Ernesto Cota - Protein-Ligand Interactions Flashcards

Question

Is the biotin-avidin interaction stable?

Answer 1

Sortase – function Enzyme used to anchor secreted proteins on the external cell membrane of Gram+ bacteria 1. Recognizes and cleaves the LPXT-G motif on the C terminus of secreted proteins - forming acyl-enzyme intermediate 2. Transfer - Forms isopeptide (amide) bonds between C-terminus of secreted proteins and NT of peptidoglycan (lipid II)

Answer 2

Engineer erythroid precursors → new RBCs that expressed a protein called Kell with a modified C-terminus (LPXTG-HA) → sortase recognizes motif and binds - sortase modified RBCs Addition of target protein with an aminoglycine nucleophile - nucleophilically attack and replace sortase – tethering it to the cell surface

Answer 3

Most amino acids are neutral pH are relatively chemically inert, but we can… **Add new functionalities to proteins** using reagents that: 1. Covalently modify biomolecules in specific, desired sites 2. Produce minimal side reactions 3. Work in ‘gentle’ solution conditions (i.e. aqueous, near neutral pH) 4. Are safe to use outside ‘chemistry’ labs 5. Are commercially available 6. Have been tested in different systems – research papers

Answer 4

1. Nucleophilic attack of primary amine on ester of Sulfo-SMCC cross linker 2. Activated Maleimide ring double bond reacts with HS-antibody to conjugate antibody to carrier

Answer 5

**Photoaffinity Labeling (PAL)** – only reactive when exposed to light - Photo-reactive reagents are chemically inert compounds - reactive when exposed to UV or visible light - Proteins modified with PAL reagents can covalently bind their target(s) after activation by light - Unlike other methods, PAL can label low abundance and low affinity proteins Active field of research, with increasing number of commercially available compounds

Answer 6

STAT3 and HSP90 - binding partners Ku70 and ku80 - binding partners

Answer 7

We need to have a **theoretical understanding of protein-ligand interactions** Basically, we need to codify binding behavior mathematically --\> understand binding mathematicall/binding data - DG, K_on_/off, etc. We will examine models for: 1. Single site binding 2. Multiple site binding (independent sites)

Answer 8

Single site binding model - We measure the formation of ML – readout **Assumptions** 1. Binding is dynamic, reversible – non-covalent 2. Measurements when thermodynamic eq. is reached - i.e. Conc. of complex doesn’t change over time **Important to support assumptions with structural data:** 1. Number of ligand binding sites? 2. Any Cofactors? 3. Inhibitors present? 4. Conformational changes upon ligand binding – are they reversible?

Answer 9

Ka - equilibrium association constant

Answer 10

1. Create equations for total and free protein and Ligand – rearrange to my M/LFree subject 2. Insert into KD equation 3. Rearrange 4. Expand brackets 5. Move KD[ML] to the RHS to make the equation = 0 6. Factorize [ML] 7. Substitute into quadratic formula to solve for [ML] – our x value

Answer 11

Strong non-covalent interaction - Useful for purification allows strong binding to biotin tag

Answer 12

Rarely happens – transformation efficiency in yeast cells is low Plus when scanning a whole library, only a small selection of prey will bind the bait – hence, increasing the unlikely nature of transfecting two plasmids with prey that bind the bait

Answer 13

Yes we use SDS-Gel since we don’t really care about keeping the complex intact, rather the ligands will be radiolabelled and can thus be identified in the autoradiogram

Answer 14

The prey is only radiolabelled – identify ligand

Answer 15

**GST** - we need to identify ligand identity via mass spec **Y2H** - simply sequence the DNA of the inserted plasmid

Answer 16

a) **Separation of RNAX and MS2 in space** – preventing their interaction b) **Dimerization** – increases effective concentration + reducing KD of interaction (two binding regions on the same protein) c) **Increase expression of ProtY-AD fusion -** increases signal Generally, more efficient and likely to produce a true signal

Answer 17

Can use either but most of the time KD is used + easier units (M instead of M-1)

Answer 18

Examine fluorescence methods with particular focus on… Differential Scanning fluorimetry Microscale thermophoresis

Answer 19

1. Sensitive 2. Can be quantitative - K_D and T_m 3. Equilibrium measurements (in vitro) 4. Use to examine protein-protein interactions in living cells (FRET) 5. May require special reagents 6. GFP-fusion proteins 7. Chemical attachment of fluorophores to your protein

Answer 20

RU = resonance units

Answer 21

Nuclear Magnetic Resonance (NMR) Modern methodology – in constant development Provides a series of techniques to identify PLI’s.. a) Combines binding constants and structural information b) Able to observe/measure interactions with a wide range of affinities Useful for non-crystallisable complexes or as complement to XRD/EM studies Three different techniques using NMR will be discussed

Answer 22

Problem - Not possible to separate CSPs from direct ligand binding and CSPs due to allosteric/ conformational changes upon ligand binding (possible with other NMR techniques) - especially for big dynamic proteins

Answer 23

Using this technique as we can map the residues involved in binding the ligand and thus create an interaction surface

Answer 24

As you move away from the resonance angle, less light will be absorbed by film and instead it is reflected onto the detector - resulting in gradient

Answer 25

Lack of experimental phenomenon that may reduce the reliability of data e.g. Insensitive to problems like protein/ligand aggregation - no visible on the spectra Hence, Chemical shift change/peak comes from soluble proteins Note - important factor that you have to control is pH as it effects protonation state – especially in amides

Answer 26

Example, grow in E. Coli cells in a deuterated media Result? - Everything becomes deuterated BUT upon purification we use normal buffers (normal hydrogen) and all the amides will readily exchange their deuterated hydrogen whereas Carbons will not If you want to exchange the amides within the core of the proton (still deuterated) you would have to (partially) unfold the protein in order to allow for exchange in normal buffer

Answer 27

Problem based 1. Have a strategy to find out what the ligand is for a newly identified protein 2. Once identified, how can you validate the ligand binding 3. After that outline how you can determine binding constants and binding sites

Ernesto Cota - Protein-Ligand Interactions Flashcards

(143 cards)