The formation of disulfide bridges by oxidation of the sulfhydryl groups on cysteine is an important aspect of the stabilization of protein tertiary structure, allowing different parts of the protein chain to be held together covalently.
Additionally, hydrogen bonds may form between different side-chain groups. As with disulfide bridges , these hydrogen bonds can bring together two parts of a chain that are some distance away in terms of sequence. Salt bridges, ionic inter- actions between positively and negatively charged sites on amino acid side chains, also help to stabilize the tertiary structure of a protein.
Many proteins are made up of multiple polypeptide chains, often referred to as protein subunits. These subunits may be the same, as in a homodimer, or different, as in a heterodimer. The quaternary structure refers to how these protein subunits interact with each other and arrange themselves to form a larger aggregate protein complex.
The final shape of the protein complex is once again stabilized by various interactions, including hydrogen-bonding, disulfide-bridges and salt bridges. The four levels of protein structure are shown in Figure 2. Due to the nature of the weak interactions controlling the three-dimensional structure, proteins are very sensitive molecules.
The term native state is used to describe the protein in its most stable natural conformation in situ. This native state can be disrupted by several external stress factors including temperature, pH, removal of water, presence of hydrophobic surfaces, presence of metal ions and high shear. The loss of secondary, tertiary or quaternary structure due to exposure to a stress factor is called denaturation. Denaturation results in unfolding of the protein into a random or misfolded shape. A denatured protein can have quite a different activity profile than the protein in its native form, usually losing biological function.
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. The properties of amino acids also influence the tertiary structure or overall shape of the protein. One type of interaction that plays a major role in the correct folding of a protein is the hydrophobic interaction. As a polypeptide folds into its correct shape, amino acids with nonpolar side chains usually cluster at the core of the protein, staying away from water.
Once the nonpolar amino acids have formed the nonpolar core of the protein, weak van der Waals forces stabilize the protein. Furthermore, hydrogen bonds and ionic interactions between the polar, charged amino acids contribute to the tertiary structure.
These are all weak interactions in the cellular environment, but their cumulative effect helps give proteins their unique shape. Disulfide bridges, covalent bonds formed between two cysteine residues, further reinforce the shape of a protein. Disulfide bridges form when the sulfhydryl groups of two cysteine residues come into close contact because of protein folding.
Covalent bonds are not a weak interaction. Primary structure determines tertiary structure and protein function. The most important proof of this came from experiments showing that denaturation of a protein is reversible. Certain proteins denatured by heat, extreme pH, or denaturing reagents will regain their native structure and original biological function when conditions return to the state in which the native conformation of the protein was stable. For example, ribonuclease denatures in the presence of urea and mercaptoethanol, two denaturing reagents.
The disulfide bridges break apart in the reducing environment. Ribonuclease regains its native conformation after the removal of urea and mercaptoethanol. This is just one of the many examples that have shown that primary structure determines the tertiary structure.
Most proteins probably go through several intermediate structures on their way to the most stable shape, and there is no way of knowing these intermediate forms by just looking at the final, folded protein. Crucial to the folding process are chaperonins, protein molecules that assist in the proper folding of other proteins. Chaperonins keep the protein away from the disruptive chemical conditions in the cytoplasmic environment and allow the polypeptide to fold spontaneously.
Defects in protein folding provide the molecular basis for a number of genetic disorders. The structure of proteins determines their function. Therefore, an incorrectly folded protein in the human body can have catastrophic effects on the individual.
The tertiary structure of a protein can be affected by misfolding of a protein or by a change in the primary structure of the protein.
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. The normal protein has lots of alpha helical regions and is soluble.
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