The structure of a peptide is dependent on its amino acid make up, post translational modifications (such as disulfide bonds) and interactions formed between each side chain. Proteins are translated at the endoplasmic reticulum (ER) as long unfolded peptide chains which then assemble into a 3D structure. Although peptide folding is heavily influenced by sequence this is not the sole determining factor, particularly in the case of larger proteins. For example human proteins expressed in E.coli for medicinal or research purposes often require refolding following purification.
The normal secondary and tertiary structure of a protein is called the "native" structure. This structure can be destroyed, for example by heat, extremes of pH, organic solvents and chaotropic agents. A protein in this state is described as "denatured." A protein which returns to its native shape when the denaturing influence is removed is called "renatured," however renaturing many proteins is not possible. The structure of a peptide can be broken down into four components:
The primary structure of a peptide reflects the evolutionary history and in many cases is highly conserved between species. The secondary structure can be predicted from analysis of the primary structure using programs such as Rosetta and hydropathy plots. The tertiary structure can be predicted using analytical methods such as Ab Initio folding, and 'threading' of one protein onto another. These predictive methods are continually improving, and are often used to support experimental data. However, at present, only experimental techniques such as X-ray crystallography and NMR can accurately predict structure.
Protein folding requires an overall negative free energy change. This requires the sum of the entropy of hydrophobic effects and the enthalpy change with hydrogen bonds and Van der Waals forces to be negative.
Protein folding is a cooperative process, meaning it is an 'all or nothing' transition. If the folding of the protein provides a negative free energy change, then folding will proceed. If not, the protein will remain unfolded. Protein folding has a very sharp transition due to interactions within the protein. Once one domain begins to fold, this stimulates interactions between other domains of the protein causing them to fold too. A domain in its correctly folded state will be very stable. This intermediate will then be retained, allowing other domains to reach their correct fold. As each domain beings to fold, there will be an increase in stability of the protein, until the overall fold has been achieved. For this reason, in a normal cell, you will only find a protein either completely unfolded, or fully folded.
Formation of an alpha-helix is driven by a repeat sequence of hydrophobic amino acids every 3.6 residues. For example, in the sequence below, hydrophobic residues at amino acids 1 and 4 would drive formation of an alpha-helix:
This leads to coiling of the strand with hydrophobic residues on one side and hydrophilic on the other. In globular proteins this is driven by the need to reduce unfavourable interactions with water. This causes hydrophobic residues to be found on the inside of a peptide, and hydrophilic on the exterior surface. Amino acids have a certain degree of preference in the secondary structure that they form. Methionine, Alanine, Leucine, Glutamate and Lysine are commonly found in alpha-helices.
There are several forms of helix which can be formed, with the right handed alpha-helix being the most common:
The stability of an alpha-helix is affected by:
The formation of beta-strands is driven by hydrophobic interactions between R groups of neighbouring amino acids. Alternating hydrophilic and hydrophobic residues lead to a 'one up one down' pattern of amino acids. At the surface of a protein, the alternating residues lead to a gathering of hydrophilic residues on the exterior and hydrophobic on the interior of the protein.
Stacking of beta-strands to form beta-sheets is driven by Van der Waals forces. If a single beta-strand has hydrophilic residues on one side of the strand, this will attract hydrophilic residues of neighbouring strands. This leads to grouping of hydrophilic/hydrophobic amino acids, causing intermeshing of amino acids from consecutive beta-strands. Beta-sheets can be parallel or antiparallel, in reference to the directionality of adjacent beta-strands. Parallel strands form slightly distorted hydrogen bonds, needing hydrophobic interactions for stability.
In order to retain the compact structure of a protein, loops and turns are required to connect alpha-helices and beta-strands. These are usually found on the surface of the protein. Therefore they contain mainly hydrophilic residues and are often binding sites. Mutations often occur at these residues to allow for evolution of new function without disturbing the protein fold. For example all TIM barrels evolved from the same ancestor but have evolved into distinctly functional proteins due to mutations in their loops and turns.
A turn reverses the direction of a helix/strand. This is stabilised by a hydrogen bond between the backbone CO of one amino acid with the NH of the next.
A loop is a much larger sequence which changes the direction of the helix/strand. There is no regular stricture or sequence of amino acids in these regions.
The supersecondary structure of a protein is formed by the combination of several neighbouring alpha-helices/ beta-sheets into a particular geometric arrangement. Several examples of these are shown below:
Once a secondary, and in some cases supersecondary, structure has been formed, the protein can then begin to fold into its tertiary structure. Included in this are several individual protein domains which may represent different functions within a protein. Domains may interact with each other or function completely independently of one another. For example, serine proteases contain two structurally diverged beta-barrels, with the active site found between them. The presence of protein domains aids protein folding, as each domain can fold individually, accelerating folding.
Beta-sheets form various tertiary structures, depending on their biological function. Included in this are: Beta-barrels forming transmembrane channels and beta-propellers. A further example is the EF hand which is found in Calmodulin and other structurally related proteins. This consists of a helix-loop-helix structure, which binds calcium at the loop region, acting as a messenger protein.
The majority of proteins are folded at the endoplasmic reticulum, as soon as they have been transcribed. However some proteins are required to maintain an unfolded state until they reach a particular place at a particular time. Proteins may also need to be partially unfolded to cross membrane barriers. Chaperone proteins assist the folding and unfolding of proteins, ensuring they form the correct folded state. Chaperones also help to prevent denaturation of proteins in response to stress signals.
Chaperone proteins can be separated into three groups:
These are found at the endoplasmic reticulum and aid the folding of newly synthesised proteins. An example of this is BiP which is involved in protein folding and ensuring proteins remain folded during transport throughout the cell.
Lectin Chaperones aid the folding of glycoproteins. For example Calnexin is a chaperone which prevents movement of incorrectly folded proteins through the secretory pathway.
This group of chaperones is involved in ensuring the correct bonds are formed within a folded protein. This may include aiding the breaking of existing bonds as well as the formation of new bonds. An example of this is Protein Disulphide Isomerase.
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