Pulsed EPR spectroscopy is a form of electron paramagnetic resonance spectroscopy that excites unpairedelectrons with short, strong microwave pulses and records how they respond overtime, instead of sweeping a weak, constant microwave field. This article is for protein scientists, structural biologists and drug discovery teams. It explains what pulsed EPR measures, how it differs from continuous-wave EPR, which experiments it can run, and how pulsed dipolar spectroscopy turns two spinlabels into a distance distribution.
What is pulsed EPR spectroscopy?
Pulsed EPR spectroscopy is amagnetic resonance method that applies nanosecond microwave pulses to unpairedelectron spins in a magnetic field and then detects the echo or signal thespins give off. The unpaired electrons can be native to the sample, such as ametal center or an organic radical, or added on purpose as spin labels, such asa nitroxide attached to a cysteine in a protein. Because the experimenter setsthe timing between pulses, each experiment can pick out one interaction at atime: the coupling to a second electron spin, the coupling to nearby nuclei, orhow fast the spins lose their alignment.
The method has grown quicklyover four decades. Van Doorslaer and Schweiger wrote in a 2000 review in Naturwissenschaften that pulse EPR applications were rare in the early 1980s and had become commonin chemistry, physics, materials science, biology and mineralogy, mainly because of better instruments and pulse-sequence design. For a reference-leveltreatment, see the article in Wiley's Encyclopedia of Analytical Chemistry. If you are new to the technique as awhole, start with our overview of electronparamagnetic resonance spectroscopy.
How does a pulsed EPR experiment work?
A pulsed EPR experiment tips theelectron spin magnetization with one microwave pulse, lets it evolve, and then refocuses it with later pulses so the spins produce a measurable echo. The basic two-pulse (Hahn echo) experiment runs like this:
1. The sample is frozen into a glass and held at acryogenic temperature, which slows relaxation enough for the signal to lastmicroseconds.
2. A first pulse rotates the spin magnetization by 90degrees, into the plane where the detector can see it.
3. During a delay, spins with slightly different resonancefrequencies drift apart and the signal fades.
4. A second pulse rotates the magnetization by 180degrees, so the spins move back together and form an echo after a second, equaldelay.
5. The spectrometer records the echo while one delay orpulse setting is stepped. Fitting or Fourier transforming that time trace gives a spectrum, a relaxation time, or a distance distribution.
More advanced experiments add pulses, or pulses at a second microwave frequency, to this basic echo.
Which frequency bands does pulsed EPR use?
Most pulsed EPR on proteins isrun at X-band and Q-band: X-band uses microwaves near 9.5 GHz with a magneticfield of about 0.33 T, and Q-band uses about 34 GHz with a field of about 1.2T. Q-band gives more signal from small sample volumes, which is why Schmidt andcolleagues chose it for their long-distanceDEER work in Angewandte Chemie (2016). X-band instruments are more commonand are the frequency many published protocols are written for.
What is the difference between CW and pulsed EPR?
The difference between CW andpulsed EPR is how the microwaves reach the sample: continuous-wave EPRirradiates it with a weak, constant microwave field while the magnetic field isswept, and pulsed EPR fires short, strong pulses at a fixed field and recordsthe spins' response over time. CW EPR gives the full absorption spectrum in onesweep and works at room temperature in liquid samples, so it is the standardway to check spin labeling and to study spin-label mobility. Pulsed EPRseparates interactions that overlap in a CW spectrum. Pulsed methods can untangle overlapping resonance lines, which are common in disordered protein samples.
Here is how the two methods compare:
• Microwave excitation: continuous-wave EPR uses a weak, constant field, and pulsed EPR uses short, high-power pulses lasting nanoseconds.
• What is recorded: CW EPR records field-sweptabsorption, usually as a first derivative, and pulsed EPR records echoes orfree induction decays as a function of time.
• Sample temperature: CW EPR runs from roomtemperature to cryogenic temperatures, and pulsed EPR usually runs at cryogenictemperatures in a frozen glass.
• Main information: CW EPR gives g values, hyperfine splittings, spin-label mobility and spin counts, and pulsed EPR givesdistance distributions, couplings to nearby nuclei and relaxation times.
• Distances between spins: CW EPR sees only shortdistances, through line broadening, and pulsed EPR measures about 1.5 to 6 nmin protonated samples and longer with deuteration.
• Typical first use: CW EPR is used to check labeling and dynamics, and pulsed EPR is used for structure and conformationalensembles.
Most labs use both. A quick CWspectrum confirms that a protein is labeled and the label is mobile, and a pulsed measurement then answers the structural question.
What is time-resolved EPR (trEPR)?
Time-resolved electronparamagnetic resonance (trEPR or TREPR) records the EPR signal as a function oftime after a laser flash, to follow short-lived species such as triplet statesand radical pairs. The classic version uses continuous microwaves with fastdirect detection and no field modulation, so it sits between CW and pulsed methods. Pulsed, echo-detected versions also exist. TREPR is mainly used inphotochemistry and photosynthesis research, where the spin polarization of thesignal shows how the excited state formed. It is rarely the method of choicefor protein distance measurements.
Which experiments can pulsed EPR perform?
Pulsed EPR can perform four mainkinds of experiments: distance measurements between electron spins,measurements of couplings to nearby nuclei, relaxation measurements, andecho-detected spectra. Prisner, Rohrer and MacMillan list ESEEM, PELDOR (DEER),relaxation time measurements, transient EPR, high-field EPR and pulsed ENDOR as the core pulsed methods used on biological samples in their 2001 Annual Reviewof Physical Chemistry article.
These are the pulsed EPR experiments protein scientists use most often, with what each one measures:
• Echo-detected field sweep: records the EPRspectrum through the echo, used to check the spin label and choose pulsepositions.
• Hahn echo decay: measures the phase memory time(Tm), which sets the longest usable DEER trace.
• Inversion or saturation recovery: measures thelongitudinal relaxation time (T1), used to choose temperature and repetitionrate.
• Two-pulse and three-pulse ESEEM: detects weakcouplings to nearby nuclei, used with deuterium to measure solvent exposure andmembrane depth.
• HYSCORE: maps nuclear couplings in twodimensions, used to identify ligands at metal sites.
• Pulsed ENDOR (Davies, Mims): measures hyperfinecouplings, used to study the electronic structure of radicals and metalcenters.
• DEER, RIDME, DQC and SIFTER: measure the dipolarcoupling between electron spins, used for distance distributions andconformational states.
What is ESEEM?
ESEEM (electron spin echoenvelope modulation) is a pulsed EPR experiment in which the echo intensityoscillates as the pulse spacing changes, because the electron spin is weakly coupled to nearby magnetic nuclei. A Fourier transform of the oscillations givesthe nuclear frequencies, which identify the nuclei close to the electron spin.In protein work, deuterium ESEEM on a nitroxide label in D2O shows how exposedthe label is to water, which helps place a residue inside or outside amembrane. HYSCORE spreads the same information over two dimensions, whichseparates overlapping signals at metal sites.
How does pulsed EPR measure relaxation?
Pulsed EPR measures relaxationby recording how an echo recovers or decays as a delay is lengthened: inversionrecovery or saturation recovery gives the longitudinal relaxation time T1, andHahn echo decay gives the phase memory time Tm. Tm matters most for distancework, because it limits how long a DEER trace can run and so sets the longestdistance you can measure. Hydrogen nuclei in the buffer and protein speed updephasing. Replacing them with deuterium lengthens Tm, which is why deuteratedbuffer and cryoprotectant are standard in DEER samples.
What is pulsed dipolar spectroscopy?
Pulsed dipolar spectroscopy is agroup of pulsed EPR experiments that measure the magnetic dipolar couplingbetween two or more electron spins and convert it into a distribution ofdistances between them. The coupling falls with the cube of the distance, sothe oscillation frequency in the time trace tells you how far apart the spinsare. The group includes DEER (also called PELDOR), RIDME, double-quantumcoherence (DQC) and SIFTER. The result is a distance distribution, not a singlenumber, so a flexible protein that samples several conformations shows severalpeaks. Our pulseddipolar spectroscopy whitepaper covers sample design, analysis and workedexamples in more depth.
What is DEER spectroscopy?
DEER spectroscopy (doubleelectron-electron resonance) is the pulsed dipolar experiment most labs use forproteins: it records an echo from one group of spins at one microwave frequencywhile a pump pulse at a second frequency flips the partner spins, so the echois modulated at the dipolar frequency between them. The four-pulse DEERsequence is the standard form because it records the start of the trace withoutinstrument dead time. Analysis removes the background from spins on othermolecules and then fits the remaining signal, usually with Tikhonovregularization or a model-based fit, to give the distance distribution P(r).
What distance range can DEER measure?
DEER measures distances fromabout 1.5 to 6 nm in typical protonated protein samples, and longer distancesafter deuteration. Schmidt and colleagues reported in Angewandte Chemie (2016)that DEER on protonated multimeric proteins is generally limited to 15 to 60 Å,and that sparse labeling, deuteration of protein and solvent, and Q-banddetection extended the trace to about 80 µs and resolved distances up to 160 Åin the GroEL tetradecamer. The upper limit in any one sample depends on tracelength, which depends on Tm, spin concentration and the degree of deuteration.
What samples does pulsed dipolarspectroscopy need?
Pulsed dipolar spectroscopyneeds a molecule carrying two or more spin labels, frozen into a glass so themolecules cannot tumble. The most common label for proteins is a nitroxide suchas MTSL on an engineered cysteine, which forms the R1 side chain. Other optionsinclude spin-labeled unnatural amino acids, Cu(II) and Gd(III) complexes, andtrityl radicals. The buffer usually contains a cryoprotectant such as glycerolso the sample forms a glass, and both should be deuterated where the budget andsample allow.
For a typical DEER measurementon FATHOM, High Q Technologies recommends 20 to 30 µL of sample at 50 µM, whichcovers duplicate or triplicate runs. Each position in the five-sample SQBAcartridge takes 3.5 µL. Nitroxide samples are measured at 50 K, and at 50 µMmost samples reach a signal-to-noise ratio above 50 within 30 to 180 minutesafter a 30-minute automated calibration, depending on the distance.
How can you run pulsed dipolarspectroscopy without an EPR lab?
You can run pulsed dipolarspectroscopy without an EPR lab by sending samples to a measurement service orcore facility, or by installing an automated instrument that does not need anEPR specialist to operate. High Q's SampleMeasurement Service measures your samples on FATHOM and returns a distancedistribution with a sample characterization report for each sample. FATHOM itself is an X-band pulsed EPR spectrometer with a superconducting resonatorand a closed-cycle cryostat, so the only cryogen users handle is the liquidnitrogen used to freeze the sample. For aqueous samples it measures distancesof 1.8 to 6.0 nm reliably, and the longest distance measured on FATHOM so faris 7.5 nm, on a model biradical in a perdeuterated glass. For the structural questions these measurements answer in membrane proteins, see how EPR reveals membrane protein structure and dynamics.
Key takeaways
• Pulsed EPR spectroscopy uses short microwave pulses andecho detection to measure unpaired electron spins one interaction at a time.
• Continuous-wave EPR is the quick check for labeling and mobility, and pulsed EPR answers distance and structure questions.
• Pulsed EPR experiments fall into four groups:electron-electron distances, couplings to nuclei (ESEEM, HYSCORE, ENDOR),relaxation, and echo-detected spectra.
• Pulsed dipolar spectroscopy, mainly DEER, gives adistance distribution between two spin labels, which shows conformationalensembles.
• DEER covers about 1.5 to 6 nm in protonated samples,and deuteration has extended it to 16 nm in published work.
• Phase memory time (Tm) limits the DEER trace, sodeuterated buffer and cryoprotectant are standard.
Frequently asked questions
What is pulsed EPR?
Pulsed EPR is a type of electronparamagnetic resonance that excites unpaired electron spins with short, strongmicrowave pulses and records the echo they produce, instead of sweeping a weakcontinuous field. By changing the timing between pulses, it isolates singleinteractions such as electron-electron distances, couplings to nearby nucleiand relaxation times. Protein scientists use it mainly for distancemeasurements between spin labels, which run on instruments such as High Q'sFATHOM.
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What is the difference between CW andpulsed EPR?
CW EPR applies a weak, constantmicrowave field and sweeps the magnetic field to record an absorption spectrum,while pulsed EPR applies short, strong pulses at a fixed field and records thespins' response over time. CW works at room temperature and is the quick checkfor labeling and spin-label mobility. Pulsed EPR usually needs frozen samplesand separates overlapping interactions, which makes it the method for distancedistributions and nuclear couplings.
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Which experiments can pulsed EPR perform?
Pulsed EPR can perform distancemeasurements between electron spins (DEER, RIDME, DQC, SIFTER), measurements ofcouplings to nearby nuclei (ESEEM, HYSCORE, pulsed ENDOR), relaxationmeasurements (inversion recovery, saturation recovery, Hahn echo decay) and echo-detected field-swept spectra. In protein science, DEER is the mainapplication because it gives distance distributions between two spin labels.Relaxation measurements support it by setting how long the DEER trace can be.
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What is pulsed dipolar spectroscopy?
Pulsed dipolar spectroscopy is aset of pulsed EPR experiments that measure the dipolar coupling betweenelectron spins and convert it into a distance distribution. DEER is the best-known example. In protonated protein samples it typically covers about 1.5to 6 nm, and Schmidt and colleagues reported distances up to 160 Å afterdeuteration (Angewandte Chemie, 2016). The distribution shows everyconformation the labeled sites sample, not one average structure.
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