Single Molecule Studies Flashcards
(21 cards)
Are folding and chemical turnover governed by different physical chemistries?
No
Both governed by the Eyring equation
Can treat both as chemical reactions.
What is static disorder?
There being a distribution of rates across all enzymes
What is dynamic disorder?
When single enzymes can change their speed over time. Changing shape over time which can alter its ability to bind and alter substrates.
Will the same chemical reaction always proceed at the same rate?
No, there is a distribution of different energies etc that cause differing rates
Will the same enzyme reaction always proceed at the same rate?
No
No issues with orientation, but with energies
What is the Boltzmann (or Gibbs) distribution?
A probability distribution that gives the probability that a system will be in a certain state for a given energy and temperature.
Examples of direct single molecule detection
o FAD (cofactor) turnover; cholesterol oxidase
o Fluorescent substrates (e.g. NADH)
o Attach to coverslip, so they stay fixed, or track them as they move
Examples of indirect single molecule detection
o Molecular probes (FRET labels (fluorescence microscopy)
o Label free methods monitoring conformational change
Why bother with single molecule studies?
o Potentially accesses intrinsic rate constants.
o Identify rare or transient states
Intrinsic fluorophore detection
Fluorescence depends on whether FMD/FAD, or NAD(P)H are in their oxidised or reduced forms
Fluctuation in signal is associated with chemical change
Can indicate each successful redox.
FRET detection
Forster resonance energy transfer
Energy from one emissive species is taken up by another absorbative species
Fluorescence of one factor interact with another species
Links to enzyme tunnelling
Whispering gallery mode detection
Attach to a gold nanoparticle
Track shifts in absorbance spectrum
Can track changes in conformation
Force based methods for studying folding
The AFM tip is lowered to the protein and attached either specifically or non-specifically.
When the tip is retracted from the surface, the structural segments of the protein are unfolded.
Unfolding energies are calculated from the force constant associated with the ‘pulling’ of the AFM tip
How do unfolding energies appear on force-extension curves?
As sawtooth-like patterns
How limited are routes of enzyme turnover and protein folding?
Not very
Can take a bunch of different pathways and/or proceeding from different starting points.
How do we keep enzymes positioned under the microscope?
One method is to attach the enzyme to an anchoring protein or to encapsulate enzymes within virus molecules
Alternatively some microscope slides will have small wells
TIRF
Total internal reflection fluorescence microscopy
Illuminates only molecules close to the surface
When does Michaelis Menten become less useful?
When the number of substrate molecules is low
When the enzyme is switching between different forms a lot.
What effects are more important when Michaelis Menten becomes less useful?
Random fluctuations - stochastic effects
True or False
Protein folding has two stages: folded and unfolded
False
It has many intermediate states with individual energy barriers
Study of Galactosidase
Attached single enzyme molecules to beads and tethered the beads to a surface and monitored as the enzymes broke down the substrate.
At low substrate concentration the enzymes were predictable, but at higher concentrations the waiting times started to show more complicated patterns.
It became clear that there were multiple processes that were determining the rates.
