BIOC2306 NMR 2

its called the activation energy which acts as a barrier

the rate of diffusion 

so the chances of the molecules colliding 

there is no net change between Koff and Kon

this doesn't mean there is no binding

just means tht they're both happening at the same rates

Kd can be worked out with

Kd=Koff/Kon

surface plasmon resonance

SPR measures binding affinity and kinetics

it's an SPR machine

Gold plate has a immobilized protein on it

Flow solution of protein which can bind to the protein

Same time shining light on the plate

Angle of light depends on the mass of the plate

As the protein binds to the immobilized protein the angle of the light will change

SPR can measure the binding affinity and the kinetics such as (Kon and Koff)

antibodies are immobilized on the golden plate

Look at different antibodies binding to different antigens

Can see if antibodies bind to the same antigen but at different parts

So can see if antibodies bind to the same antigens and where on the antigen they bind to

Can compare two different antibodies and look at the Koff and Kon rates and their affinity

Amine coupling

NH2 groups on chip and react them with CH2 groups in the protein and the protein is covalently attached so won’t leave the chip but you aren’t able to control the orientation of the protein

His tag

chip with nickel and protein has His tag protein will bind to nickel

with His tag and are able to control the orientation able to wash off the protein

Don’t have strong covalent bond (more likely to have dissociation of the protein)

so can wash off protein and use the chip again

Can only attach the tag to the N terminal

Streptavidin on chip and biotin on protein can attach biotin to specific areas on the protein

Strong covalent bond

These are the different phases SPR

Association phase) adding ligand to chip and its binding steepness determines association rate

Steady state region) no more net addition of ligand binding

still flowing ligand over the protein but there is no net change In binding and unbinding (can determine the Kd from this directly)

Dissociation phase) stop injecting ligand and start adding buffer ligands dissociate shape of curve determines dissociation rate

Need to consider mass transport effect

Got two rates

the flow of ligand over the protein and the rate of association

If the flow rate is too low this’ll be your limiting factor

So must either increase rate of flow or concentration of ligand or amount of protein on your chip

the last part of the SPR graph 

This involves partially denaturing the protein to dissociate the ligand

Must make sure not to damage the protein too much for the next experiment

This is where nickel chips are important since buffers can be used to wash off the protein in its entirety and just immobilize another batch of protein

NMR is able to detect weak bindig unlike SPR 

Binding causes change in chemical shift or peak which you are able to determine which amino acids it has bound to since you know which peak corresponds to which amino acid

Uses a lot more protein in NMR and you won’t get kinetic like Koff and Kon unlike SPR NMR can only detect the binding, where the binding is happening and the Kd of it

What we see on the graph depends on the exchange between the bound and the unbound states so Kex

If the exchange is much lower than the change in the chemical shifts than it is classed as slow and we’ll have two peaks in the graph

If the exchange is much higher than the difference in the chemical shift than it is classed as fast and there will be one peak between the two peaks in the slow rate of exchange

If the rate of exchange is between the slow and fast then there’ll be a broad peak in the middle (difficult to interpret)

Can use this information to map the active site

You know which amino acids are being used in the binding

Must assign amino acids to peaks first thou

To measure the Kd

Measure the change in chemical shift and the concentration of the ligand to get the graph

Only works for fast exchange

Can minus the spectrum with no binding from the spectrum that contains protein (since we saturated it with protein) so we get a spectrum of only the amino acids that are involved in binding

Small fragments of parts of drugs and will bind weakly bind to the antigen

NMR can detect this

Can identify fragments then bind closely to each other

Can combine fragments that bind near each other to develop a better drug