Protein Dynamics Flashcards
Give an overview of protein dynamics?
Proteins are dynamic - which is vital for their function
Critical for ligand/inhibitor binding
Protein dynamics are characterised by the timescale of fluctuations (kinetic) and the amplitude/directionality of the fluctuations
What is some empiracle evidence of small/large scall conformational changes in proteins?
- Atoms in proteins undergo small-scale vibrational and rotational motions that are detected by infra-red and Raman spectroscopies (6 x 1012 to 1014 /s)
- Large multi-domain proteins undergo large movements relative to each other upon ligand binding
e. g. myosin/actin cycle or GroEL subunit and in complex with GroES - Proteins spontaneously (but very rarely) undergo transient but complete unfolding with a frequency of 10-4 to 10-12 /s depending on barrier height - proteins are metastable
- Oxygen release from oxyhaemoglobin - the protein must undergo Å-scale ‘breathing’ motions that allows O2 access to the haem group
What evidence can we acquire from quenching reagents?
Fluorescence of aromatic amino-acids is instantly quenched by close proximity of certain small molecules such as O2, I- and acrylamide
Many internal residues are also efficiently quenched
This means that side-chain and backbone regions of the protein that are buried in the native state can interact with reagents in solution
Therefore either the solute can ‘dissolve’ into the protein OR proteins undergo ‘breathing’ motions that transiently expose buried regions of the protein to the solvent
However there are problems with interpretation - quenchers may bind non-specifically etc (old technique)
What is hydrogen exchange?
NH groups of backbone can exchange rapidly with solvent (e.g. D2O) As deuterium (2H) resonates at a completely different frequency the signal for each amide proton (NH) will reduce and eventually disappear
Exposed hydrogens are exchanged fast, whereas the buried hydrogens are protected and therefore take much longer for exchange
What does the rate of HX depend on?
The rate of HX depends on:
pH (10x increase in rate per pH unit increase above ~ pH 3)
Location of each amide within the protein
On the degree of burial from solvent and hydrogen bonding in the native state/frequency of partial unfolding
Rate of exchange of some amide protons in native protein are significantly retarded (by factors of 10^6)
These NH groups are buried and /or involved in H-bonding
Describe hydrogen exchange by 1D hydrogen NMR?
The signal for protons in a protein are very sensitive to their environment
Each peak relates to protons in a particular chemical environment
Used for smaller proteins as with larger proteins:
There are so many proton signals with similar chemical shift
The width of each peak increases (line broadening)
Describe 2D 25N-1H NMR?
In two dimensional methods, overlapping or clustering of signals are minimised by using two parameters to sort out the data
E.g. separating proteins by 2D-electrophoresis
For HX using 2D NMR, overlapping 1H resonances of the amide bond are resolved by also measuring the chemical shift of nitrogen on the same amide bond
The protein is grown in 15N labelled medium
This is called an HSQC spectrum
This generates a fingerprint of (ideally) all the NH groups (except Pro) in a protein
This is a powerful method capable of revealing residue-specific information about conformational dynamics
Different residues show vastly different exchange rates
However amide protons which are buried or involved in hydrogen bonding networks do exchange
Proteins undergo extensive breathing motions
What evidence can we get from aromatic ring flipping?
The location of each peak reflects the functional group but also the environment of the that group
If the residues are fixed then each proton ‘feels’ a different environment and separate peaks are observed
If flipping occurs faster than the measurement of the NMR spectrum, only a single peak will be measured for 2 H’s (> 10,000 ring flips / s)
By measuring the separation of these two peaks we can work out how fast the aromatic ring is flipping around
Example: frequency of 180 degree rotation at different temperatures
Most proteins give averaged spectra for Phe and Tyr suggesting that even fully buried rings are quite dynamic despite the fact that the interior of a protein is closely packed (crystalline density)
Therefore flipping suggests movement of surrounding atoms to allow motion
What can modern NRM techniques show?
They show a richness of dynamics from ps to seconds E.g. Side chain rotamers Methyl rotation Loop motion Bond vibration Larger domain motions
What are the types of evidence we can obtain from crystallography?
No density and B-factors
Small proteins can give different structures
Describe the evidence of No density and B-factors - achieved from crystallography?
For many proteins the electron density obtained by X-ray diffraction is smeared out to different extents in different parts of proteins
One reason for ‘blurring’ of the position of the protein atoms is due to the local flexibility of the polypeptide
The extent of smearing is expressed as the ‘temperature factor’ B
Cold areas (blue) have low B-factors and are static
Hot areas (red) have high B-factors and are dynamic
Sometimes these area are so smeared out that no density is visible
The binding sites of many proteins become more rigid upon binding their ligand
The initial flexibility facilitates specific complex formation - conformational selection
Describe the evidence of small proteins can give different structures - achieved from crystallography?
The structure of BPTI and other small proteins can vary by an average of 0.4 to 0.5 Å in the relative positions of the backbone Ca atoms in different crystal lattices
Also different conformations may result by crystallising the protein at different temperatures
Low temperatures may ‘trap’ the protein in a different state - different sub-conformations from the native state
This suggests that the energy landscape may be rough and many conformations of similar stability are present in conditions where the native state is stable
Describe molecular dynamics?
We can start with the crystal structure and apply thermal energy to see the protein move - using computers
This can reveal the nature of protein motion
On a graph the larger the root mean square positional fluctuation - the more the chain has moved from its original position
Gives highly detailed information at the molecular level
What can we see from molecular dynamics?
Rich pattern of atomic movement
Fluid-like in the centre of a protein allowing structural displacements of up to ~2 Å
More mobile in loops and terminal regions and less mobile in areas of defined secondary structure
Can also examine the rates of interconversion and relative stabilities of each conformation:
A protein’s native structure really consists of a large collection of conformational sub-states that have approximately equal stabilities
These sub-states that have slightly different atomic arrangements randomly interconvert at rates that increase with temperature
What are energy landscapes?
These landscapes for proteins are rough
Going along energy to the transition state the protein has to overcome many barriers and traps - this is why we can have slightly different conformational states
That native well still has some roughness and is therefore still dynamic
As temperature increases the thermal energy available to the protein increases, making it easier to jump between states (ps-ns)
The resulting dynamic behaviour of proteins makes some regions of proteins flexible which in turn is a major influence on activity
So at low temperatures proteins may not bind ligands tightly
