Experimental structure
measurement
Nuclear Magnetic Resonance
Nuclear Magnetic Resonance
Physics of NMR
Many nuclei have nonzero spin S
The spin leads to a magnetic moment
µ = γS
! is the gyromagnetic ratio, which varies from nucleus to nucleus
Isotope
Spin ( )
! (107 rad/s per T)
1H
1/2
26.75
2H
1
4.1
12C
0
N/A
6.73
13C
1/2
14N
1
?
15N
1/2
-2.71
16O
0
N/A
17O
5/2
?
19F
1/2
25.18
31P
1/2
10.84
32S
0
N/A
Nuclear Magnetic Resonance
In the absence of external field, µ’s point randomly.
In the presence of a field B, there is an interaction energy –µ!B which tend to align " with B.
Assuming B points up, this increases the numbers of µ’s pointing up (nu) at the expense of those pointing down (nd)
The energy involved is small compared to kBT, so the net magnetization is small:
nu − nd ∝
eµB/kB T − e−µB/kB T
= tanh (µB/kB T ) ∼ µB/kB T
eµB/kB T + e−µB/kB T
typically "B ~10-5 kJ/mole (at B = 1 Tesla) so "B/kBT~10-6
The net magnetization not aligned with B0 will rotate around B0 at the Larmor frequency
ν = γB/2π Hz
Every unique ! has its own unique #.
NMR is just a measurement of how much of each ! is present in a sample.
Since the #’s are often very close together, sophisticated tricks may be needed to resolve them.
Sometimes you excite one # and measure another to probe the coupling between them.
Like in X-ray crystallography, determining the structure of a protein from raw NMR data is a non-trivial task.
This is a very expensive, low-sensitivity way to determine the elemental composition of a sample.
(but it’s used in extreme places like at the bottom of oil wells)
Nuclear Magnetic Resonance
The NMR signal
As the magnetization rotates, it can be detected in a pickup coil.
Magnetize the sample and then
tip the magnetization 90° to get a “large” net magnetization transverse to B0
7'%()%*+,"8./01*"93%+4'0
7'%()%*+,"8./01*"93%+4'0
!"#$%"&'%()%*+,"-./01*"23%+4'0"'%0-15,"'%3.'4"4$%"&'%()%*+1%2"&.'"1*-161-)05"231*2"
This is called free
induction decay;
its signal looks like:
!"#$%"&'%()%*+,"-./01*"23%+4'0"'%0-15,"'%3.'4"4$%"&'%()%*+1%2"&.'"1*-161-)05"231*2"
single #:
7#
7#
power spectrum
a few #’s:
7#
7#
7#
many #’s:
7#
The “amount” of signal at frequency # is proportion to the number of nuclei with Larmor
frequency #.
Signal ! nnet # ! B2. NMR is usually signal limited, so NMR uses the largest possible B0.
Nuclear Magnetic Resonance
NMR and chemistry
The effective Blocal is slightly altered from the externally applied B0 by local shielding
Therefore the observed #=!Blocal/2! is different from the the baseline #0=!B0/2!
Blocal is usually lower than B0, but not always.
The amount of shielding varies with the local chemical environment
This is what makes NMR structure determination possible.
Protons in different bonds have marginally different Larmor frequencies.
The shift in resonance is small: usually several ppm (a few x 10-6)
Protons are the nuclei of choice because they have the highest !.
Unconvincing classical
explanation of shielding
here as a mnemonic device
Nuclear Magnetic Resonance
Clever pulse sequences can detect particular #–# couplings
A short pulse sets all µ’s rotating
Equivalent to the impulse response.
A pulse at a particular frequency sets only that particular # rotating.
Equivalent to a forced harmonic resonator.
2D NMR uses variable time lags between pulses to probe coupling between spins
Usually along peptide backbone, but sometimes through hydrogen bonds
Dipole-dipole interaction between spins transfers spin polarization from one nucleus to another
This is called the Nuclear Overhauser Effect (NOE)
Like FRET, efficiency ~ 1/(1+(R/R0)6), so this is a sensitive distance ruler for R ~ R0 ~ 5 Å.
We will talk about the FRET distance dependence in a few weeks.
Two peaks “talk” to each other only if they are close. They are either
close in sequence (coupling along backbone), or
close in physical space (coupling through NOE or similar effect)
A measurement of nonlocal interactions.
This is the key to figuring out structure.
Not so useful if you already
have the aa sequence
Nuclear Magnetic Resonance
COSY: COrrelation SpectroscopY
The existence of a cross peak tells you that two H are coupled.
The COSY spectrum for isopentyl acetate
The COSY spectrum for a protein is much more complicated
Nuclear Magnetic Resonance
Solving the protein structure
Start from
(1) Covalent sequence
usually most easily obtained some other way
(2) Steric hindrance rules
knowledge of size of residues and allowed dihedral angles along backbone
(3) List of which residues are “close” to each other
NMR coupling data gives a range for H-H distances, but not usually a firm number.
Construct the 3D structure that is “most” consistent with (1)-(3).
Conceptually, this is quite similar to the X-ray crystallography problem.
Since this is a multidimensional minimization, picking a reasonable starting point is often critical to success.
Optimization routine often starts from homologous known structure.
For ab initio structure determination, you need very high quality data
Lots of protein: 1/2 ml @ 1 mM
Lots of time: days of data acquisition to discriminate between similar chemical shifts
Unless protein is small (less than about 10 kDalton) unique solution can be hard to find because there’s just too
much overlap between spectra.
NMR often gives several conformations that are almost equally good at satisfying (1)-(3).
Unclear which of these is “the” structure, or whether several conformations exist simultaneously.
Some people use the lowest energy (best figure of merit) structure
Some people use a spatial average of all the good structures
Nuclear Magnetic Resonance
Variations on structure determination with NMR.
You can use less sample / shorter times if all you care about is following a couple of peaks
from a particularly strong NMR signal.
711
Microscale NMR
Andrew M Wolters, Dimuthu A Jayawickrama and Jonathan V Sweedler*
Single-cell NMR:
Analytical techniques
Figure 1
Figure 2
(a)
Betaine
Helmholtz coil
Microcoil 600 MHz 1H-NMR spectra obtained
from two single A. californica neurons in 8 min
with 256 scans. The major peaks of choline
and betaine, which function as osmolytes, are
observed. Reproduced from [32••] with
permission of Wiley–Liss, Inc., a subsidiary of
John Wiley and Sons, Inc., © 2000.
1.0
0.8
0.8
0.6
NMR microcoil
SI
NMR spectroscopy is increasingly being used to characterize
microliter and smaller-volume samples. Substances at
picomole levels have been identified using NMR
spectrometers equipped with microcoil-based probes. NMR
probes that incorporate multiple sample chambers enable
higher-throughput NMR experiments. Hyphenation of capillaryscale separations and microcoil NMR has also decreased
analysis time of mixtures. For example, capillary
isotachophoresis/NMR allows the highest mass sensitivity
nanoliter-volume flow cells to be used with low microliter
volume samples because isotachophoresis concentrates the
microliter volume sample into the nanoliter volume NMR
detection probe. In addition, the diagnostic capabilities of
NMR spectroscopy allow the physico-chemical aspects of a
capillary separation process to be characterized on-line.
Because of such advances, the application of NMR to smaller
samples continues to grow.
712
Addresses
Department of Chemistry and the Beckman Institute,
University of Illinois, Urbana, Illinois 61801, USA
*e-mail: [email protected]
(b)
Choline
0.4
0.4
0.2
Current Opinion in Chemical Biology 2002, 6:711–716
0.2
0.0
1367-5931/02/$ — see front matter
© 2002 Elsevier Science Ltd. All rights reserved.
Abbreviations
CE
capillary electrophoresis
CEC
capillary electrochromatography
cITP
capillary isotachophoresis
FWHM full-width at half-maximum
0.6
0.0
4.5
4.0
3.5
3.0
2.5
4.7
ppm
Introduction
Individual-cell studies and single combinatorial bead
analyses demonstrate that measurement techniques must
be refined to successfully scrutinize minute sample quantities.
Both sensitivity and sampling become critical in the smallvolume, mass-limited regime, particularly when full
structural identification is required. NMR spectroscopy
can provide structural information unattainable by other
techniques. However, the relatively low sensitivity of
NMR often precludes its application to such mass-limited
samples. Considerable attention has consequently focused
on means to enhance NMR sensitivity. As NMR sensitivity
scales to the 7/4 power of the magnetic field strength [1],
the primary strategy has concentrated on increasing field
strength. Unfortunately, the development of high-field
magnets possessing both excellent field homogeneity and
stability has proven to be difficult and expensive.
Therefore, alternative avenues to improve NMR sensitivity
such as exotic polarization transfer schemes [2–4] and
cryogenic probes [5,6] have been explored.
One particularly attractive approach to enhance NMR
sensitivity for small-volume, mass-limited samples uses
Helmholtz NMR coil. (a) A standard Helmholtz NMR coil (6.7 mm
diameter) designed for 5 mm NMR tubes (~220 µl observe volume)
and an NMR microcoil wrapped around 200/360 µm inner
diameter/outer diameter capillary (~30 nl observe volume).
(b) Enlarged view of microcoil with US penny in background.
(Courtesy of MRM Corp. Savoy, IL).
reduced-diameter NMR coils (see Figure 1). As predicted
theoretically [1,7] and verified experimentally [7,8], the
mass sensitivity, defined as signal-to-noise (S/N) per sample
quantity, of an NMR coil depends inversely on its diameter.
Further gains in sensitivity can be achieved by optimizing
coil geometry. Conventional NMR probes typically employ
coils possessing Helmholtz geometry (a saddle-shaped coil)
to facilitate easy loading of samples contained within glass
tubes (Figure 1a). However, for a given coil diameter,
solenoidal coils exhibit a several-fold enhancement in NMR
sensitivity compared with that of Helmholtz coils [1].
External loading (probe outside of magnet bore) or flow
injection can be used for sample introduction into solenoidal
coils. Because of the different advantages afforded by the
two coil geometries, the specific application determines the
best choice for the NMR probe.
4.2
3.7
3.2
2.7
ppm
Wolters, Jayawickrama and Sweedler, Microscale NMR,
As the first researcher
to capitalize
these6:711
principles
Curr Opinion
ChemonBiol.
(2002)injection protocol [23••]. With estimated analyte quantities
for mass-limited samples, Odelblad [9] in 1966 employed
of only 540 +/− 170 picomoles per bead (~180 picomoles in
solenoidal NMR microcoils possessing diameters as small
NMR coil), 600 MHz NMR spectra with acceptable S/N
as 200 µm (~30 nl observe volume) to obtain continuouswere acquired in only 1 h. The authors estimated that
wave 1H-NMR measurements of mucus excreted from
analysis time could be decreased to ~15 min per bead by
improving sample handling and loading.
human cervix cells. Early solenoidal microcoil NMR
spectra suffered from low resolution due to the proximity
Single biological cells probably represent the most
and orientation of the coil to the sample. Olson et al. [8] in
intriguing mass-limited sample for NMR analysis.
1995 acquired high-resolution Fourier-transform NMR
Numerous NMR microimaging experiments of individual
spectra from solenoidal microcoils by developing appropriate
cells have been conducted over the past 16 years [24–31].
magnetic susceptibility matching technology. With reducedIn these studies, properties of intracellular water such as
diameter NMR coils specially designed for flow-through
spin density, relaxation times, and diffusion were measured.
applications, NMR has been employed as an on-line
However, the ability to obtain NMR information about
detector for microscale separations including HPLC
other components within single cells remains appealing.
[10–14], capillary electrophoresis (CE) [10,11,15–20] and
Grant et al. [32••] successfully identified several major
capillary electrochromatography (CEC) [10,11,17,18,21].
Lacey et al. [22] provided a comprehensive summary on
osmolytes and metabolites present in individual
microscale NMR, defined by observe volumes of 10 µl or
~300 µm diameter Aplysia californica neurons from spatially
less, from its genesis to early 1999.
localized 1H-NMR spectra by utilizing a 700 µm diameter
solenoidal microcoil. Obtained with specially designed
Several major advancements in both the application and
pulse sequences, 600 MHz NMR spectra were acquired
development of microscale NMR have occurred over the
from 220 × 220 × 220 µm volumes (~10 nl) of the cell in
past three years. Of particular significance, microscale
only 8 min (see Figure 2).
NMR has enabled the successful NMR analysis of single
cells and combinatorial chemistry beads, the on-line
In another study, Grant et al. [33] employed NMR
integration of NMR to a greater variety of microscale
microimaging to spatially measure the water diffusion
separations, and the emergence of multiple coil probes, all
coefficient in single neurons. Interestingly, whereas water
of which are reviewed below.
in the nucleus appears to possess a single diffusion
value, water in the cytoplasm seems to have two distinct
values, which the authors attribute to compartmentation.
Mass-limited sample analysis
Seeber et al. have developed 100 µm diameter solenoidal
Utilizing their high sensitivity, microcoils have enabled
microcoils [34] and accompanying hardware [35,36] with
NMR analysis of small-volume, mass-limited samples that
the intent of obtaining images of smaller cells (10–100 µm
previously could not be performed in a reasonable time.
diameter) at 1–2 µm resolution. To adapt microcoil
Lacey et al. successfully acquired 1H-NMR spectra from
technology for in vivo tissue metabolite studies, Berry et al.
synthesized products cleaved from single combinatorial
[37] have created an implantable 200 µm diameter
chemistry beads by using an 800 µm diameter solenoidal
solenoidal microcoil probe.
microcoil (~800 nl observe volume) and a unique flow
Nuclear Magnetic Resonance
Time-resolved NMR
Harper et al, Conformational Changes in a Photosensory LOV Domain Monitored
by Time-Resolved NMR Spectroscopy, J. Am. Chem. Soc. 126:3390 (2004)
Spatially resolved NMR
MRI
Uses a deliberately nonuniform B to limit the H signal to a small volume
Looks at the number of H in that voxel and infers tissue density
fMRI is MRI that only looks at H shifts due to oxyHb and deoxyHb
This depends on local oxygen levels which are assumed to relate to
neuronal activity.
Exact mechanism of contrast and relation to neuron function are unclear.