TB6 Flashcards
Give the equation to determine Kd
Kd = [reactants]/[products]
Give the 2 equations that can give Gibbs free energy
DeltaG = -RTlnKd = delta H - T(delta S)
Give the Langmuir equation
n~ = [free ligand]/(Kd + [free ligand])
Equation for multiple, independent and identical binding sites
n~ = n[free ligand]/(Kd + [free ligand])
Give the equation used to create a Scatchard Plot
n~/[free ligand] = (number of binding sites)/Kd - (n~)/Kd
Describe isothermal titration calorimetry
A label-free method for measuring binding thermodynamics of any two molecules that release or absorb heat upon binding.
Describe surface plasmon resonance
Real-time, label-free detection of biomolecular interactions using a phenomenon that occurs when polarized light strikes an electrically conducting surface at the interface between two media. This gives k-on, k-off, and thus Kd.
Describe thermal shift assay
Measures the changes in the thermal denaturation temperature and hence stability of a protein under varying conditions. When something binds to the protein, you’ll see a shift in Tm and this can be used on small ligands to determine Kd.
Describe differential scanning calorimetry
A thermoanalytical technique in which the difference in the amount of heat required to increase the temperature of a sample and reference is measured as a function of temperature. The heat capacity will change upon ligand binding and can determine Kd.
Describe fluorescence polarisation
A small, fluorescently labeled molecule is excited with plane-polarized light and emits mostly depolarized light because the molecule tumbles rapidly during the time between excitation and emission. However, when the molecule binds a much larger molecule (i.e., protein), it rotates more slowly and the emitted light remains largely polarized.
Define polarization anisotropy, A
The degree of polarization divided by the total fluorescence output; it’s dimensionless and sits between 0 and 0.5.
A = ( I-parallel - I-perpendicular) / (I-parallel + I-perpendicular)
List the 3 types of protein interactions
Protein-protein, protein-ligand, protein-nucleic acid
Describe the solvent accessible surface area
When a probe sphere the size of a water molecule is rolled around molecules X and Y, and complex XY, the SA is the difference between these:
BSA = (Ax + Ay) - Axy
What is a hot spot?
A subset of the interface core that are very important for the stability of PPIs, such that substitutions here dramatically reduce affinities.
List the 3 amino acids commonly found at hot spots
Trp, Arg and Tyr
What are the two most common types of interactions at interfaces?
Pi-cation and pi-pi
Describe alanine scanning
SDM to replace single residues with alanine and thus determine their contribution to the stability or function of a protein
Give the chemical formula for a simple bimolecular association:
A + B <–> AB
Give the chemical formula for an induced fit association
A + B <–> AB’ <–> AB
Give the chemical formula for pre-equilibrium association
A <–> A’ <–> A’B
Describe Y2H experiments
DNA binding domain recognises and binds the upstream activating sequence. Together they bind the activation domain to recruit RNA pol.
Fuse AD to bait protein X and DBD to prey protein Y. The reporter gene will only be transcribed if X and Y form a complex.
Describe in vivo chemical cross linking
Photo-activatable chemical groups (photo-Leu or -Met) into the bait protein and activate with UV light to cross-link to the prey protein. Isolate and identify using mass spectrometry.
Describe BRET
Uses chemiluminescence: protein X is bound to luciferase, which oxidises in the presence of luciferin. This triggers a transfer of energy, and if bound to a YFP-tagged protein, fluorescence will occur.
Describe split protein sensors
The protein is split into two domains: X and Y. Each are attached to either the N- or C-termini of a protein with a detectable signal. If X and Y interact, the signal will be detected. Can be used with membrane proteins.
Describe EMSA
DNA bound with a protein is larger than free DNA so will move more slowly through the gel matrix. This can be quantified using programmes.
Describe antibody-based supershift EMSA
If you think you know which protein is binding DNA, add an antibody to that protein and this should slow down the migration even more.
Describe microscale thermophoresis
An IR laser introduces a microscopic temperature gradient across a capillary tube, and molecules diffuse away from it. The movement is dependent on various parameters that change upon binding of a ligand. Detection is either through fluorophores or intrinsic fluorescence.
Define ambiguous interaction restraints (AIRs)
Ambiguous distance between all residues shown to be involved in an interaction i.e., their chemical shifts are perturbed by addition of a ligand.
Describe saturation-transfer difference NMR (STD-NMR)
STD-NMR is typically run as a 1D difference of two spectra: one in which the protein is irradiated and one in which the protein is not (reference spectrum). You take the difference between the experimental and reference spectrums, allowing you to see residual signals that indicate a binding interaction. As you’re detecting the ligand, you will almost always get chemical shifts and be able to tell from the shift locations which ligand is bound. The protein is selectively irradiated with a radiofrequency pulse, saturating the magnetization and some of this is transferred to the ligand upon binding.
Describe waterLOGSY
- Bulk water is selectively irradiated with RF, saturating the water magnetization
- Some of this is transferred to ligand
- NOEs are generated when the ligand binds, and these depend on tumbling time of the molecule. Ligands (small) that bind protein (large) will have the sign of the NOE flipped.
Describe relaxation dispersion (editing)
This relies on the fact that linewidths (1/T2) are broader for larger molecules, and so the spectra will differ largely from a ligand and a ligand-protein. The binding of small molecules is therefore observed through line-width changes upon the addition of a binding protein. This experiment is based on the CPMG to convert linewidth into signal intensity.
What is Smoluchowski’s limit and why is it rarely observed?
The kon values for uncharged, uniformly reactive spheres, in which there is no chemistry. In reality, the observed kon values are far lower for two main reasons:
1. The molecules must often collide with appropriate geometry
2. The molecules may require energy in the collision for conformational rearrangement
Describe stopped flow fluorescence
Small volumes of solutions are rapidly and continuously driven into a high-efficiency mixer. This mixing process then initiates an extremely fast reaction. Often, fluorescence is used to measure this time: one of the molecules is tagged such that the fluorescence changes upon binding, and this change is what’s measured.
Give the pseudo-first order equation
k_app=k_on [B]+k_off
Describe fluorescence equilibrium titration
Take the individual samples from the EMSA and look at them in a fluorometer. This means the concentration must remain the same within the cuvette and you stand a greater chance at spotting non-specific interactions. Kd is extracted in the same way as EMSA.
What does a binding logo show?
height of the letter indicates how often the base is found at that position. Different environmental conditions can change this.
What is direct readout of DNA?
DNA-sequence recognition, arising from protein-base contacts
What is indirect readout?
recognition of intrinsic DNA structure, or its ability to be deformed upon protein binding
Describe facilitated diffusion
The answer to the target search problem: it involves several processes, including ‘one dimensional’ sliding back/forth DNA. It can also jump and hop via specialized versions of 3D diffusion or move via intersegmental transfer.
Describe collision induced dissociation
acceleration of ions in a gas environment that leads to generation of mainly b and y ions.
Describe higher-energy collisional dissociation
specific to orbitrap instruments. Similar to CID but performed in an HCD collision cell and accumulation of fragments in the C-trap generates mainly b ions.
Describe electron-transfer dissociation
ions are exposed to high energy electrons that lead to dissociation into c and z ions.
What is a quadropole?
is a mass filter: ions enter the 4 rods and with changing frequencies/amplitudes you can isolate certain ions. Those that don’t have a certain mass can be sent out of the rods to filter the sample.
What is electrospray ionisation?
where a high voltage is applied to a liquid to create an aerosol
What is a quadropole ion trap?
stabilizes ions as a ‘packet’ depending on the voltage set. These are then ejected and hit the detector. It can also break peptides apart in the presence of gas molecules.
What is a TOF?
contains a tube that ions are ejected into and the length of time they spend in the tube is used to determine their size; the heavier the ion, the slower it moves through the tube. This has high resolution and sensitivity, but requires a filter first.
What is an orbitrap?
has electrodes where ions are concentrated into a small packet and injected. These packets form rings and start to move around at a certain frequency, dependent on the mass of the ion. This data is deconvoluted using Fourier Transformations.
Give the 5 classes of IDPs
Entropic chain activities, effectors, scavengers, display sites, and assemblers
Describe Type VI secretion
large harpoon that injects effectors into a contacted neighboring bacterium
Describe toxins on a stick
on the cell surface are toxins that wait for neighbors to touch before translocating the toxin effectors to kill the neighboring cell
Define bacteriocins and give an example
diffusible substance that binds the competitor cell surface and self-translocates into the cell.
e.g., KlebC
