Module 3- Protein Dynamics Flashcards
Differences of GPCRs
Occur in different tissues and mediate different responses
Distinguished by sequence and response to different ligands
Features of GPCRs
7 transmembrane helices
Undergoes conformational change when ligand binds to allow for g-protein binding and effects
Can cause signal amplification or signal damping
GPCR conformational change
Helix 6 moves away from the core, bringing helix 5 with it a bit which opens up the inside for g-proteins to bind
Biased signalling and GPCRs
An agonist leads to one response (G-protein or arrestin signalling) more than the other
Therefore, GPCR must not be switching between R and T state- not one or other
Ligands have different effects so two state model doesnt match
Has multiple conformations
Ways to observe protein dynamics and movement
Fluorescence
NMR
Molecular dynamics (MD)
What does NMR tell us
Atom type
Amino acid type
Chemical (spatial) environment
Based on chemical shift (varies relative to chemical atom, hard to measure absolutely and measure is relative to standard) and peak height (how many nuclei interact with oscillating magnetic field
What is needed for NMR
Atoms with an odd number of protons and neutrons so need to set up experiment with their isotypes
HSQC (heteronuclear single-quantum correlation) NMR
Most common type
Signal for each 15N-H or 13C-H (distinguish different H depending on what its bound to)
Reduced overlap of spectral peaks- can distinguish one from the other
Exchange spectroscopy and how NMR works
Discusses how conformational exchange can occur between adjacent atoms during the delay in exchange spectroscopy, called cross-peaks- movement of energy to different atom
NMR: magnetic probe generates spin on atom nucleus due to absorbance of energy. Conformational change causes a change in chemical shift
Molecular dynamics force field calculations
Determine time progression in fs steps for atoms
Newtons laws used to figure out what happens with movement
V=dx/dt
F=ma- compute all forces then find acceleration, gives a new velocity for each time stamo
Can put motions together and see the movement occurring
Molecular dynamics in practive
Have detailed 3D structure, include water, counter ions and thermal energy
Calculate F and a using Newton and force fields from protein structure prediction
Calculate v and position for every atom in next 1 fs
Repeat millions of times recording new structure at intervals
Analyse resulting trajectory with RMSD and RMSF
Validate results with wet lab experiment
Uses of molecular dynamics
Represent explicitly the motion of all atoms of biomolecule
Derive kinetic and equilibrium properties and compare with experimental data
RMSF
Root mean square fluctuation residue by residue- time averaged indicates flexibility of different regions related to crystallographic B factors
Show what happened- what residue/ atom and how far does it move on average- peaks show which residues move- doesnt say how often
RMSD
Root mean square difference between two structures at each time
Averaged over all atoms, how protein structure differs from a reference 9starting point) over time
Can trace events and tell how often movement occurs
Other ways to see dynamics- experimentally
Crystallography gives snapshots
NMR shows specific residues moving and can probe speed of change
MD stimulates changes in entire structure- shows structural details
smFRET tracks changes at specific positions across time
Equilibrium between conformations can be expressed as free energy diagrams
