PPI

Question 1

Size-Exclusion Chromatography(SEC)

Answer 1

FUNCTION:
-separates based on hydrodynamic volume and molecular weight
-used to purify proteins

MECHANISM: uses a gel filtration which has small pores allowing smaller beads to get stuck within them while larger beads flow out to the bottom before

Question 2

Protein-protein interactions(PPI)

Answer 2

TRANSIENT:
-complex can assemble and fall apart
-weak-high affinities
-function can be turned off and on
ex: signaling molecules
OBLIGATE:
-only/mainly function as complex or oligomer
-high collective affinities
-remain associated until they are damaged or cells no longer have use for them
ex: large machines like proteasome or ribosome

Question 3

Interfaces in PPI

Answer 3

DEFINITION: a surface that makes contact with PPI

NOTE:
-most interfaces are fairly flat
-interfacial patch 1,200-2000 angstroms
-packing density similar to interior of a protein
-Most prevalent residues are 1.Leu and 2.Arg and aromatics
STRUCTURE:
-CORE: central solvent excluded region
-75% of buried surface area(BSA)
-majority of binding energy
-typically more hydrophobic than surface in general but less than interior of protein

-RIM: surrounds the core by partly buried outer ring that includes water-mediated interactions
-similar composition to protein surface
-major role is exclusion of water from core

Question 4

Favorable binding interactions

Answer 4

-Shape complementarity:
-maximize favorable VDW interactions(contact surfaces at correct distances)

-Charge/dipole complementarity:
-close distance between opposite charges/dipoles
-optimal geometry for H-bonds\

-Conformation: lowest energy conformation in bound states

-Hydrophobicity: maximize non-polar surfaces excluded from water

Question 5

PPI “hot spots”

Answer 5

A group of residues that make large contributions to binding

-Trp, Arg, Tyr: able to form hydrophobic, aromatic, and polar interactions(pi-cation interactions between Arg and either Phe, Trp, Tyr are found at more than 50% of hot spots)

Question 6

Detecting PPI

Answer 6

-SEC
-Biolayer interferometry(BLI)
-cross-linking mass spec

Question 7

Biolayer interferometry(BLI)~Octet

Answer 7

optical technique analyzing interference patterns of white light reflected from surface of biosensor tip

FUNCTION:
-determine kinetics and affinity of interactions

MECHANISM:
-one molecule is bound to biosensor and analyte(in solution) is measure
-done in 96 well plate
-the change in bound molecules to biosensor tip causes shift in interference pattern

Question 8

Cross-linking mass spectrometry(XLMS)

Answer 8

chemical cross-linking of proteins coupled with mass spec which gives information on which amino acids are near each other and thus overall structure

MECH:
1. Protein sample are combined with reagents that form covalent bonds between amino acids and side chains in solution
2. sample is digested and peptide pairs identified by mass spec.

CROSSLINKERS:
-composed of reactive end groups and a spacer
-lysine side chains are very reactive and abundant on protein surfaces(good target for cross-linkers)
-BS3 most common type of reagent, cross links primary amines
-can use different spacers