kinetic and thermodynamic analysis of intermolecular interactions 6/12 Flashcards
what is bimolecular kinetics
how fast a complex is formed between 2 molecules and how fast it breaks down into is components
why?
understanding mechanism for pharmaceutical development - the lifetime of the drug target has to be long (low Kd) for the drug to be efficient
high Ka for the formation of the complex
what is the derivative
the derivative of the concentration of AB
d[AB]/dt
how quickly the conc of aB changes
the rate of complex formation (forward reaction) is given by = the product of the molar concentrations of A and B and the association constant Ka
forward = d[AB]/dt = Ka[A][B]
backwards reaction is the dissociation of th complex which is the product of the molar concentration of the complex and the dissociation rate constant
d[AB]/dt=-Kd[AB]
equivalent rate equations telling us about AB
net rate of complex formation = difference between the two
d[AB]/dt = Ka[A][B] - Kd[AB]
Ka and Kd are fixed under a given set of interactants under a given set of conditions
kinetics at equilibrium
rate of forward and backward reactions equal(association = dissociation)
and produce concentration stabilises
displayed on a graph one can see the time dependent accumulation of AB
when equilibrium is reached - the equilibrium dissociation constants is now defined by the concntration of 8 times h
kinetics at equilibrium
rate of forward and backward reactions equal(association = dissociation)
and produce concentration stabilises
displayed on a graph one can see the time dependent accumulation of AB
when equilibrium is reached - the equilibrium dissociation constants is now defined by [A][B] /[AB]all values are the same = Kd = x
a lowed ka and kd equilibrium Kd is the same
just measuring the kd doesn’t tell us about how fast bimolecular complexes form
surface plasmon resonance monitor process of binding between biomolecules in real time
how?
in a BIAcore T200 instrument
chip slots into instrument
computer to monitor
buffer and sample solutions that flow over chip
analyte flows over the surface and ligand and interacts with stationary ligand
sensor which is positioned above this surface detects the change in mass concentration as analyte molecules from the solution bind to the immobilised ligand to form complexes on the surface process - accossiation
when the sample flows over the surface it I replaced by buffer to measure the dissociation process - mass changes as molecules leave the surface
SPR measures the refractive index change cost to the sensor chip surface as molecules bind to the sensor chip
change in refractive index is proportionate to the mass of the material that has bound. measured in resonance units RU) units same for most proteins and similar for other kinds of biomolecules
to visualise SPR signal, polarised light reflects from class side of sensor chip (opposite side of solution ) SPR response is the angle at which he reflected intensity is lowest watch the movement to monitor change in refractive index
sensor chip made up of a thin layer of gold and glass slide with a chemically derivatives matrix on the surface of the gold censorship
why is the amount of energy reflected off of he chip surface actually different from the amount of energy that impinges on it
because the electrons in the metal sheet of gold propagate a plasmon (a wave of energised electrons in the surface of the gold layer)
this diminishes the amount of light that is returned to the detector (a diode array) which enables you to look at a number of intensities .wavelengths because one can use diff types of metal as the sensor
the diminution of the amount of energy thats reflected off the chip sa dip in the energy reaching the detector
this dip shifts
appears as a change in refractive index as molecules bind onto the surface and dissociates off
sensorgram = recording of the process plot of sPR response against time
SPR monitored continuously = sensor gram provides real time record of the ligand analyte binding of analyte from solution t ligand result in a change in conc at the surface
sensor grab = recording of the process plot of spring response against time
tells us the ligand light interaction
at equilibrium and when we wash away analyse as the complex at he surface dissociates
binding of analyte from solution t ligand result in a change in conc at the surface
phases:
1. baseline - close to 0t starts to ligand immobilised on the chip
association phase =
equilibrium
wash away the ligand and then we go int dissociation
washed up regeneration of the chip ready for the next sample
baseline period of sensorgram
wash period before analyse is infected establishes the baseline from which the binding responses can be measured
+ reliability needs to be flawed and stable
can take time to restore baseline
association after injection
observed rate of binding is he difference between the association and dissociation rates
analyte to complex
observed binding rate is highest at start of injection when conc of available ligand is highest and the concentration of the complex is 0
rate decreases as injection proceeds as ligands sites become occupied
and conc of complex increases
if injection increases for steady amount of time steady state is reached where
rates of dionssociation and associate are equal
dissociation phase after injection
complex ligand and analyte
wash away free analyte from running buffer = dissociation
dissociation = exponential decay
regeneration
refractive index differing largely from running buffer
provides bulk response which may be positive or negative
sensorgram shape not important
response level and activity of the ligand afterwards are v important
wash the chip with suitable solution to ensure removal all of analyte
crucial to careful kinetic measurements
what is the relationship between kinetic constants ka and kd and the eq dissociation constant Kd
considering an alate injection that is long enough to reach eq
calculating kd from kinetic rate constants
the rate of AB formation equal to the rate of AB breakdown
at eq rate of AB dissociation is 0
therefore Kd = kd/ka
Kd is the ratio of the on rate to the off rate to quote the stability of a complex
the fate the rate of the association (slower the rate of dissociation)
the lower the value of Kd and thus the higher the affinity of the interaction
1:1 binding kinetics
during the sample injection the ]analyte] is kept constant by continuous supply of new sample
[AB] on the chip surface is measured in resonance units by the respnse above the baseline
[B] free ligand is not measured directly.
however given as a Rmax-R
Rmax is the maximum analyte binding capacity
so the response units at maximum minus response units any time
dR/dt=kaA-kdR
R and Rmax are measured in resonance units therefore doesn’t mater that [B] and [AB] are unknown
[A] must be known
after the sample has passed over the surface the [free analyte] is 0
rate of dissociation=
dR/dt=-kdR
independent of he conc of analyse in the sample
only depends on amount of complex on the chip
what may cause the experimental situation to deviate significant from the ideal binding mechanism
mass transport limitation
heterogenous samples
multivalency
complex interaction mechanisms
advantages of the detection method
instantanoeous
continuous real time monitoring of molecules binding t the surface
no radioactive or fluorescence labels are required on any of the molecules involved
the dependence of the refractive index on concentration is similar for practically all biological molecules so SPR can be used equally for proteins carbohydrates, nucleic acids, lips, pharmaceutical compounds, cells and viruses
light is refracted from the back of the sensor surface and doesn’t penetrate the sample, calyces can be performed on coloured or opaque samples alike w no interference from absorption or scattering
characteristics which allow us to decide which of the interaction partners would be the ligand or the binding partner
molecular weight
maximum concentration (i.e. solubility limitations)
no. of binding sites
compare the binding properties of diff interactants w a common partner
i.e. mutated proteins binding to a particular immobilised ligand if there is a common partner same sensor chip can be used to save money on pricy sensor chips
molecules with multiple independent binding sites e.g. IGG to be used as ligand rather than an analyte since this leads to multiple ligands on the surface
avidity is seen as slower dissociation compared with one cell binding
method of coupling ligands to surface of the chip
most commonly used sensorchip
= CMS = carboxymethylated
negatively charged carboxyl groups on a dextran matrix used for covalent immobilisation of the
ligand via amine, thiol
what is amine coupling
the easiest way to immobilise a protein - covalent attachment to the surface through lysine residues on the protein
carboxylated activated with awater soluble carbodiamide using hydroxy susinamide
EDC/NGS
then amino groups on a ligand will displace this group and form an amide bond between the ligand and the dextran on the chips surface
other covalent coupling methods incase amine coupling is unsuitable if ligand has essential Lys residues that ma be inactivated in amine coupling
A surface thiol coupling= introduces carboxyl residue ester reacted with systamine peptide bond linkage onto the resin
get a disulfide bridge formed between between ligand and residue
protein needs in free Cys
B ligand thiol coupling similarly carboxyl gorup suscinamide ester made disulphide containing compound added onto chain and through cytemine
provide the ligand in SH form will form a disulphide bridge displacing the activating ring to give us an SS coupling
C
Aldehyde coupling
N hydroxy succinamide activation and then treat with hydrazine = chip in. hydroxide form which will react w aldehyde function to give shift base coupling which can be reduced to a stable ligand attachment to the chip
High affinity, non covalent capture
Amine coupling
not suitable for DNA or RNA an alternative immobilisation method is needed
called biotinylation
where ligand is captured with a Streptavidin (SA) chip
relies on a tight interaction between the ligand and a capturing molecule immobilised on the sensor surface
Strptavidin (SA) capturing molecule is attached to chip
ligand attached to straptavidin because of biotin attached to it
analyte recognises free ligand
important that ligand does dissociate from capturing molecule during analysis
can use anti mouse IGG antibodies
useful for capturing monoclonal antibodies when present as analtye
tag specific capture mechanism
NTA-nickel complex
will capture recombinant His tagged proteins
can be trapped on nickel nth surface
regeneration of chip conditions between samples
to get all of the analyte off of the ligand
curve will show if the preparation of the chip was good enough
cleaning includes using 10 millimoler mM glycine at pH 2.5 to disrupt interactions
can use 0.1@ SDS removes tightly bound analytes
wash step is crucial
for robust analysis use many [Analyte]s
> 100 fold range of concentration to draw sensor grams
model=
curve saturated then clear dissociation rate
if graphs shoots up or too low and never reaches steady rate or saturation not suitable
protocols for fitting curves into 1:1 interactions or heterogenous ligand situations or 2 state reactions
1:1 interaction
A+B= AB
