What is Isotopic labelling?
Isotopic labelling is defined as a methodology that is widely used to introduce less common isotope of carbon, hydrogen, nitrogen and sometimes oxygen into the proteins or polypeptide chains . All isotopes of an element exhibit the same chemical properties because of the same atomic number but different physical properties due to a different number of neutrons. Isotopes are being used in a variety of applications including isotopic labelling .
Isotopes are broadly used in isotopic labelling. In this method, rare variants of elements are used as tracers or indicators in chemical reactions . Atoms of a given element are frothy and they cannot be distinguished from each other. But, with the usage of isotopes having different atomic masses , mass spectrometry or infrared spectroscopy can be used to differentiate uniform and diverse nonradioactive stable isotopes . Example includes stable isotopic labelling with amino acids in cell culture (SILAC). In this method, stable isotopes are being used to quantify proteins .
Background of Isotopic Labelling and NMR:
The process of isotopic labelling of proteins was first started in the late ’60s and it resulted in the manufacturing of isotopically labelled proteins that had been isolated from organisms such as bacteria and plants . The proteins from these organisms were labelled by placing the proteins in a media rich in isotopically labelled nutrients . In the previous years, the proper explosion of labelling strategies and labelling production approaches had been carried out to permit nuclear magnetic resonance spectroscopic studies of biologically bulky proteins and networks of proteins of size greater than 10kDa .
Approximately 30% of all the proteins synthesized in organisms were integral membrane proteins (IMP) and demanded a lipid atmosphere to work and fold properly . Membrane proteins were released only when the membranes were disrupted. Membrane proteins have been used as a host for various important functions such as they can work as receptors, transporters, channels, electrical and photo-transducers, and so forth .
Although membrane proteins were involved in the various vital process occurring throughout the body such as signal transduction process, transportation of molecules through the membranes and transmission of ions , instead of these important function, protein data bank (PDB) had deposited only 308 membrane proteins as of February 2009. This amount was very small in size as compared to the thousands of high power resolution structures that had been determined for their soluble corresponding part .
There were various considerations for the scarcity of high-resolution membrane proteins structures . The first reason was that the expression, as well as purification of membrane proteins with proper folding, was difficult to obtain . And the second reason was that the membrane proteins required lipids and detergents for structural and functional studies .
Membrane stimulated environment enamel the proteins in such a way that proteins form huge and moderately stumbling complexes. These complexes then confound nuclear magnetic resonance spectroscopic analysis . However, the success in the study of membrane proteins had been made possible through various strategies such as manufacturing system of proteins, hardware of NMR, pulse sequences and isotopic labelling strategies .
The process of determination of the structure of proteins was accelerated during the era of 2009 with the help of X-Rays crystallography . Both the types of NMR including SSNMR and solution NMR had made tremendous progress for scrutinizing membrane-bound proteins .
Nuclear Magnetic Rasonance (NMR) Spectrometery:
Spectroscopy is a branch of science which concerns with electromagnetic waves and materials. In this field, we can determine that the wavelength and frequency of waves have an influence on the matter . Scientists have been using spectroscopy in order to originate the characteristics of materials at the molecular level. Nuclear Magnetic Resonance is considered as special branch of spectroscopy. It has been widely used to manipulate the magnetic characteristics of atomic nuclei .
The main objectives of NMR spectroscopy include:
- With the NMR we can determine a different chemical and physical characteristic of compounds 
- NMR can be applied to the study of naturally occurring large molecules such as proteins 
Nuclear magnetic resonance experiments have been used to determine resonance energies, 3D structures of compounds as well as a contact of protein molecule .
Basic Principle of NMR:
NMR works on the principle that the nucleus is contained by all-atom. Electrons in the atoms circulate around the nucleus . The nucleus can spin and due to the spinning of the nucleus and charge on the nucleus, it can behave like a magnet . Nuclear magnetic resonance spectroscopy works by concerning a magnetic field to the nucleus and after applying a magnetic field, it quantify the quantity of sufficient energy which is required to place several nuclei in resonance . All nuclei are surrounded by different electronic environment i.e shielded or deshielded . These different atmospheres demand diverse quantity of energy to achieve resonance. Then an NMR spectrum is obtained and this spectrum delivers a sign or peak . These peaks symbolize the amount of energy which is required to place nuclei in resonance .
Isotopic labelling is a vital process that has been used to simplify the overlapped spectra made by nuclear magnetic resonance spectroscopy and made it easier to identify the structure of biological molecules such as proteins . Nuclear magnetic resonance has made numerous developments in structure determination and isotopic labelling is a vital part of these developments. It has been widely used for the representation of biological macromolecules at the atomic level .
Major Application of Isotopic Labeling for NMR Structural Analysis of Proteins:
Isotopic labelling of proteins has various applications to analyze the complete three-dimensional structures of proteins through nuclear magnetic resonance spectroscopy. The major application is being the identification of protein-protein associations.
Identification of Protein-Protein Associations:
The knowledge of homo-oligomeric membrane proteins by NMR had been an extremely helpful application of methyl labelling as well as uniform isotopic labelling. These particles showed symmetry, therefore, NMR signals were chemically equal. In this way, a single set of resonance had been seen . However, asymmetric labelling techniques had been introduced to find out the structural knowledge about the symmetric oligomers. This technique was based on the introduction of isotopic asymmetry in these complexes .
The purpose of this technique was the introduction of isotopic asymmetry in these complexes . This had been achieved by labelling one of the protomers with specific isotopic strategy and the remaining protomers with other specific isotope strategy. Then complexes/oligomers were formed in which the dipolar contacts between two protomer regions had been detected by pulse sequences .
Two asymmetric isotopic methodologies had been proposed in order to find out the inter-protomer relation present in pentameric phospholamban (PLN) for both solution and solid-state nuclear magnetic resonance spectroscopy .
PLN is a homo-pentamer which is made up of five indistinguishable protomers (52 residues each). The transmembrane segment of each protomer comprised of for the most part hydrophobic amino acids Isoleucine, Leucine and Valine , which were associated with keeping the oligomer together by hydrophobic communications. The primary labelling plan was invented to test protomer contacts in cleanser micelles by using solution NMR .
In this plan, half of the protomers were marked [U-2H, 12C, 14N] and 13CH3 at the Isoleucine sigma1 (utilizing 2-ketobutyric corrosive 4-13C,3,3-D2 as ancestor), while the other half was labelled [U-2H, 12C, 14N] and 13CH3 at the Leuδ1/2/Valγ1/(utilizing 2-keto-3-(methyl-D3)- butyric corrosive 4-13C as forerunner) . Utilizing a methyl-methyl NOESY pulse succession, it was conceivable to effectively distinguish and unambiguously relegate inter-protomer contacts, which were utilized for structure findings .
Several chemical methods had been proposed to amend reactive amino acids present inside chain cluster after the expression and purification of proteins. By utilizing reagent containing isotopic labels, possibilities had been made to specifically supplement amino acids along with molecules which contain NMR active isotope. cysteines, tyrosine and lysines were most frequently used to modify their side chains .
In proteins, the sulfhydryl group (-SH) present in cysteine could efficiently react with various chemical groups under mild situations. Two applications such as the incorporation of fluorine atoms and location-focused methyl group replacement had made use of extraordinary nucleophilicity of free thiol moieties in cysteines . Free thiols in cysteines reacted with trifluoromethyl derivatives including BFTA (3-bromo1,1,1-trifluoroacetone), SETFA (S-ethyl-trifluorothioactetate) and TFASAN ( trifluoroacetamidosuccinicanhydride) . This type of labelling method had been effectively used for the study of various proteins including citrate synthase, G-actin, Myosin S-1/F-actin complex, SH3 domain, rhodopsin and β2-Adrenergic Receptor .
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