences have high sequence similarity they are likely to have the same fold
75
how does homology modelling work
break sequence down into likely domains and find stuctures of homologues for each of these
Align imput sequence with homologue
model parts that align well by copying template
use theoretical methods to fill gaps
optimise geometry to adhere to ideal torsion angles and ramachandran
76
what is alpha fold
AI-based protein structure predicition tool that uses deep learning (no human feature extraction gets better over time with more data) to accurately predict three-dimentional protein structures
coevolves - derives relationships between the sequence and infer shared evolution between the sequences
makes a number of models which it then relaxes and decides the best model >90% confidence is almost as good as an experimental models
better than homology modeling
77
what are pLDDT and PAE
they are confidence testers
pLDDT - predicted local distance difference test between real and predicted
PAE - predicted aligned error value differences between residues difference bwtween predicted and actual
low pLDDT usually means disorder or bias in seqience databases: pathogens vs mammalians
78
what proteins arent covered by alpha fold
shorter than 16 / longer than 2700 AA
containing non standard AA
from viruses
79
downsides of alpha fold
unlikely to reflect signle disrupting mutations
or
alterations to folding caused by modification
or
doesnt predict quaternary very well
also
biased towards bound forms doesnt produce many ligand free models
also
based off of PDB which is primarily human and organisms of interest such as SARS covid so organism bias
80
Why are X-rays used to study proteins
the scale of proteins
as they are so small nanometers are not usually a good enough resolution to effectively visualise
measures in angstroms (same as interatomic distances)
81
why cant you have an xray microscope
microscopes use lens and we currently have very poor xray lenses
so xrays show scatter patterns via a detector
Even with a theoretical xray lens the energy required for microscopy woould destroy the molecule via radiation
82
What is NOE spectroscopy
used to transfer magnetism between nuclei.
shows correlation between two atoms via intensity of crosspeaks being proportional to the separation of the two nuclei
records the chemical shift of the atoms surrounding one particular nuclei
F1 is the chemical shift of all protons
F2 Chemical shift of X
F3 chemical shift of the proton connected to X
additional spikes can be caused by proteins close in space to the subject
83
how do xrays interact with atoms
interact with the electrons - the oscillation of the xray causes a similar oscillation in electrons
doesnt interact with H well as they are undetectable because the electron density is pulled towards the carbon
Understanding the whole atom required adding up the scattering of each individual atom called the fourier transformation
84
how are electron density and the fourier transformation related
they are the inverse of one another
85
what is the phase problem that arises in relation to the fourier transform
we can only measure amplitudes so the relativity of waves to one another is unknown without the correct phase
86
why do we need to grow crytals for xrays
-crystals amplify weak scattering
-> general scattering is inefficient as low probability of e- interaction
-regular pattern allows measurability
-minimise radiation damage from x rays
-secondary radiation damage can be avoided using cryocooling
87
what is a crystal and how are proteins arranged in this
a unit lattice + motif
array of points arranged so the environment of any lattice point is adentical in another
proteins are irregularly shaped so have to form a regular arrangement which leaves gaps to achieve symmetry
tends to be parts of multiple proteins (or multipul proteins in general) per cell
mirror symmetry isnt permitted so this relies on protein chirality
88
how does x ray diffraction work in regards to protein crystals
-scattering arises from individual atoms
-crytal introduces gaps into the pattern as convolution of object and lattice cause diffraction
-pattern phase is identical to scattering pattern
-can have discrete spots as planes must have correct interplane distance .
-> as correct distance in plane is required some xrays have to move extra distance (explained by Braggs law) causing spots from the reflection of braggs planes
89
how do proteins physically form crystals
a mixture of induced dipole dipole, polar, hydrophobic, pi stacking and electrostatic interactions
- highly order H2O molecules support H bond formation
-when mixed with a chemical cocktail of precipitants and pH buffer solubility is altered to reached isoelectric point
methods used: hang drop vapour diffusion or using machinery
90
how does hang drop vapour diffusion work
-cryoprotectant is added to prevent water crystal forming
-nylon loop used to collect a single crystal
-plunged in liquid nitrogen
-use a synchrotron radiation for the protein cryallography
91
how to interpret x-ray crystal structures
electon density is produced from strong electron density in a single place
-> therefore mobile elements wont be visible blurred out as noise
-some disordered amino acids appear well ordered via electron density in the N terminus interacting with itself to form trimer structures in solution
92
how can crystallography be used as a tool to study enzyme mechanism
-can visualise the structure of enzymes (first done with lysozymes
-allows visualisation of binding pocket and active site
-can visualise how the active site distorts (in lysozymes case to half chair conformation) upon binding
-can conform the position of theorised bonds
-can use a diffusion trap
-> decrease the pH from 4.6 to 6.8 and decrease the rate of reaction in order to trap ESC
_>being left in diffusion buffer can show catalytic activation allowing proposal of mechanisms
93
crytallographys use as a tool for drug design
you can you to discover new inhibitors
via ultra high throughput random chemical fragment screening
(>2000 chemical fragments tested)
-tested kinetically for Kf - the concentration of each substance required to produce half maximum inhibition (smaller number better)
-complete a literature search for mimicks of desired product
94
how are images formed in EM and what do they tell you
thin sample in a vacuum, images form based on the density between different biological material and the buffer solvent
-whether theres contaminants
0symmetry
-conformational changes
-dense cofactors(like metal ions)
95
What signifcance does the vitreous ice phase have on EM
preparation of samples
rapid cooling of water doesnt form an ordered structure doing so preserving the biological strucuture
if this is cooled with liquid ethane creates thin samples that allow electrons to pass through easily that have a large surface area to volume ratio
96
how are grids prepared for EM
a grid is supported using tweezers 2-3 microlitre of sample added
blotted to remove excess
plunged into liquid ethane
97
the problem with EM
can only take 2D images as to reduce noise in images so many 2D images at different positions and angles must be taken snf combined to construct a 3D image
98
how to conclude resolution of single particle maps
split the data in =to two treating each half as separate
see how far the reflections reach into the Fourier space
resolution measured by where the signal is swamped by the noise
particles are then sorted into different classes based upon their states
->randomly assigned at first
->compare the particles to reference translate and rotate
->average the particle images together to improve signal and cancel out noise and move them to the best suited class
99
what is mas Kd proportional to?
- length and flexibility of the linker
-separation of the binding motif
100
what is the KIX domain and how does it interact
the KIX domain comprised of 3 alpha helices and 2 3(10) helices bundles
this is a bad target for crystallography
-> interacts with short linear motifs
-> interacts with 2 different binding sites with some
allosteric character
101
what is differential scanning fluorimetry and how is it used
its a technique used to determine protein stability
-> it works well with tryptophan as it reacts differently depending if its in hydrophobic or hydrophilic conditions
allows you to plot the ratio of folded to unfolded protein
the cross over point between folded and unfolded is the melting temperature of the protein.
can be used experimentally to:
-> check for the presence of folding
-> the impact of mutation to determine stability can work out which regions are important for stability
102
what is circular dichroism spectroscopy and how is it used
based upon chirality and exploits the circular polarisable light
creates a plot of of absorbance/CD against wavelength which creates different types of structure that can be compared against known environments
csn be used to release structural elements by plotting CD against elements like temperature
used to characterise secondary protein structure
103
What is SAXS and how is it utilised
small angle xray scattering
gives a series of R values that can help characterise proteins
Rr is the maximumm hard sphere radius
Rh is the hydrodynamic radius
-> radius of sphere that diffuses at the same rate as the solute
Rm is the equivalent mass radius
-> radius is it had uniform radius
Rg is the radius of gyration
->root mean squared average of the distance of scattering elements from the centre of mass of the molecules
104
what processes are studyable using NMR spectroscopy
-molecular recognition
-conformational changes
-catalytic mechanisms
-post translational modificiation
-regulation
-protein folding
- affinity and dissociation constants
105
what affect does the what you feed bacteria have on NMR
dictates the experiments open to you
15N - Ammonium chloride
->ligand binding
->biomolecular interaction
->dynamics
->everything the others can do
13C - Glucose
->resonance assignment
->3D structures
->larger proteins (>20 kD)
2H - 2H2O, glucose
-> larger proteins (>20 kD)
106
charactaristic spikes in 1H NMR spectrum
-small spike at 0.0 indicates interaction with aromatic ring indicating folded protein
-spike from 3.6-5 indicate alpha helices
-5-6 indicated H in beta sheet
-6-7 indicates aromatic group
-7-10 indicates amide group
-above 10 small spike shows tryptophan side chain
107
why use a multidimentional NMR spetra and what do folded proteins appear like as opposed to unfolded
NH in proteins are more complex than isolated as there are many in different positions
folded - dispertion because of different amino acid character
unfolded less dispersed as less environments
bound ligands will have different chemical shifts
108
how interactions can be studied with NMR spectroscopy
NMR spectra chemical shifts change in response to an increasing amount of binding partner
(*this binding partner is invisible to the spectra but the effect on the protein isnt*)
the changes depend in kinetic rates
- high affinity = slow chemical exchange
- low affinity = fast chemical exchange
single site, 1:1 binding changes in position are linear
109
how can you tell if there is interaction between two amino acids or things
if no interaction the NMR spec for the orignial amino acid will remain the same with just the presence of extra spikes for the other amino acid
if interaction movement
110
how to transition from 1D to multidimentional data
you need to transfer magnetism beitween nuclei via:
- j coupling (through bond)
->larger coupling more effective transfer
-nuclear Overhaus effect (through space)
111
advantages of NMR comparitively to Xray
crystals not required
information about secondary structure available earlier
rapid method for checking fold of mutants
can study unstructured regions
x ray has higher resolution
analyses much smaller proteins than Xray
112
what does NMR measure
chemical shift
signal intensity
(intensity of peaks)
linewidth
(dynamic properties)
113
measuring molecular motion why
affects the rate at which NMR singal relaxes
the magnetic field at the 15N spin is influenced by the bond 1H spin
reorientation of the bond with respect to B0 causes fluctuations in the local field
the variations in the filed cause fluctuations in the local field lead to a relaxation of the excited site
we can then measure this rate
dependent on the tumbliing rate of the molecule via N-H group
As size increases molecular tumbling rate decreases
