![]() ![]() Techniques for Studying Tertiary Structure Another biophysical method, Fourier transform infrared (FTIR) spectroscopy, can also be used to estimate secondary-structural components in a protein by measuring the wavelength and intensity of infrared radiation (IR) absorption by a protein sample. These spectra can then be used to quantify the fraction of each secondary structural element in the protein. ![]() It measures different secondary structural elements in a protein, including α helix and β sheet, and random coil, based on their characteristic spectra in the far-UV region of the spectrum. To study a protein’s secondary structure, the most common method is circular dichroism spectroscopy (CD). Techniques for Studying Secondary Structure The primary structure of a protein can also be analyzed by means of peptide mapping, another MS-based method well suited for verifying sequence and identifying sequence variations like point mutations. Nowadays, the advancement of MS-based proteomics has led to the advent of de novo protein sequencing, allowing for rapid and accurate sequence determination of any given protein. ![]() The former is particularly useful to generate full sequences of large proteins (more than 40 amino acid residues). This can rely on two types of methods: 1) mass spectrometry (MS)-based protein sequencing, and 2) Edman degradation. Structural analysis of a protein usually starts with the determination of its primary structure: the amino acid sequence. Techniques for Studying Primary Structure Current methods for the study of the four levels of protein structure. In this section, we will discuss some of the widely adopted techniques for protein structural analysis. Various analytical, biophysical, and computational tools are currently available to study the four levels of protein structure, as highlighted in Table 1 below. These bonds hold the subunits together and arrange themselves to form a larger protein complex. The quaternary structure is stabilized by the same bonds as for the tertiary structure, including different noncovalent bonds and disulfide bonds. The complete structure of such a protein is designated its quaternary structure, and each polypeptide chain is referred to as a subunit. Many proteins are made up of more than one polypeptide chain to perform their function. They are abundantly found in secretory proteins and extracellular domains of membrane proteins. 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. In addition, there is one type of covalent bond that can also contribute to tertiary structure: the disulfide bond. Bonds between side chains (R groups) of amino acids-including hydrophobic interactions, hydrogen bonds, and ionic bonds -contribute to the tertiary structure. 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. For example, α helices are especially abundant in membrane proteins and hair cells (i.e., α-keratin), and β sheets are the main component of amyloid fibers in both animals and bacteria. Most proteins contain α helices and β sheets. A β sheet is generated when multiple segments of a polypeptide chain lie side by side, creating a sheet-like structure held together by hydrogen bonds. An α helix is made when the polypeptide chain turns around itself, forming a structural motif that resembles a spiral staircase. The most common types of secondary structure are α helix and β sheet. The formation of the secondary structure is mainly driven by hydrogen bonding between amino groups and carboxyl groups on the polypeptide chain. The secondary structure of a protein refers to the local folding patterns on the polypeptide chain formed by intramolecular interactions between atoms of the backbone. Each type of protein possesses a distinct primary structure, which drives the bonding and folding of the linear amino acid chain, and ultimately determines the protein’s unique three-dimensional shape. Therefore, the structure of a protein begins with its amino acid sequence, which is considered its primary structure. Proteins are made up of amino acids linked together by peptide bonds in different orders and different lengths, ranging from 30 amino acids to more than 100,000. ![]()
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