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Analysis of Tertiary Order Structure by Nuclear Magnetic Resonance (NMR) (CAT#: STEM-B-0377-CJ)

Introduction

Structure and conformation of a biological molecule is key for its function. The higher order structure of a biopharmaceutical molecule is, thereby, often directly connected to the quality, stability, safety, and efficacy of a therapy. The higher order structure is considered a critical quality attribute and, thus, a detailed understanding of the higher order structure of a biopharmaceutical compound is critical in every research and development phase. Characterizing the secondary, tertiary and, if present, quaternary structure of a biopharmaceutical compound requires multiple analytical techniques.

The overall three-dimensional conformation of a single polypeptide chain (a protein molecule) is referred to as the tertiary structure, which typically includes different elements of secondary structures such as α helices, β sheets, random coils, and loops. Bonds between side chains (R groups) of amino acids—including hydrophobic interactions, hydrogen bonds, and ionic bonds —contribute to the tertiary structure.

In addition, there is one type of covalent bond that can also contribute to tertiary structure: the disulfide bond. Disulfide bonds are a type of post-translational modification (PTM) formed between sulfur-containing side chains of cysteine residues, allowing distant parts of the protein to be held together. They are abundantly found in secretory proteins and extracellular domains of membrane proteins.




Principle

The principle of NMR usually involves three sequential steps: 1. The alignment (polarization) of the magnetic nuclear spins in an applied, constant magnetic field B0. 2. The perturbation of this alignment of the nuclear spins by a weak oscillating magnetic field, usually referred to as a radio frequency (RF) pulse. The oscillation frequency required for significant perturbation is dependent upon the static magnetic field (B0) and the nuclei of observation. 3. The detection of the NMR signal during or after the RF pulse, due to the voltage induced in a detection coil by precession of the nuclear spins around B0. After an RF pulse, precession usually occurs with the nuclei's intrinsic Larmor frequency and, in itself, does not involve transitions between spin states or energy levels.

The two magnetic fields are usually chosen to be perpendicular to each other as this maximizes the NMR signal strength. The frequencies of the time-signal response by the total magnetization (M) of the nuclear spins are analyzed in NMR spectroscopy and magnetic resonance imaging. Both use applied magnetic fields (B0) of great strength, often produced by large currents in superconducting coils, in order to achieve dispersion of response frequencies and of very high homogeneity and stability in order to deliver spectral resolution, the details of which are described by chemical shifts, the Zeeman effect, and Knight shifts (in metals). The information provided by NMR can also be increased using hyperpolarization, and/or using two-dimensional, three-dimensional and higher-dimensional techniques.

Applications

Biopharmaceutica

Procedure

1. Place the sample in a magnetic field.
2. Excite the nuclei sample into nuclear magnetic resonance with the help of radio waves to produce NMR signals.
3. These NMR signals are detected with sensitive radio receivers.
4. The resonance frequency of an atom in a molecule is changed by the intramolecular magnetic field surrounding it.
5. This gives details of a molecule’s individual functional groups and its electronic structure.
6. Nuclear magnetic resonance spectroscopy is a conclusive method of identifying monomolecular organic compounds.
7. This method provides details of the reaction state, structure, chemical environment and dynamics of a molecu

Materials

• Sample: Proteins
• Equipment: NMR spectroscopy
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