EPR and methologies

Question 1

What is EPR?

Answer 1

Electron Paramagnetic Resonance.

It is a spectroscopic technique that detects species that have UNPAIRED electrons.

Question 2

What is ES(M) R ?

Answer 2

Electron Spin (Magnetic) Resonance

Question 3

List some

Unpaired e- species:

Answer 3

1) Radicals
2) Transition metals
3) Defects in material

Question 4

How does EPR work? Differences between

Answer 4

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

Question 5

What is spectroscopy

Answer 5

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.

Question 6

What is the Boltzmann distribution?

Answer 6

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

Question 7

Describe the difference between the two energy states.

Answer 7

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,

Question 8

What exactly is detected by the EPR spectrum?

Answer 8

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.

Question 9

How can an electron can be brought into a state of resonance?

Answer 9

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

Question 10

describe CW EPR type resonance spectrums

Answer 10

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

Question 11

describe pulsed EPR type resonance spectrum

Answer 11

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.

Question 12

In Pulsed ERP

Answer 12

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.

Question 13

In pulsed EPR

Answer 13

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

Question 14

In Pulsed EPR

Answer 14

DEADTIME is the length of time for excess energy to DISSIPATE from the system before detection; the time in between initial pulse and measurement

Question 15

Pulsed EPR relies on spin echo experiment; spin echo recovery sequence (90x-t-180y-t- echo)

Answer 15

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

Question 16

How is EPR different from NMR?

Answer 16

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.

Question 17

List the outline of instruments used in EPR

Answer 17

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.

Question 18

What are some uses of EPR?

Answer 18

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

Question 19

Paramagnetic

Answer 19

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

Question 20

How does spin labeling work?

Answer 20

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.

Question 21

Nitroxides

Answer 21

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

Question 22

Spin labeling in DNA

Answer 22

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

Question 23

Sonogashira reaction is a cross-coupling reaction

Answer 23

used in organic synthesis to form carbon–carbon bonds.

It is very specific and high yeilding. The spin label attached with RIDID LINKER

Question 24

Spin labeling in proteins

Answer 24

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

Question 25

How do the techniques determine the success of labeling

Answer 25

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)

Question 26

What is PELDOR (deer)

Answer 26

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

Question 27

the nucleosome core octamer by spin-labeling and pulsed EPR

Answer 27

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

Question 28

What are histone chaperones?

Answer 28

They act to assemble the histones.

Asf1 is a chaperone

They are labeled approximately 44 Angstroms apart when in complex with a dimer

Question 29

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

Answer 29

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

Question 30

List three different types of spin labels

Answer 30

1) mobile
2) intermediate
3) immobile

cystine attached to MTSSL label shows it mobility hrough the different peak shapes

Question 31

EPR and DNP

Answer 31

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.)

Question 32

List other APPLICATIONS of EPR

Answer 32

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)

Question 33

What is ENDOR?

Answer 33

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.

Question 34

What is ESEEM?

Answer 34

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

Question 35

EPR

Answer 35

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