In addition to becoming denatured, proteins can also form aggregates under certain stress conditions. Aggregates are often produced during the manufacturing process and are typically undesirable, largely due to the possibility of them causing adverse immune responses when administered.
In addition to these physical forms of protein degradation, it is also important to be aware of the possible pathways of protein chemical degradation.
These include oxidation, deamidation, peptide-bond hydrolysis, disulfide-bond reshuffling and cross-linking. The methods used in the processing and the formulation of proteins, including any lyophilization step, must be carefully examined to prevent degradation and to increase the stability of the protein biopharmaceutical both in storage and during drug delivery.
The complexities of protein structure make the elucidation of a complete protein structure extremely difficult even with the most advanced analytical equipment. An amino acid analyzer can be used to determine which amino acids are present and the molar ratios of each. The sequence of the protein can then be analyzed by means of peptide mapping and the use of Edman degradation or mass spectroscopy.
This process is routine for peptides and small proteins but becomes more complex for large multimeric proteins. Peptide mapping generally entails treatment of the protein with different protease enzymes to chop up the sequence into smaller peptides at specific cleavage sites. Two commonly used enzymes are trypsin and chymotrypsin. Mass spectroscopy has become an invaluable tool for the analysis of enzyme digested proteins, by means of peptide fingerprinting methods and database searching.
Edman degradation involves the cleavage, separation and identification of one amino acid at a time from a short peptide, starting from the N-terminus. One method used to characterize the secondary structure of a protein is circular dichroism spectroscopy CD. These spectra can be used to approximate the fraction of the entire protein made up of each type of structure.
A more complete, high-resolution analysis of the three-dimensional structure of a protein is carried out using X-ray crystallography or nuclear magnetic resonance NMR analysis.
To determine the three-dimensional structure of a protein by X-ray diffraction, a large, well-ordered single crystal is required. X-ray diffraction allows measurement of the short distances between atoms and yields a three-dimensional electron density map, which can be used to build a model of the protein structure.
The use of NMR to determine the three-dimensional structure of a protein has some advantages over X-ray diffraction in that it can be carried out in solution and thus the protein is free of the constraints of the crystal lattice.
Many different techniques can be used to determine the stability of a protein. For the analysis of unfolding of a protein, spectroscopic methods such as fluorescence, UV, infrared and CD can be used. Thermodynamic methods such as differential scanning calorimetry DSC can be useful in determining the effect of temperature on protein stability.
HPLC is also an invaluable means of analyzing the purity of a protein. Other analytical methods such as SDS-PAGE, iso-electric focusing and capillary electrophoresis can also be used to determine protein stability, and a suitable bioassay should be used to determine the potency of a protein biopharmaceutical.
The variety of methods for determining protein stability again emphasizes the complexity of the nature of protein structure and the importance of maintaining that structure for a successful biopharmaceutical product. Notice: JavaScript is required for this content. Davies represent the tertiary structure of the antigen-binding portion of an antibody molecule.
Each circle represents an alpha carbon in one of the two polypeptide chains that make up this protein. The filled circles at the top are amino acids that bind to the antigen. Most of the secondary structure of this protein consists of beta conformation , which is particularly easy to see on the right side of the image.
Do try to fuse these two images into a stereoscopic 3D view. I find that it works best when my eyes are about 18" from the screen and I try to relax so that my eyes are directed at a point behind the screen. Where the entire protein or parts of a protein are exposed to water e. If a protein does not fold correctly it will not function properly. Therefore, researching a protein's structure is very important when trying to understand what it does and how it works.
When scientists study a protein they must first determine the sequence of amino acids in the protein chain primary structure. They use this sequence to predict the presence of any alpha helices or beta sheets secondary structure. They can then use X-ray crystallography and NMR to determine a protein's full 3-dimensional shape tertiary structure. Knowing the tertiary structure of a protein is often crucial to understanding how it functions and how to target it for drug therapy or other medical uses.
Tertiary Structure Protein Structure Tutorials. Interactive Molecular Window click and drag to rotate.
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