X-ray
- crystals with many molecules
- density down to Angstrom resolution: high resolution structure
- b-factor (or Debye-Waller factor): reports on local atomic mobility
- large scale flexibility cannot be assessed
- main limitation: you need a crystal, which is particularly difficult for membrane proteins
The crystal structure is high res, but is the crystal structure (not physiological condition)!
-> really hard to determine for membrane proteins (are really hard to crystallize)
SAXS -> small angle X-ray scattering
- molecules studied under biological conditions
- global shape (envelope) of proteins/assemblies
- SAXS is biased towards extended structures!
Solution structure, but bad resolution, limited dynamics information
-> you can determine how fast the exchange between the two shapes is
.> you get kind of a grid (Gitter) that you can model a structure inside
Cryo EM (Electron Microscopy)
- imaging many shadows of single molecules in amorphous ice (vitreous ice) at low temperature with different orientations
- using a computer for calculation of the structure, so far not atomic resolution, but coming closer
- no dynamic information
Frozen structure, ensemble, medium/high resolution, limited dynamics information
-> again NO physiological conditions
NMR
- NOEs (nuclear Overhauser effect) provide information on distances between atoms (distance restraints)
- all restraints provide structure with resolution comparable to x-ray
- no NOEs between atoms belonging to different flexible domains
- domain dynamics can be assessed by transverse relaxation dispersion experiments, if chemical shift of atoms is changing as a result of domain motion
Solution structure, almost as detailed as X-Ray + dynamics information, still ensemble
-> physiological conditions
-> average structure
-> time consuming and expensive
SM FRET (single molecule)
ONE distance with 2-3 Angstrom resolution (for single molecules)
-> beyond structure biology
-> interesting for changes in protein structures
-> transfer of energy from excited donor to the acceptor (when they are in close proximity -> can happen because of spectral properties that only/mostly the donor gets excited
-> but noise is occurring due to the photon emission process (the less photons the lower the noise)
WHY? Distribution vs Average
Heterogeneity
STATIC: certain time -> individuals are different
DYNAMIC: ONE individual is changing some property in time
-> ensemble average = Ergodic = time average of one individual
Ensemble dynamics
Synchronization necessary
SM dynamics
Synchronization not necessary
SM force
tether -> spring -> pull
-> both directions possible (unfolding, folding)
Energy levels and photo physics
- Limits the number of photons per time interval
- Extremely important in dynamics experiments: accessible time scale
Jablonski diagram -> time scales
RISC = µs
ISC = µs
-> both need a spin flip which takes long because it is unprobable
IC =10 ns
Abfall der Energie = ps
fluorescence transmission = ns
phosphorenge transition = µs
Electronic transitions -> extremely fast (immediate)
Excitation rate (or the inverse) depends on PHOTON FLUX DENSITY (photons/area time)
R = average photon emission rate (photons per time)
Fluerescence quantum yield
-> tell us about the quality of the fluorophore
- percentage of Emissive transitions out of all excitations
Signal/Background
- General problem: fluorescence of a single dye is very weak
- needs to overcome background
- WE ARE COUNTING PHOTONS!
Background
- Laser induced (proportional to laser power):
- Scattering (instantaneous, scales with concentration of scatterers)
- Rayleigh (same wavelength as excitation)
- Raman (redshifted to excitation)
- Reflexion at interfaces (instantaneous)
- Background fluorescence (delayed)
- Background Without any excitation laser:
- Detector dark signal (thermally)
-> fluorescent contaminations in the solvent
Photo bleaching
- ähnlich wie bei einem Poster an der Wand bei dem die Farbe verblasst -> für ein Photon ein 1/0 Prozess (alive or dead)
Background reduction
- Excitation wavelength (absorption / filter)
- Use the best (and appropriate) filters!
- Temporal filtering (pulsed excitation)
- Detectors with low dark count rate
FRET
- via fluorescence, measure one distance as a function of time
- two fluorophores: donor is excited, energy is transferred to the acceptor of it is close to the donor
- distance turns into color:
-> long distance, low transfer, most light emitted from donor
-> short distance, high transfer, most light from the acceptor - fluorescence is very weak, some pW
- typically single photons are detected and counted
- photon counts are subject to shot noise, unavoidable
- noise is the most limiting problem in single molecule fluoresence
Quenching
- fluorescence quenching can also be used as a reporter on conformational changes (labelling with fluorophore and quencher)
Fluorogenic substrates, products or co factors
- to study single enzyme activity, substrates or products that fluoresce can be monitored
- co-factors of enzymes might also change their fluorescence properties during turnover
Force measurement
- molecule is held by tethers and pulled from one or both sides, until e.g. unfolding occurs
- the recorded force-distance curve provides information on the conformation energy landscape
