A protein or polypeptide is comprised of monomer amino acids chained together via peptide bonds. This sequence of amino acids (residues) defines the 1° structure. By convention a polypeptide starts on the end of the amino acid with its amine group exposed (the N-terminus) and finishes at the opposite end of the chain on the amino acid with its carboxyl group exposed (the C-terminus).
The 2° structure of a protein is characterized by local structures formed of the underlying polypeptide sequence. Hydrogen bonding is largely responsible for holding these structures, which include alpha-helices and beta-sheets.
A protein's 3° structure comprises its three-dimensional structure or geometric shape, largely due to interactions between the side chains on its constituent amino acid residues during the process of protein folding.
Proline is unique among the amino acids in that its side chain is a cyclic structure that forms a second alkyl group to its amine. Two consequences of having this five-membered ring that twice connects to the amino acid's backbone are conformational rigidity and an inability to act as a donor for hydrogen bonding (due to lack of an available hydrogen once proline has been incorporated into a polypeptide). These restrictions translate to proline acting as a destabilizing element in the middle of 2° structures (e.g. kinks in an alpha-helix) but useful in accommodating tight turns and in the folding of the protein.
Disulfide bonds between two cysteine residues act as a bridge to connect different parts of the folded protein. This cysteine dimer, called cystine, aids in the stabilizing the protein's final conformation.
Hydrophobic bonding influences protein folding by bringing together portions of the polypeptide with hydrophobic side chains, generally into the interior of the protein, and protecting them from an aqueous environment.
4° structure reflects the final protein's composition of multiple polypeptides. These folded subunits can be identical or non-identical, involve multiple subunits (e.g. dimer, tetramer), and are held together by non-covalent interactions and disulfide bonds.
A protein takes its unique conformation firstly from the sequence of amino acids comprising its 1° structure as well as the process of folding that it undergoes. The stability of this conformation can be described by ΔG (the difference in Gibbs Free Energy) between its various states of conformation (e.g. folded vs unfolded). Hydrogen bonding and the hydrophobic effect play significant roles in the conformational stability of a protein, which is dependent on temperature, pH, salt concentration, and the presence of chaperones (other proteins that assist in folding and unfolding).
To be biologically active, a protein must adopt and maintain a specific conformation under physiological conditions.
The process of protein folding often begins while the polypeptide sequence is still being translated and can be assisted by chaperones. Problems with chaperone-assisted folding or inappropriate conditions (i.e. temperature, pH, salt concentration, solvent) can cause misfolding, which leads to an inactive or even toxic protein. Similar to misfolding, a change in conditions can also cause denaturing, the loss of 4°, 3°, and 2° structures as a protein unravels.
|Condition||Potential Effect||Potentially affected structures|
|Temperature||spontaneous rearrangement at "melting temperature"||2°, 3°, 4°|
|pH||disruption of ionic bonds and hydrogen bonds||2°, 3°, 4°|
|Salt concentration||disruption of non-covalent interactions||2°, 3°, 4°|
|Solvent||disruption of hydrogen bonding||2°, 3°, 4°|
|Enzymes||Hydrolysis of peptide bonds||1°|
Hydrophobic interactions utilize both repulsion and attraction (a push and a pull) to contribute to a protein's conformational stability. The push comes from the thermodynamically favorable shielding of hydrophobic residues afforded by their location inside the protein. The pull results from van der Waals forces between nonpolar side chains on the polypeptide, especially London dispersion forces, which are amplified in the close quarters of a hydrophobic core and produce a greater affect in larger proteins.
The solvation layer (or shell) describes the structured organization of a solvent (e.g. water) around a solute (e.g. a polypeptide or protein). In the case of a protein which displays hydrophobic residues on its surface, the surrounding water will orient into a highly structured organization to optimize hydrogen bonding among water molecules (as hydrogen bonding with the presented hydrophobic side chains is not an option). This highly ordered rearrangement has a much lower entropy and is less favorable than if polar side chains were present on the surface of the protein. Thus, a conformation that buries its hydrophobic residues inside the protein leads to less disruption of water's hydrogen bonding, allowing for less structure and higher entropy, which increases the protein's conformational stability.
Proteins have a number of characteristics based on the variety of attributes and interactions of their constitutive amino acid residues. These characteristics can be harnessed by separation techniques to isolate and analyze proteins. The characteristics of particular proteins, such as size, charge, and solubility, will influence the choice of separation technique.
An isoelectric point (pH(I)) is the pH at which the protein has no net electric charge. The isoelectric point is influenced by the anionic or cationic character of the protein's amino acid side chains at a certain pH. Separation can be performed by the movement of proteins over a pH gradient in a gel electrophoresis. Proteins at their isoelectric point also have lower solubility and may precipitate out of solution.
Electrophoresis focuses on separating proteins mainly by size or charge in the course of moving across an electric field, usually with a support medium (e.g. a gel). At the end of the migration, the proteins can be stained to show the location of various protein samples, and conclusions can be drawn about the characteristics of the protein. For example, a small protein will travel farther than a larger protein, and a positively charge protein will be pulled towards the cathode (-) while a negatively charged protein will be pulled towards the anode (+).
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