Spectroscopic Methods and Analyses: NMR, Mass Spectrometry, and Metalloprotein Techniques (Methods in Molecular Biology)

389 Pages·1993·20.32 MB·English

by  Christopher Jones

CHAPTER 1
Introduction
to Nuclear Magnetic Resonance
Christopher Jones and Barbara Mulloy
1. Introduction
This brief guide is not intended as a full explanation of the theory
and practice of nuclear magnetic resonance (NMR), on which there are
a large number of excellent texts (I-3), but as an introduction to the
terms used in the subsequent chapters. The section as a whole does not
provide a comprehensive outline of the NMR of organic compounds,
which would be out of place in this volume, but is a selection of
particular applications likely to be of use to molecular biologists and
biochemists. Over the last few years, the number of publications deal-
ing with NMR determinations of protein and peptide conformation in
solution has increased dramatically, and this is reflected in the amount
of space given here to the subject in Chapters 2 and 3. The use of NMR
m the study of internal mobility in proteins and in interactions between
molecules is covered in Chapter 7. Chapters 5 and 6 deal with struc-
tural studies on complex carbohydrates, which have thrived on recent
advances in NMR. Nucleic acids and their interactions are covered in
Chapter 4.
2. Basics of NMR
When the sample is placed in a magnetic field, the nuclei of some
of its constituent atoms (usually ‘H, but r3C, 15N, 19F,3 1P,a nd 2H are
also commonly encountered in biomedical research) are forced into
From Methods m Molecular &o/ogy, Vol 17 Spectroscopic Methods and Analyses
NMR, Mass Spectromefry, and Mefalloprotem Techmques Ed&d by C Jones, B Mulloy,
and A H Thomas Copynght 01993 Humana Press Inc , Totowa, NJ
1
2 Jones and Mulloy
alignment with the field. In this state, the absorption or emission of
electromagnetic radiation with a suitable, resonant frequency becomes
possible. The frequency of the absorbed energy is directly propor-
tional to the strength of the magnetic field, so the resonance condition
can be achieved either by scanning the frequency of the electromag-
netic radiation at constant field (as effectively happens in modern
Fourier transform [FT] spectrometers) or by scanning the magnetic
field at a constant irradiation frequency (as usually happened in older
continuous-wave [CW] instruments). The nomenclature of NMR is
complicated by the fact that both options are enshrined in the terminoi-
ogy independently of the experimental setup used.
3. Fourier Transform NMR
The older CW instruments utilized a monochromatic irradiation
frequency and observed an absorption spectrum, whereas FT machines
use a broad-band pulse of radiation to equalize the populations of the
high- and low-energy states and then observe an emission spectrum.
This pulse methodology has proven extremely powerful in its practi-
cal application, and the subsequent chapters assume that such instru-
ments are available.
A significant advantage of the FT method is that, since the whole
spectrum is acquired in a few seconds following a single pulse, data
from many such acquisitions may be added together to give much
improved signal-to-noise ratios and sensitivity. Without this advan-
tage, the use of relatively insensitive nuclei, such as i3C, in studies of
biological samples would be impossible.
4. The 1D Spectrum
The NMR peak as usually seeni n the one-dimensional (1D) spectrum
can be characterized by four basic parameters: the frequency (or field) at
which resonance occurs, the intensity of the peak, couplings to other
nuclei as revealed by the multiplicity of the peak, and a series of para-
meters, such as linewidth, based on relaxation behavior (seeF ig. 1).
4.1. Chemical Shift
The resonance frequency is usually quoted as the difference, in parts
per million, from that of a reference standard arbitrarily set to 0 ppm.
The advantage of this scale is that it is Independent of the base oper-
Introduction to NMR 3
-B)-a-ManNAc-(l-OPO,-
;OAc
Chmkal ShlH (ppm)
B
Spin-spin coupling
4’5 -‘---- Chemical shift
Fig. 1 Four parameters that can be measured from the ID NMR spectrum of a
brological macromolecule (A) Expansions of the 500-MHz NMR spectrum of the
menmgococcal type A polysaccharrde (recorded at 343 K = 70°C). (B) A further
expansion of the H2 doublet. The frequency at which resonance occurs is the chemi-
cal shift; the intensity of the peak 1s usually measured by mtegratron, the multrplic-
ity of the srgnal reflects spin-spin coupling, and the drfference between the
frequencies of the two lures gives the spin-spin coupling constant; the lmewrdth IS
related to the rate at whrch the nucleus relaxes from Its exerted state
4 Jones and Mulloy
LOW FREQUENCY HIGH FRECUENCY
LOW FIELD HIGH FIELD
DESHIELDED SHIELDED
TMS
reeonance
+ .
I
10 ppm 0 mm
498.8e5,ooo Hz Reeonance frequency 500.000,000 Hz
Fig. 2. The chemical shift scale for protons in a 500-MHz spectrometer (in which
the magnetic field is 11.744 T). The frequency of the tetramethylsilane (TMS)
resonance is taken as an arbitrary reference point, and other frequencies are expressed
in terms of parts per million (ppm) of this frequency.
ating frequency of the instrument (i.e., the strength of the field pro-
duced by the magnet), and is equally applicable to field or frequency
scanning. The primary standard chosen for ‘H work, trimethylsilane,
resonates at higher frequency than most other nuclei, and the scale
runs in the opposite direction to increasing frequency (or field). The
ends of the spectrum are often referred to as high field (or high fre-
quency) or low field (or frequency). Individual ‘H nuclei resonate at
different frequencies, becauset hey are shielded from the applied mag-
netic field by the electrons around them. Thus, an effect that moves a
resonance to low field corresponds to an additional deshielding of the
nucleus, and movement to high field is a shielding (see Fig. 2). Aro-
matic systems with a cyclic x-electron system generate a magnetic
Introduction to NMR 5
field of their own when placed in an external field, which affects the
chemical shift of nearby nuclei in a manner that depends on the geom-
etry of the system, This is referred to as ring-current anisotropy.
In a given magnetic field, different types of nuclei resonate at dif-
ferent frequencies, and instruments are usually described by therr pro-
ton frequency (e.g., a500-MHz instrument). In such an instrument 13C
nuclei resonate near 125 MHz and 31Pn uclei near 202 MHz. Thus, the
individual spectra arew idely separatedi n frequency. The typical widths
of the spectra of different elements can be very different, too: Most of
the ‘H spectrum 1sb etween 0 and 10 ppm, whereas the r3C spectrum
occupies 200 ppm. The chemical shift of a resonance is dependent
primarily on its local chemical environment and less critically on
geometric factors.
4.2. Intensity
With reasonable care in the choice of experimental conditions, the
intensity (integral) of a resonance is proportional to the number of
nuclei contributing to it. Thus, integration of parts of the spectrum can
be used to show how many of each type of nucleus are present within
a complex molecule, or to quantify the amounts of two or more distinct
chemical entities.
4.3. Coupling
A single resonance may be split into several separate lines by the
influence of the spins of nearby nuclei. This interaction, called J- or
scalar-coupling, is small and independent of the applied magnetic
field (and so is quoted in Hertz), and operates throughchemical bonds.
The magnitude of the splitting depends on the number of chemical
bonds involved and the geometry of the interaction.
The most commonly measured and used coupling constant is that
between ‘H nuclei separated by three bonds (written as 3.1r.ruw) hich
depends on the dihedral angle about the central (usually C-C) bond.
This relationship can be fitted to an equation, called the Karplus curve,
relating the coupling (typically l-10 Hz) to the dihedral angle between
the two C-H bonds, although the coefficients depend on the nature of
other substituents on the carbon skeleton. An example, showing a
Karplus curve characterized for the fragment H-N-Ca-H in peptides,
is shown in Fig. 3 (4). Because the difference in energy caused by this
6 Jones and Mulloy
3Jc~m,~~ = 2 17 - 127COd + 5 41COS%
Fig. 3 The Karplus relatlonshlp between the three-bond proton-proton coupling
constant (3JH,H) between the a and NH protons of ammo acids in peptldes, and the
dihedral angle between the C-H and N-H bonds as characterized m ref. 4 The
curve IS symmetrical about 180”
coupling is small, the populations of the two levels are only slrghtly
different. Thus, the two peaks in a resonance (split into a doublet) have
almost identical intensities, although the situation may appear more
complex if a resonance is coupled to more than one other nucleus.
Conventionally, the signal arising from a particular type of nucleus is
usually described as a single resonance even if it is split by scalar
coupling.
When two coupled resonances have very similar chemical shifts,
multiplets become distorted, and it is no longer possible to measure
coupling constants directly from the spectrum. This is “strong cou-
plmg.” Spectroscopists use an alphabetical convention to describe
systems of coupled spms, in which adjacent letters in the alphabet
Introduction to NMR
denote strong couplmg (an AB system has two strongly coupled spins)
and distant letters in the alphabet denote weak coupling (an AMX
system has three weakly coupled spins). 13Cs pectra would be greatly
complicated by coupling to protons, except for the fact that they are
usually recorded with some form of broad-band irradiation of the fre-
quencies absorbed by protons, which effectively decouples the proton
and carbon nuclei.
4.4. Relaxation
The equalization of the population of the nuclear spin states caused
by the RF pulse (in modern instruments) creates a high-energy system
that relaxes back to thermal eqmlibrium by a varrety of mechanisms,
and analysis of the relaxation rates and pathways provides a great deal
of information about the geometry and dynamics of the system. The
characteristic rates (I?,, R2) of relaxation resulting from these mecha-
nisms, or their reciprocal relaxation times (T, = l/R,, T2 = 1/R2), are
important not only as data, but because their values determine optimal
conditions for the acquisition of spectra. There are many NMR experi-
ments for which it is important that a delay between pulses is incorpo-
rated sufficient to give effectively full relaxation. The spin-lattice (or
longitudmal) relaxation time (T,) cannot be deduced from a simple 1D
spectrum, but must be measured in a separate experiment, usually the
inversion recovery experiment. The spin-spin (or transverse) relaxation
time (T,) can be estimated from the width of lines in the 1D spectrum
or more accurately measured by special experiments, usually basedo n
the “spin-echo” experiment. Thesee xperimentsa red escribed m ref. 2.
The nuclear Overhauser enhancement (NOE) is also a relaxation
phenomenon. Nuclei close to each other in space transfer energy to
each other during relaxation, and the extent of this transfer is related
to the distance between the nuclei. The NOE from protons to then
attached carbons is conveniently exploited to increase the intensity of
the r3C spectrum, by u-radiating the sample at a frequency absorbed by
protons while the 13Cs pectrum IS accumulated (a routine measure in
any case to remove multiplicity in the carbon spectrum owing to J-
coupling between protons and r3C). The NOE between two protons
can be used to estimate the drstance between them and 1s of great
importance m the determination of three-dimensional structures of
brologrcal macromolecules (see Chapter 2).
8 Jones and Mulloy
All theser elaxation parametersa res trongly influenced by the mobility
in solution and, hence,t he molecular size of the compound of interest. For
large molecules, TI and T2a rer educed, andt he interproton NOES become
negative in sign. The magnitude of all threeo f theser elaxation effects can
be expressed in terms of the correlation time (Q, which is itself a charac-
teristic of the rate of random reorientation of a molecule in solution.
In NMR studies of biological molecules, it is usually assumed that
relaxation by a single mechanism, known as dipolar relaxation, takes
place between directly bonded nuclei with magnetic spins. There are
other relaxation mechanisms that become important m specific cir-
cumstances, for example, relaxation via a paramagnetic nucleus (see
Chapter 2, Section 7.).
5. Transfer of Magnetization
Transfer of magnetization from one resonance in the spectrum to
another may be the result of other mechanisms than the NOE. If a
nucleus is involved in a chemical reaction while it is excited, it will
take its remaining magnetization with it to its new environment. This
mechanism, known as chemical exchange, can be used to study the
reaction concerned (see Chapter 7). In systems where both NOES and
chemical exchange are taking place, it can be difficult to tell them
apart without the use of elaborate two-dimensional (2D) techniques.
For experiments of this kind, it is necessary to irradiate one reso-
nance and observe the results elsewhere in the spectrum. FT spectrom-
eters use a very short, intense pulse of RF radiation (a “hard pulse”)
that has a bandwidth (related to the reciprocal of the pulse length)
considerably wider than the spectral width. Continuous irradiation at
the desired frequency can be used to saturate a smgle resonance. This
is the method used in saturation transfer experiments and sometimes
for measurement of NOES. For some experiments, however, it is nec-
essary to deliver a pulse to an individual resonance, for example, where
selective inversion (of the spin of a particular nucleus) is required.
This can be done using a relatively long, low-power pulse (a “soft
pulse”) that gives excitation of a narrow bandwidth, but is otherwise
identical to the “hard pulse.“The selectivity of the pulse can be further
improved (5,6) if, instead of a square pulse in which the transmitter is
switched on at the final power, the pulse is “shaped” as a Gaussian or
half-Gaussian (Fig. 4).
Introohction to NMR 9
-
Fig 4. Shaped pulses. (A) A simple square pulse, a long, “soft” pulse (1 e , of
low power) with this shape will excite one part of the spectrum selectively, but ~111
cause artifacts (B) A Gaussian pulse will be as selective as the square pulse and
avoid some of the artifacts There are many other possible shapes for pulses
6.2D NMR Experiments
Modem spectrometersu sing pulse methods do not record the spectrum
directly, but rather use an mterferogram of magnetization vs time, which
is digitized and Fourier transformed to a spectrum of magnetization vs
frequency (Fig. 5). Excitation of the sample need not be by a single pulse,
and multipulse sequencesh ave been introduced to give a wide variety of
informative experiments, including the 2D methods.
If a delay between the pulses is introduced and a series of spectra are
collected at various values of this delay, a second FT of intensity vs
incremented delay generates a second frequency axis. This is the basis
of 2D NMR. The spectra are usually plotted as a contour map with
intensity as the z axis. The most common series of 2D spectra has the
original spectrum occupying the diagonal (frequency 1 = frequency 2)
and a number of off-diagonal peaks with frequency coordinates con-
necting two peaks in the original spectrum, The position and intensity
of these peaks generate additional information and extend the power
of the NMR methods. Chapter 2 in this section gives a more detailed
account of the principles behind 2D NMR spectroscopy, particularly
of 2D NOE spectroscopy or NOESY, here we give an overview of the
range of methods available and the information they provide.
1D NMR spectra are usually recorded in the “phase-sensitive” mode,
which is to say that the real and imaginary data points resulting from
Jones and Mulloy
Free lnductfon decay (FID)
Fourier transform F(w) = lm f(t)ezp( -wt)dt
J-m
I
B
Spectrum
,I ” I ,, & ” * * m
Frequency (w)
Fig 5 (A) An mterferogram of magnetlzatlon vs time (or free mductlon decay
[FID]) recorded on a pulse FT NMR spectrometer This IS drgltahzed and Fourier
transformed to give (B) a spectrum of magnetization vs frequency (m this case a
13C spectrum)