Carr - NMR Flashcards
What are the two most widely used systems for examining protein-protein interactions? Give some examples of other experimental methods well.
1) Yeast two-hybrid system
2) Affinity purifications
Chemical crosslinking
Chemical footprinting
Protein arrays
Fluorescence resonance energy transfer (FRET)
Fluorescence cross-correlation spectroscopy
How does the yeast two-hybrid system work?
A bifunctional transcription factor (usually GAL4) is split into its DNA-binding domain (DBD) and its activation domain (AD).
Each segment is then fused to a protein of interest, and if these two proteins interact, the activity of the transcription factor is reconstituted.
The system has been scaled up and applied in genome-scale screens.
How does affinity purification work?
A protein of interest (bait) is tagged with a molecular label to allow easy purification.
The tagged protein is then co-purified together with its interacting partner, which are usually identified by mass spectroscopy.
How may protein-protein interactions be examined computationally?
Through comparison of complete genome sequences
Protein-protein interactions may be permanent or transient. Give some examples of permanent interactions.
They are found in most homomers in PDB, and molecular machines such as the Fo subunit of ATP synthase
Transient protein-protein interactions may be either weak or strong. Give some examples of each of these.
Weak: these proteins exist in a bound-unbound equilibrium
e.g. electron transport, and cell surface receptors
Strong: these interactions may be triggered by a chemical modification, a change in conformation or localisation
e.g. G-protein alpha and beta-gamma subunits
What are the two most commonly used techniques for obtaining high resolution 3D structures of interacting proteins?
X-ray crystallography
NMR
X-ray crystallography is the most commonly used technique for obtaining a high resolution 3D structure of interacting proteins. Give one advantage and one disadvantage.
Advantage = no size limit
Disadvantage = difficult to obtain sufficient material for the crystallisation of large complexes
Give one advantage and one disadvantage of NMR
Advantage = Proteins can be characterised in solution, closer to their native conformation
Disadvantage = limited to proteins that have approx. 300 residues
Two experimental methods for determining how molecules interact include X-ray crystallography and NMR. Name three computational methods.
Inference from known structures
Domain signature
Linear-motif detection
Energy (E) = hv. What does this mean?
h = Planck constant v = NMR resonance frequency
What are the 4 key observable features of an NMR spectra?
Peak position
Peak splitting
Peak intensity
Peak shape
What are the 4 key observable features of NMR spectra, and what information does this give you?
1) Peak position - chemical (electronic) environment of a nucleus
2) Peak splitting - neighbouring nuclei (torsion angles)
3) Peak intensity - nuclear count (ratio) - T1 dependent
4) Peak shape - molecular motion, chemical exchange, uncertainty principal, uncertainty in energy
What is the name for Peak Position in an NMR spectra?
Chemical shifts
What is the name for Peak Splitting in an NMR spectra?
Coupling Constant (J) Hz
