Nuclear magnetic resonance final

Nuclear magnetic resonance (NMR)

By: Dr. Ashish C PatelAssistant ProfessorVet College, AAU, Anand

• Nuclear magnetic resonance spectroscopy, most commonly known as NMR spectroscopy, is a research technique that exploits the magnetic properties of certain atomic nuclei.

• This type of spectroscopy determines the physical and chemical properties of atoms or the molecules in which they are contained.

• Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique that can be used to investigate the structure, dynamics, and chemical kinetics of a wide range of biochemical systems.

• The first NMR derived three dimensional solution structure of a small protein was determined in 1985 means NMR is about 25 years earlier than X-ray crystallography

• NMR spectroscopy can provide information about conformational dynamics and exchange processes of biomolecules at timescales ranging from picoseconds to seconds

• Efficient in determining ligand binding and mapping interaction surfaces of protein/ligand complexes.

• Nowadays, three-dimensional structures can be obtained for proteins up to 50 kDa molecular weight, and NMR spectra can be recorded for molecules well above 100 kDa.

• This technique relies on the ability of atomic nuclei to behave like a small magnet and align themselves with an external magnetic field.

• When irradiated with a radio frequency signal the nuclei in a molecule can change from being aligned with the magnetic field to being opposed to it.

• The instrument works on stimulating the “nuclei” of the atoms to absorb radio waves. The energy frequency at which this occurs can be measured and is displayed as an NMR spectrum.

• The most common atomic nuclei observed using this technique are 1H and 13C, but also 31P, 19F, 29Si and 77Se NMR are available.

History• 1946 Bloch, Purcell introduced about nuclear magnetic

resonance• 1955 Solomon gave concept about NOE (nuclear Overhauser

effect)• 1966 Ernst, Anderson introduced Fourier transform NMR• 1975 Jeener, Ernst gave concept about 2D NMR• 1985 Wuthrich first solution structure of a small protein

(BPTI) from NOE derived distance restraints• 1987 3D NMR 13C, 15N isotope labeling of recombinant

proteins

• Two common types of NMR spectroscopy are used to characterize organic structure:

– 1H NMR:- Used to determine the type and number of H atoms in a molecule

– 13C NMR:- Used to determine the type of carbon atoms in the molecule

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• The source of energy in NMR is radio waves which have long wavelengths having more than 107nm, and thus low energy and frequency.

• When low-energy radio waves interact with a molecule, they can change the nuclear spins of some elements, including 1H and 13C.

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In a magnetic field, there are two energy states for a proton: a lower energy state with the nucleus aligned in the same direction as Bo, and a higher energy state in which the nucleus aligned against Bo.

When an external energy source that matches the energy difference between these two states is applied, energy is absorbed, causing the nucleus to “spin flip” from one orientation to another.

The energy difference between these two nuclear spin states corresponds to the low frequency RF region of the electromagnetic spectrum.

When a charged particle such as a proton spins on its axis, it creates a magnetic field. Thus, the nucleus can be considered to be a tiny bar magnet.

Normally, these tiny bar magnets are randomly oriented in space. However, in the presence of a magnetic field B0, they are oriented with or against this applied field.

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More nuclei are oriented with the applied field because this arrangement is lower in energy.

The energy difference between these two states is very small (<0.1 cal).

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• Thus, two variables characterize NMR: an applied magnetic field B0, the strength of which is measured in tesla (T), and the frequency n of radiation used for resonance, measured in hertz (Hz), or megahertz (MHz).

The energy difference between two nuclear spin states (v) needed for resonance and the applied magnetic field strength (B0) are proportionally related:

The stronger the magnetic field, the larger energy difference between two nuclear spin states (v) and higher the needed for the resonance.

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A nucleus is in resonance when it absorbs RF radiation and “spin flips” to a higher energy state.

BO

• Both liquid and solid type of samples can be used in NMR spectroscopy.

• For liquid sample, conventional solution-state NMR spectroscopy is used for analysing where as for solid type sample, solid-state spectroscopy NMR is used.

• In solid-phase media, samples like crystals, microcrystalline powders, gels, anisotropic solutions, proteins, protein fibrils or all kinds of polymers etc. can be used.

• In liquid phase, different types of liquid solutions, nucleic acid, protein, carbohydrates etc. can be used.

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Electromagnet

1.Sample holder :- Glass tube with 8.5 cm long,0.3 cm in diameter 2.Permanent magnet :- It provides homogeneous magnetic field at 60-100 MHZ 3.Magnetic coils :- These coils induce magnetic field when current flows

through them. 4.Sweep generator :- To produce the equal amount of magnetic field pass

through the sample

Radiofrequency Transmitter

Sweep Generator

Radiofrequency Amplifier

Audio Amplifier

Detector

Oscilloscop and / or

Recorder

• Radio frequency transmitter:- A radio coil transmitter that produces a short powerful pulse of radio waves

• Radiofrequency :- A radio receiver coil that detects Receiver radio frequencies emitted as nuclei relax to a lower energy level

• Readout system :- A computer that analyses and record the data

• All subatomic particles (neutrons, protons, electrons) have the fundamental property of spin. This spin corresponds to a small magnetic moment.

• In the absence of a magnetic field the moments are randomly aligned. When a static magnetic field, Bo is applied this field acts as a turning force that aligns the nuclear spin axis of magnetic nuclei with the direction of the applied field

• This equilibrium alignment can be changed to an excited state by applying radio frequency (RF) pulses.

• When the nuclei revert to the equilibrium they emit RF radiation that can be detected

Principles of nuclear magnetic resonance

• The nuclei of many elemental isotopes have a characteristic spin (I). Some nuclei have integral spins (e.g. I = 1, 2, 3 ....), some have fractional spins (e.g. I = 1/2, 3/2, 5/2 ....), and a few have no spin, I = 0 (e.g. 12C, 16O, 32S, ....).

• Isotopes of particular interest and use are 1H, 13C, 19F and 31P, all of which have I = 1/2.

• A spinning charge generates a magnetic field and resulting spin-magnet has a magnetic moment (μ) proportional to the spin.

• In the presence of an external magnetic field (B0), two spin states exist, +1/2 and -1/2.

• The difference in energy between the two spin states is dependent on the external magnetic field strength, and is always very small.

• Strong magnetic fields are necessary for nmr spectroscopy. • Modern nmr spectrometers use powerful magnets having fields of

1 to 20. Even with these high fields, the energy difference between the two spin states is less than 0.1 cal/mole.

• For nmr purposes, this small energy difference (ΔE) is usually given as a frequency in units of MHz (106 Hz), ranging from 20 to 900 Mz, depending on the magnetic field strength and the specific nucleus being studied.

• For spin 1/2 nuclei the energy difference between the two spin states at a given magnetic field strength will be proportional to their magnetic moments.

• In order to induce nmr, a oscillatory magnetic field has to be applied at the frequency which corresponds to the separation (ΔΕ) of the two spin energy levels.

How does it work

• To get the nuclei in a molecule to all align in the same direction, a very strong magnetic field is generated using a superconducting electromagnet, which requires very low temperatures to function.

• The coils of the magnet are surrounded by liquid helium (-269°C), which is prevented from boiling off too quickly by a surrounding layer of liquid nitrogen (-196°C). These coolants are all contained in double-layer steel with a vacuum between the layers, to provide insulation just like a thermos.

• There is a narrow hole through the middle of the magnet, and the sample tube and radio frequency coils ("probe”) are located there.

• A solution of the sample in a uniform 5 mm glass tube is oriented between the poles of a powerful magnet, and is spun to average any magnetic field variations.

• Radio frequency radiation of appropriate energy is broadcast into the sample from an antenna coil (colored red). A receiver coil surrounds the sample tube, and emission of absorbed rf energy is monitored by dedicated electronic devices and a computer.

Radio Frequency Transmitter

Magnet Pole

Sweep Generator

Sweep Coils

Sweep Coils

Spinning Sample

tube

Magnet Pole

Radio Frequency Receiver & Amplifier

Control Console and

Recorder

• An nmr spectrum is acquired by varying the magnetic field over a small range while observing the rf signal from the sample. An equally effective technique is to vary the frequency of the rf radiation while holding the external field constant.

Chemical shifts• The exact resonance frequency depends on the chemical

environment of each spin, such that for example the NMR spectrum of a protein will show NMR signals with slightly different frequencies. These differences are called chemical shifts.

• The first step of a structure determination by NMR consists of assigning the chemical shifts of all the atoms/spins of the molecule which are observed in an NMR spectrum.

• The resonance frequencies are called chemical shifts and are measured in parts per million (ppm) in order to have chemical shift values independent of the static magnetic field strength.

• Backbone amide protons HN in a protein resonate around 8 ppm, while Hα spins have resonance frequencies between 3.5-5.5 ppm.

Factors affecting chemical shift:• Electronegative groups • Magnetic anisotropy of π-systems • Hydrogen bonding • Electronegative groups:- Electronegative groups attached to the C-H system

decrease the electron density around the protons, and there is less shielding (i.e.deshielding) and chemical shift increases

• Magnetic anisotropy of π-systems:- The word "anisotropic" means "non-uniform". So magnetic anisotropy means that there is a "non-uniform magnetic field".

• Electrons in π systems (e.g. aromatics, alkenes, alkynes, carbonyls etc.) interact with the applied field which induces a magnetic field that causes the anisotropy. It causes both shielding and deshielding of protons. Example:-Benzene Hydrogen bonding:- Protons that are involved in hydrogen bonding are typically change the chemical shift values. The more hydrogen bonding, the more proton is deshielded and chemical shift value is higher.

Proton NMR • The most common form of NMR is based on the hydrogen-1 (1H), nucleus or

proton. It can give information about the structure of any molecule containing hydrogen atoms.

Nuclear overhauser effect• NOE is the transfer of nuclear spin polarization from one spin to

another spin via cross-talk between different spins (normally protons) in a molecule and it depends on the through space distance between these spins.

• The local field at one nucleus is affected by the presence of another nucleus. The result is a mutual modulation of resonance frequencies.

• NOEs are typically only observed between protons which are separated by less than 5-6 Å.

• NOE is related to the three-dimensional structure of a molecule. For interproton distances > 5 Å, the NOE is too small and not observable.

J coupling constants

• Provide information about dihedral angles, and thus can define the peptide backbone and side chain conformations.

• Mediated through chemical bonds connecting two spins. The energy levels of each spin are slightly altered depending on the spin state of a scalar coupled spin (α or β).

Structure determination by solution NMR

NMR spectrum• An NMR spectrum appears as a series of vertical peaks/signals

distributed along the x-axis of the spectrum. • Each of these signals corresponds to an atom within the molecule

being observed• The position of each signal in the spectrum gives information

about the local structural environment of the atom producing the signal.

• As we move towards bigger molecules with more and more atoms, the 1D spectra become very complex, and 2D and 3D spectroscopy becomes important in understanding the relationships and interactions between different atoms in the molecule.

• The information contained in 1D spectra can be expanded in a second (frequency) dimension - 2D NMR

• In a 1D experiment a resonance (line) is identified by a single frequency: NH(f1nh)

• In 2D spectra, a resonance (cross-peak) is identified by two different frequencies: NH (f1nh, f2ha), NH (f1nh, f2ha)

• Usually, the second frequency depends on how the NMR experiment is designed.

Resonance assignment• The crosspeaks in NOE spectra cannot be interpreted without knowledge

of the frequencies of the different nuclei• The frequencies can be obtained from information contained in COSY

(correlation spectroscopy) spectra• The process of determining the frequencies of the nuclei in a molecule is

called resonance assignment (and can be lengthy...)• Two-dimensional COSY NMR experiments give correlation signals that

correspond to pairs of hydrogen atoms which are connected through chemical bonds.

• COSY spectra show frequency correlations between nuclei that are connected by chemical bonds

• Since the different amino acids have a different chemical structure they give rise to different patterns in COSY spectra. This information can be used to determine the frequencies of all nuclei in the molecule. This process is called resonance assignment

• Modern assignment techniques also use information from COSY experiments with 13C and 15N nuclei

• NOE Spectroscopy experiments give signals that correspond to hydrogen atoms which are close together in space (< 5A), even though they may be far apart in the amino acid sequence.

• Structures can be derived from a collection of such signals which define distance constraints between a number of hydrogen atoms along the polypeptide chain.

Example: short distance (< 5 A, NOE)correlations between hydrogen atoms in a helix

Multi-Dimensional NMR:Built on the 3D Principle

• NMR is used in biology to study the Biofluids, cells, organs and macromolecules such as Nucleic acids (DNA, RNA), carbohydrates Proteins and peptides and also Labeling studies in biochemistry.

• NMR is used in physics and physical chemistry to study High pressure diffusion ,liquid crystals, Membranes.

• 3D structure determination of proteins, nucleic acids, protein/DNA complexes, ...)

• NMR is used in pharmaceutical science to study Pharmaceuticals and Drug metabolism.

• NMR is used in chemistry to determine the Enantiomeric purity. Elucidate Chemical structure of organic and inorganic compounds.

• 1H widely used for structure elucidation. Inorganic solids- Inorganic compounds are investigated by solid state 1H-NMR.eg CaSO4 H2O. ⋅Organic solids- Solid-state 1H NMR constitutes a powerful approach to investigate the hydrogen-bonding and ionization states of small organic compounds.

Applications of NMR

• Direct correlation with hydrogen-bonding lengths could be demonstrated, e.g. for amino acid carboxyl groups.

• Polymers and rubbers- Examine hydrogen bonding and acidity. • In vivo NMR studies- concerned with 1H NMR of human brain. Many

studies are concerned with altered levels of metabolites in various brain diseases.

• To determine the spatial distribution of any given metabolite detected spectroscopically IS (image selected in vivo spectroscopy).

• MRI is specialist application of multi dimensional Fourier transformation NMR for Anatomical imaging, for measuring physiological functions, for flow measurements and angiography, for tissue perfusion studies and also for tumors.

Limits• Molecular weight limits for protein structure calculation (monomer):

5-15 kDa: routine15-20 kDa: usually feasible20-30 kDa: long term project40-50 kDa: in the next future?

• Molecular weight limits for peptide/protein, protein/protein interactions (MW of the AB complex, A < 10 kDa):20-30 kDa: routine30-50 kDa: feasible50-100 kDa: in the next future

Advantages of NMR Limitation of NMRObtain angles, distances, coupling constants, chemical shifts, rate constants etc. These are really molecular parameters which could be examined more with computers and molecular modeling procedures.

This is good for the more accurate determination of the structure, but not for the availability of higher molecular masses

With a suitable computer apparatus we can calculate the whole 3D structure

The resolving power of NMR is less than some other type of experiments (e.g.: X-ray crystallography) since the information got from the same material is much more complex

There are lots of possibilities to collect different data-sets from different types of experiments for the ability to resolve the uncertanities of one type of measurements

The highest molecular mass which was examined successfully is just a 64kDa protein-complex

This method is capable to lead us for the observation of the chemical kinetics

There are lots of cases when from a given data-set - a given type of experiment – we may predict two or more possible conformations, too

We can investigate the influence of the dielectric constant, the polarity and any other properties of the solvent or some added material

The cost of the experimental implementation is increasing with the higher strength and the complexity of the determination

Disadvantages1) Sensitivity• The greatest disadvantage of NMR spectroscopy and imaging compared with

other modalities is the intrinsic insensitivity of the methods. The signal that can be generated in the

• NMR experiment is small and, for practical purposes, most strongly coupled with the concentration of the nuclei in the sample.

• For example, the human body is composed of -70% water, and thus a relatively large signal can be obtained from the 1H nucleus in water that is effectively at a concentration in the tens of molar range.

• Thus, it is possible to measure signals from cubes (voxels) of tissue as small as = 0.3mm on a side from the human brain, generating the high-quality.

• The NMR signals from water will always be detectable at resolutions approximately two orders of magnitude greater than those of other NMR-sensitive nuclei.

• Thus, compounds present in submillimolar and certainly micromolar concentrations cannot practically be detected directly in tissues.

• As a result, the sample size generally dictates the choice of magnet and field strength; thus, the smaller the sample, the more sensitive the experiments.

2) Working in a High-Magnetic-Field Environment• An inevitable consequence of carrying out NMR investigations is the need to

work in a high-magnetic-field environment.• No known intrinsic risks are associated with high magnetic fields; however, the

presence of the magnetic field can affect equipment routinely used in animal research.

• For example, electronic monitors and computer-controlled devices may function improperly or not at all.

• Due to the nature of the forces involved, the result can be a scalpel, a pair of scissors, or even a gas cylinder becoming a flying object.

• Instruments that can be obtained that are non ferromagnetic, thus reducing the potential difficulties associated with working in a high-magnetic-field environment.

• Now a days, a steel passive shield or an active shield may be placed around the magnet to reduce the magnet fringe fields and minimize the risks.

• This reduction can be particularly important when the space available to site the instrument is limited.

3) Motion Sensitivity• Most MR techniques are motion sensitive. This sensitivity leads to

signal distortions that are visually most evident in artifacts on images or more subtly in quantitative measurements.

• Some MR techniques such as functional MRI (fMRI1) are particularly sensitive to motion artifact, thus great care must be taken not to minimize the distorting effects of motion and thus minimize misinterpretation of data.

• In animal studies, anesthesia is usually essential to avoid gross movement of the animal during the study.

• Cardiac gating may be necessary even when imaging other organs such as the brain because of motion caused by the pulsatile blood flow. In some cases, such as lung imaging, it may also be necessary to gate to respiratory motion or, alternatively, the subject may be controlled via mechanical ventilation.

Examples/ uses of NMR Spectroscopy• Several different NMR-sensitive nuclei can be used in the study of

biological systems, and the most common are 31P, 1H, 13C, 23Na, and 19F. • 31P, 1H, and 13C-NMR spectroscopy are typically used to investigate

cellular metabolism and bioenergetics. • Whereas 23Na NMR studies usually focus on issues related to ion

transport and regulation of ion pumps. Fluorine does not occur naturally in biological systems.

• However, it is a very sensitive NMR nucleus. Therefore, fluorine-labeled compounds can be introduced into cells and used as an indicator of a cellular process such as calcium concentration.

• 19F-NMR spectroscopy has also been used to follow the metabolism of drugs, such as 5-fluorouracil.

NMR and X-ray crystallography are complementary

• Molecules are studied in solution.• Protein folding studies can be done by monitoring NMR spectra• Denatured states of a biomolecule, folding intermediates and even

transition states can be characterized• Conformational or chemical exchange, internal mobility and

dynamics at timescales ranging from picoseconds to seconds• Very efficient in mapping interactions with other molecules• Upper weight limit for NMR is ~ 50 kDa