BIOLOGICAL APPLICATIONS

OF NMR SPECTROSCOPY

By Nandita Pathak
MSc. CHEMISTRY
(FINAL)
2022-2023
 CONTENTS
 WHAT IS NMR SPECTROSCOPY?
 GENERAL APPLICATIONS IN BIOCHEMISTRY
 BIOLOGICAL APPLICATIONS OF NMR SPECTROSCOPY
1. STRUCTURE OF PROTEINS AND PEPTIDES
2. ASSESSMENT OF PROTEIN CONFORMATIONAL EXCHANGE
BY NMR
3. HOW TO IDENTIFY BINDING SITES IN PROTEINS
 MAGNETIC RESONANCE IMAGING (MRI)
1. PRINCIPLE
2. MRI INSTRUMENTS
3. DESCRIPTION
 NMR IN MEDICAL DIAGNOSTICS
 WHAT IS NMR SPECTROSCOPY?
The phenomenon of nuclear magnetic resonance (NMR), concerned with
magnetic properties of certain atomic nuclei, was first enunciated by American
physicist, Felix Bloch and Edward Purcell in 1946 for which they shared the
Nobel Prize, in 1952.
The NMR spectroscopy depends upon the fact that most isotopes of the
elements possess gyromagnetic properties, meaning thereby that they behave
like tiny spinning bar magnets.
When a sample containing nuclei exhibiting this immutable gyromagnetism is
placed in an appropriate DC magnetic field and is simultaneously irradiated by a
weaker rotating radiofrequency magnetic field, the nuclei can be compelled to
a) Reveal their presence,
b) Identify themselves and,
c) Describe the nature of their surroundings , all by means of a minute radio
signal which they transmit to a receiver coil coupled closely to the sample.
 1.STRUCTURE OF
PROTEINS AND
PEPTIDES
 Biomolecular NMR spectroscopy can be used
to determine the structure of proteins up to a
mass of ~50kDa.
 The preparation of proteins or selected
domains for NMR requires recombinant
expression and isotopic labelling to enrich the
samples with C-13 , N-15 OR H-2.
 Cryoprobe technology has reduced the sample
amount(less than 10 mg) required for NMR
experiments.
 Heteronuclear multidimensional NMR spectra
is recorded for the assignment of all chemical
shifts (H1, C13,N15).
 2. ASSESSMENT OF PROTEIN
CONFORMATIONAL EXCHANGE BY NMR
Apart from providing the 3D structure of molecules, NMR
methods also help to identify protein-protein interaction for molar
recognition by mapping.
By saturation transfer difference NMR, protein resonance can be
selectively saturated.
It is possible to calculate PMR difference spectrum of the ligand
from ligand spectrum without saturation of protein.
Binding constants can also be determined using titration
experiments.
 HOW TO IDENTIFY BINDING SITES IN
PROTEINS?
• Beyond mapping the flexibility of residues in known protein binding
sites, NMR techniques can also be used to identify novel binding sites
in proteins.
• Protein motions on the time scale of microseconds to milliseconds are
accessible to NMR technique.
• The diffusion constants for rotation around the three principal axes x,
y, z known as rotational diffusion tensor can be determined.
• The principal components and orientation can be derived from the
analysis of the ratio of the spin-spin and spin-lattice relaxation times
T2/T1.
• Analysis of these values for the protons of rigid amide ( CONH) groups
leads to characterisation of the conformational exchange of proteins.
Residues constituting the ligand-binding interface often experience a
different environment in the bound state as compared to the free
state.
The amide signals of these residues are thus broadened due to
exchange between these two environments when the free and bound
states are in equilibrium.
This approach has been successfully applied to identify amino acids at
the binding site of a 16kDa protein that binds to and regulates the
251kDa hydroxylase of the methane mono-oxygenase protein system.
The free and bound forms of the regulatory protein exchange on the
time scale of milliseconds.
Other examples include identification of specific sites in the weak
self-association of the N-terminal domain of the rat N-cell adhesion
protein CD2 (CD2D1) using the concentration dependence of the T2
values.
 MAGNETIC RESONANCE IMAGING
(MRI)
 NMR imaging or MRI also called
Tomography or Zeugmatography is
the technique in which data from
pulsed RF excitation of solid or
semi-solid objects are subjected to
Fourier transformation and
converted to three dimensional
images of the interior of the
objects.
 In MRI, the map of density of NMR
nuclei (say 1H, 2H, 13C, 31P,) in various
regions of the objects is produced
rather than the NMR spectrum.
PRINCIPLE
1. The fundamental principle of MRI is that
magnetic field strength is varied throughout
the object to obtain profiles from different
directions.
2. The linear variation or field gradient is
created by auxiliary coils in the magnet bore
that are under the control of computer of
the magnetic resonance instrument.
3. Thus ,in order to obtain 1D image
projection, the signal is obtained in the
simultaneous presence of field gradient and
RF pulse and the intensity is plotted against
the frequency , with the frequency axis
being coded.
 MRI INSTRUMENTS
 MRI instruments used for clinical imaging operate with field
strengths of 3T but experimental instruments can operate at 20T,
allowing the imaging of whole live organisms with enough spatial and
temporal resolution to follow regenerative processes continuously at
the single cell level.
DESCRIPTION
 PROTONS IN DIFFERENT
POSITIONS IN THE SAMPLE
EXPERIENCE DIFFERENT MAGNETIC
FIELDS H1 and H2 for two of the
regions and will have different
frequencies v1= γH1 / 2π and v2 = γH2 /
2π.
 The frequency v2 will be higher than the
frequency v1 and the frequency
difference ∆v = v2 – v1.
 Thus, by changing the centre frequency
of the NMR probe pulse in increments
of ∆v, it is possible to probe
successive positions in the direction of
applied magnetic field gradient.
 Each consecutive radio frequency pulse produces FID signals that encodes
the concentration of protons at each position along the direction of the
field gradient.
 By performing the Fourier transformation on the FIDs, concentration
information can be produced as indicated by the heights of the peaks.
 Images in three directions can be made by an extension of this coding
concept using additional magnetic field gradients along the y- and x-axes.
 NMR IN MEDICAL DIAGNOSTICS
The NMR imaging (MRI) method is
generally acknowledged to be superior
to the existing imaging technique such
as X-ray CAT scans because
1. The irradiation frequencies are very
low in energy and harmless to human
tissue and organs.
2. The rapidity with which individual
images can be accumulated and
stored makes this technique an
important source of information of
physiological functions of the body
organs.
 MRI is one of the most important and
promising application of FTNMR.
 It plays a central role in routine clinical
imaging of large volume soft tissues.
 Because proton is the sensitive nuclide
and is present in all biological systems.
 1HNMR is used exclusively in clinical
environment.
 MRI is used in locating tumours
(invisible to X-ray analysis), edema,
lesions and brain disorders.
 MRI images of 3D brain reconstruction
calculated using MRI data from a young
patient is shown in Figure.
 SOME OTHER SALIENT
POINTS ARE :
 The ability to generate high resolution (mm) images from three
dimensional is a unique feature of MRI technique.
 MRI can be applied to the whole body or specific organ investigations
on head, thorax, abdomen, heart, liver, pancreas and musculoskeletal
regions.
 The use of contrast agents with paramagnetic properties has enabled
investigation of organ function as well as tissue perfusion and
transport across the blood-brain barrier and vascular anatomy.
 MRI is able to detect motion of the blood flow because the
oxygenated and deoxygenated blood carry distinct NMR signals.
 Most MRI and in-vitro studies have been performed using NMR.
 Phosphorus is another NMR sensitive nucleus which due to its natural
abundance finds numerous applications in metabolic and other
physiological studies.
 Other NMR sensitive nuclei such as H 2, C13, N15, F19, Na23 have also been
investigated in living systems.
 The study of certain functions rather than anatomy by MRI, called
functional imaging is becoming useful in medical research and diagnosis.
 NMR imaging has a potential application in the field of agriculture.
 The H1 –MRI and C13 –NMR are used to study differential distribution of
water and oil as well as the germination process in groundnut seeds etc.
REFERENC
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