Nuclear magnetic resonance (enzymology)

Nuclear magnetic resonance (enzymology)

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  Topic: Nuclear MagneticResonance Presented By: Mohsin Shad 2013-bc-005 Presented To: Dr.Muhammad Tayyab 2
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  Nuclear Magnetic Resonance NMR Spectroscopy is one of the powerful and theoretically complex analytical techniques used to determine the structure of organic compounds It allows the characterization of a very small amount of sample (10mg), and does not destroy the sample It provide vast information about a molecule's structure It is used to confirm a complicated molecule's structure by conjunction with other types of spectroscopy and chemical analysis . 3
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  Principle of NMR Theprinciple of NMR comes from the spin of nucleus Nuclear spins generates magnetic field without applied an external magnetic field The nuclear spins are random in directions When an external magnetic field (B0), is present the nuclei align themselves either with or against field of the external magnet. 4
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  The emitted radiofrequency is directly proportional to the strength of the applied field. V= 𝛾𝐵 𝑂/2𝜋 B0 = external magnetic field experienced by proton γ= magnetogyric ratio (the ratio between nuclear magnetic momentum and angular momentum) 5
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  NMR Instrumentation There areseven parts of NMR Spectroscopy which are described below: 1. Sample Holder 2. Permanent Magnet 3. Magnetic Coils 4. Sweep Generator 5. Radio Frequency Transmitters 6. Radio Frequency Receiver 7. Read out Systems 6
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   Sample Holder: Glasstube which is 8.5cm longer and 0.3cm in diameter.  Permanent Magnet: It provides homogenous magnetic field at 60-100 MHz  Magnetic Coils: These coils induce magnetic field when current flow through them.  Sweep Generator: To produce an equal amount of magnetic field pass through the sample 7
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   Radio FrequencyTransmitters: Radio Frequency Transmitters that produces a shorts powerful pulse of radio waves.  Radio Frequency Receiver: Radio Frequency Receiver that detects receiver radio frequencies emitted as nuclei relaxes of lower energy level.  Read out Systems: A computer that analysis and record the data. 8
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  Chemical Shift Scale Achemical shift is defined as the difference in parts per million (ppm) between the resonance frequency of the observed proton and that of the tetramethylsilane (TMS) hydrogen  Chemical Shift Scale is also Called d Scale It does not depend on the magnetic field strength. 9
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  How We MeasureThe Chemical Shift ? Measure the absorbance frequency of NMR standard Measure the frequency of our sample Subtract the sample frequency from that of the standard Finally divided by the frequency of the standard This gives a number called the “chemical shift 10
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  Types of NMR Thereare several types of NMR spectra, which depend upon:  The kind of instrument used  The type of nucleus involved  The physical state of the sample  The environment of the analytic nucleus  The purpose of the data collection Most NMR spectra can be categorized as either wide line or high resolution. 11
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  Wide-Line spectra: The spectrain which the bandwidth of the source of the lines is large enough so that the fine structure due to chemical environment is obscured. A single peak is associated with each species High-Resolution Spectra: Most NMR spectra are high resolution and are collected by instruments capable of differentiating between very small frequency differences of 0.01 ppm or less. But in the higher resolution spectrum Two of the three peaks can be resolved into additional peaks. 12
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  NMR Applications  Someof the applications of NMR spectroscopy are listed below:  It determine the bio macromolecules in aqueous solutions under near physiological conditions.  It determine the residual structures of unfolded proteins and the structures of folding intermediates  It determine the chemical properties of functional groups in bio macromolecules  such as the ionization states of ionizable groups at the active sites of enzymes. 13
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  Advantages of NMR With a suitable computer apparatus we can calculate the whole 3D structure of proteins and enzymes.  The motion of segments (domains) can be examined.  This method is able to lead us for the observation of chemical kinetics.  We can investigate the dielectric constant, the polarity and any other properties of the solvent or some added material. 13
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  • A powerfultool in the research of polymer chemistry and physics • A matured technique for chemical identification and conformational analysis of chemicals whether synthetic or natural. • Solid state NMR has the potential for determining atomic-resolution structures of domains of membrane proteins in their native membrane environments, including those with bound ligands • A power tool for the detection of interior water and its interaction with bio macromolecules. 15
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  Disadvantages of NMR This is good for more accurate determination of the structure, but not for the availability of higher molecular weight.  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.  Unfortunately we are just able to determine the degree of probability of being of protein segment in the given conformation.  The cost of the experimental implementation is increasing with the higher strength and the complexity of determination. 16
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  NMR Application inEnzymology  NMR is applied :  To the study of conformational dynamic processes in enzymes  To study the biological activities of enzymes  To study the exchange of more open or flexible conformational states with more closed or constrained states  NMR spin relaxation studies of the enzymes ribonuclease HI from mesophilic and thermophilic bacteria 17
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