TB6 Flashcards

Question

Describe EMSA

Answer 1

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.

Answer 2

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.

Answer 3

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.

Answer 4

Ambiguous distance between all residues shown to be involved in an interaction i.e., their chemical shifts are perturbed by addition of a ligand.

Answer 5

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.

Answer 6

1. Bulk water is selectively irradiated with RF, saturating the water magnetization 2. Some of this is transferred to ligand 3. 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.

Answer 7

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.

Answer 8

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

Answer 9

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.

Answer 10

k_app=k_on [B]+k_off

Answer 11

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.

Answer 12

height of the letter indicates how often the base is found at that position. Different environmental conditions can change this.

Answer 13

DNA-sequence recognition, arising from protein-base contacts

Answer 14

recognition of intrinsic DNA structure, or its ability to be deformed upon protein binding

Answer 15

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.

Answer 16

acceleration of ions in a gas environment that leads to generation of mainly b and y ions.

Answer 17

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.

Answer 18

ions are exposed to high energy electrons that lead to dissociation into c and z ions.

Answer 19

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.

Answer 20

where a high voltage is applied to a liquid to create an aerosol

Answer 21

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.

Answer 22

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.

Answer 23

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.

Answer 24

Entropic chain activities, effectors, scavengers, display sites, and assemblers

Answer 25

large harpoon that injects effectors into a contacted neighboring bacterium

Answer 26

on the cell surface are toxins that wait for neighbors to touch before translocating the toxin effectors to kill the neighboring cell

Answer 27

diffusible substance that binds the competitor cell surface and self-translocates into the cell. e.g., KlebC