EPR and methologies Flashcards
What is EPR?
Electron Paramagnetic Resonance.
It is a spectroscopic technique that detects species that have UNPAIRED electrons.
What is ES(M) R ?
Electron Spin (Magnetic) Resonance
List some
Unpaired e- species:
1) Radicals
2) Transition metals
3) Defects in material
How does EPR work? Differences between
A spin of an electron gives it a magnetic Moment. A magnetic field has two orientations (+1/2 or -1/2)
In DIAmagnetic species all electrons paired and so overall it has NO SPIN
PARAmagnetic species have at least one UNPAIRED electron and so have spin. EPR deals with unpaired.
Where there is no magnetic fields, the e- have SAME ENERGY and so SAME number of UNPAIRED e- in both states (one + and - means no net spin) but where there is an magnetic field (B0), the e- don’t have the same energy
What is spectroscopy
The differene beteen the 2 energy levels (low energy -e- and high energy +e-.
E difference is proportional to the magnetic field; increase field, increases difference
The absolute energy difference between the high and low energy states is what determines the population distribution between the states.
No E difference in the absence of a field.
What is the Boltzmann distribution?
This determines the the ratio/proportion of e- in high and low states; the equation shows particles partitioning between energy states.
The higher the frequency, the greater SENSITIVITY to the technique used.
The population difference in EPR is greater than NMR , so EPR is more sensitive
Describe the difference between the two energy states.
If it orients itself parallel to the field it is in a low energy state(-1/2)
If it orients itself anti-parallel to the field it is in a high energy state (+1/2) It takes (greater) energy to turn a magnet around AGAINST the field,
What exactly is detected by the EPR spectrum?
At equilibrium, there is an small excess of number of spins in the LOW ENERGY state.
The state of resonance is detected: where the electron absorbs or emits the correct amount of electromagnetic radiation (at the correct frequency) in order to move between energy levels.
ie, the movement from low to high.
How can an electron can be brought into a state of resonance?
change in energy depends on magnetic field and so e- moves if:
1) fixed frequency and vary the field- CONTINUOUS WAVE (CW) technique; magnetic field increases E change only at fixed frequency a which point it will show an absorption peak
2) fixed field, vary the frequency- DOMAIN/PULSED technique
describe CW EPR type resonance spectrums
Spectrum : x axis magnetic field, y axis absorbance.
When y=zero this corresponds to absorbance peak maximum.
the gradient of the absorbance peak (derivative of the spectrum)
Electrons are shown to relax FASTER 9than protons
describe pulsed EPR type resonance spectrum
Resonance only
occurs when correct
frequency is obtained
Frequency applied as a pulse of short high energy microwaves.
instruments produce short enough pulses to excite about 100MHz of spectrum; the size of the pulse determines on how much of spectrum to excite.
However this technique is difficult, required frequencies are hard to obtain so CW is used.
In Pulsed ERP
Each e- is represented by a vector in either +z (low energy) or -z (high energy) the NET magnetization determines the direction of the magnetic field (B0)
If field is applied, the net magnetisation will turn 45 degrees, then 90 degrees at which point it will align with B1 direction.
At B1 net magnetisation, PHASE COHERENCE happens.
In pulsed EPR
To revert the B1 state to the equilibrium state through RELAXATION
T) spin-lattice relaxation (longitudinal) where z magnetization decreases.
Tii) spin-spin relaxation (transverse) where xy magnetisation and so increase in Z
Ti always LONGER than Tii
In Pulsed EPR
DEADTIME is the length of time for excess energy to DISSIPATE from the system before detection; the time in between initial pulse and measurement
Pulsed EPR relies on spin echo experiment; spin echo recovery sequence (90x-t-180y-t- echo)
1) apply 90 degree pulse (so z direction to y direction) and wait time (t) in which they go clockwise from y
2) apply pulse in 180 degree on the y axis so e- are flipped and wait or time in which they travel back o start point y.
Tii at this stage, spread out called dephasing
3) echo- when in phase (e- direction same a SIGNAL is seen
How is EPR different from NMR?
1) Uses gigahertz frequencies instead of megahertz
2) More sensitive (Boltzmann distribution)
3) Requires weaker magnets.
4) Has much broader lines due to much faster relaxation rate.
5) Pulsed EPR must be carried out at much lower temperature (50K cf 293K)
6) Pulsed EPR experiments are based on spin echo pulses
7) At the moment can not excite whole spectrum, but NMR can.
List the outline of instruments used in EPR
1) source- microwave bridge
2) sample (in cavity in magnetic field
3) detector (microwave bridge
4) spectrum, outputted to computer
The HIPER project instrumentation aims to produce no deadtime and so maximal sensitivity, and ins pulses that excite whole nitroxide spectrum (similar to NMR) with high powered field and power.
What are some uses of EPR?
1) structural biology - determines their PRIMARY SEQUENCES of nucleic acids and proteins which are polymeric chemicals
This is anylysed through the use of :
i) X-ray Crystallography
ii) NMR (size restriction)
iii) Small angle x-ray scattering (in solution)
iv) Circular dichroism
v) EPR
EPR requires an unpaired electron, most systems do not have this so the molecule of interest must be LABELED
Paramagnetic
Paramagnetism is a form of magnetism whereby certain materials are attracted by an externally applied magnetic field, and form internal, induced magnetic fields in the direction of the applied magnetic field.
Molecule that do not contain paramagnetic species are EPR slient.
Advantage: easier to interpret simple systems by spin labels. but disadvantage as there are fewer accessible systems
How does spin labeling work?
the reactivity is unique o a specific location in molecule (RNA/DNA/Protein) and posses a chemistry that suits its chemistry; the impact of the modification and its ability to be removed is also important.
Nitroxides
they have a STABLE UNPAIRED e- where higher density on the N-O bond.
It can be inactivated and made silent by reducing agents; vitamin C reduces radicals;so where N-O molecules are radical but where N-OH molecules are nonradical
Spin labeling in DNA
DNA oligomers (60 nucleotides) are modified AFTER they are made.
Synthesis incorporates a 5-iodo 2’deoxyuridine which can be controlled for selection
Post synthetic incorporation requires 2’-aminouridine
4 amino TEMPO converted to an isocyanate
Isocyanate reacted with purified DNA; this reaction (amine and isocyante very fast and specific)
Spin label linker still has rigid peptide bond character
Sonogashira reaction is a cross-coupling reaction
used in organic synthesis to form carbon–carbon bonds.
It is very specific and high yeilding. The spin label attached with RIDID LINKER
Spin labeling in proteins
The protein must be MUTATED.
MTSL agent is used for very specific condition to form a DISULPHIDE BOND with the protein.
The linker is flexible
Label itself is SMALL
How do the techniques determine the success of labeling
if it worked?
1) Mass spectroscopy
2) CW EPR
if the new label has effected the system of the molecule
1) Functional assay
2) Structural assay (looking at the melting point and folding of protein)
What is PELDOR (deer)
PELDOR - pulsed electron Double resonance (AKA deer)
EPR experiment that uses two different frequencies to control different spins in order to find out the strength of their coupling.
The accurate distance between the spins can then be determined from their coupling strength, which is used to study structures of large bio-molecules.
The coupling is dependent on angle and distance; frequency of the coupling that is measured.
SHORT DISTANCE, MEANS LARGE FREQUENCY
The echo osilcates on graph
the nucleosome core octamer by spin-labeling and pulsed EPR
The nucleosome has different forms; tetramer assembles into an octamer which becomes a nucleosome (an octamer of two types of histones.)
Cystines found 60 Angstroms apart however in different forms of histone assembly the oscillation in pulse graphs varies; the tetramer is less defined and less stable
The distance between cystines decreased to 25 Angstroms, frequency increases
What are histone chaperones?
They act to assemble the histones.
Asf1 is a chaperone
They are labeled approximately 44 Angstroms apart when in complex with a dimer
A supercoiled form is cleaved once to produce a nicked circle. It is cleaved twice which produces a linear form. How is it cleaved? How are these Dynamics shown through CW line-shape analysis
Endonuclease binds to the cylinder DNA in a 4 way junction. The last 16 residues at the N terminus are not shown in crystal structure but must have an important function in the CLEAVAGE.
By placing Cysteine residues along the first 16 amino acids of endonuclease we can examine how dynamic the peptide is, with and without DNA
CW spectra (peaks heights) show the spin label as less mobile when bound to the DNA ; acts as a control. S29C site far way from DNA binding region doesnt change if enzyme is bound to DNA
The distance between dimer related spin labels was measured attached and not attached to DNA.; in the presence o DNA, the distances decreases.
The tail residues act as around DNA and cleave up to twice
List three different types of spin labels
1) mobile
2) intermediate
3) immobile
cystine attached to MTSSL label shows it mobility hrough the different peak shapes
EPR and DNP
Dynamic nuclear polarization (DNP) can be used to transfer polarization from electronic spins to coupled nuclear spins.
Under optimal conditions, the polarization of the nuclear spins can be increased by the ratio between the electronic and nuclear resonance frequencies. (13C nuclear spins, this ratio is 2600.)
List other APPLICATIONS of EPR
1) Electron transport processes - It is important for respiration and photosynthesis.
2) Quantum computing
3) Dynamic nuclear polarisation
4) Local structure of proteins (important interactions and functions determined through ENDOR/ESEEM)
What is ENDOR?
Electron Nuclear DOuble Resonance.
Microwave and radio frequency applied fields produce HYBRID EPR and NMR.
Sample in field and radiation used to induce TRANSITIONS between spin states.
When there is saturation at a specific EPR transition there is a EQUAL population in each spin states.
The frequency of signal is applied to NMR to induce NUCLEAR spin transitions.
The resultant change in EPR signal represents the arrangement of NUCLEI around EPR active site.
What is ESEEM?
Electron Spin Echo Envelope Modulation
This is a magnetic resonance effect which can be observed in pulsed electron resonance experiments
Modulation arises in state of MIXING HYPERFINE LEVELS; the interference between pairs of EXCITED EPR transitions.
the spacing of the modulations show the degree of couple between the electron and the nucleus
EPR
Spin labels and their impact used to determine structural information about their molecule of interest.
Direct electron nuclear or electron-electron measurement can yield hi-res local information
