192 5.4 NMR and Other Radio Frequency and Microwave Resonance Spectroscopies
in the microwave range of ~10 GHz, with ESR spectrometers normally generating B-fields
of ~1 T or less.
Unpaired electrons are chemically unstable, associated with highly reactive species such
as free radicals. Such chemical species are short-lived, which limits the application of ESR,
though this can be used to an advantage in that standard solvents do not give rise to a meas
urable ESR signal; therefore, the relative strength of the signal from the actual sample above
this background solvent noise can be very high.
Site-directed spin labeling is a genetics technique that enables unpaired electron atom
labels (i.e., spin labels) to be introduced into a protein. This uses a genetics technique called
site-directed mutagenesis (discussed in more detail in Chapter 7). Here, specific labeling sites
in the DNA genetic code of that protein are introduced. Once incorporated into the pro
tein, a spin label’s motions are dictated by its local physical and chemical environment and
give a very sensitive metric of molecular in the vicinity of the label. A common spin label is
nitroxide, also known as “amine oxide” or “N-oxide,” which has a general chemical formula of
R3N+–O− where R is a substituent organic chemical group, which contains an unpaired elec
tron predominantly localized to the N–O bond, which has been used widely in the study of
the structure and dynamics of large biomolecules using ESR.
5.4.9 TERAHERTZ RADIATION APPLICATIONS AND SPECTROSCOPIES
Terahertz radiation (T-rays) occupies a region of the electromagnetic spectrum between
microwaves and infrared radiation, often referred to as the terahertz gap, where technolo
gies for its generation and measurement are still in development. The fastest existing digital
photon detectors have a bandwidth of a few tens of GHz, so the ~1011–1013 Hz characteristic
frequencies of terahertz radiation (corresponding to wavelengths of ~30–3000 μm) are too
high to be measured digitally but instead must be inferred indirectly, for example, by energy
absorption measurements sampled at lower frequencies. However, the energy involved in
transitions between different states in several fundamental biological processes has a charac
teristic equivalent resonance frequency in this terahertz range.
These include, for example, the collective vibrational motions of hydrogen-bonded nucleo
tide base pairs along the backbone of a DNA molecule, as well as many different molecular
conformational changes in proteins, especially with a very high sensitivity to water, which is a
useful metric for exposure of different molecular surfaces in a protein undergoing conform
ational changes. Terahertz spectroscopy, only developed at around the turn of the twentieth
century, has yet to emerge into mainstream use in addressing practical biological questions
(though for a good review of emerging applications, see Weightman, 2012). However, it has
significant future potential for investigating a variety of biological systems.
Terahertz spectroscopy typically utilizes a rapid pulsed Ti–sapphire laser for generation
of both terahertz radiation and detection. The laser output is in the near infrared; however,
the pulse widths of these NIR wave packets are ~10−13 to 10−14 s, implying a frequency range
of several terahertz, though centered on an NIR wavelength output of ~800 nm (~375 THz),
which thus needs to be downshifted by two orders of magnitude.
KEY POINT 5.2
A pulse of electromagnetic radiation is an example of a wave packet, and if it has a dur
ation of Δt, it can be deconstructed into several Fourier frequency components, which
span an angular frequency range Δω, which satisfies ΔωΔt ≈ 1. This can be viewed as a
classical wave equivalent of the quantum uncertainty principle. This implies the range
of frequencies in a wave packet Δυ ≈ 1/2πΔt. An alternative depiction of this is with
position x and wave vector k uncertainty, ΔkΔx ≈ 1, for example, superposing sev
eral waves of different wavelengths generates an interference pattern, which results in
increased spatial localization of this wave train.