The function of the protein depends on its 3D conformation. Proteins are modular in nature and their interactions with other molecules in the cell rely on the presence of specific functional domains. The precise shape of the domain, resulting from the presence of non covalent bonds between residues in a polypeptide chain decides about the function. The best known example of the shape-function relationship is the “key and lock” strategy of enzymatic function. The change of enzymatic pocket, due to mutation or modification of an amino acid residue changes the affinity of the enzyme. In short, the better fit between two molecules, the better it functions, the more bonds can be made, the faster the signal can pass, or the stronger two molecules connect (think adhesion molecules). Show
The 3D conformation of the protein depends on the interactions between amino acids in the polypeptide chain. Since the sequence of the amino acids is contingent on the DNA code, the shape of the protein is encoded in the DNA. Proteins have four levels of organization. Primary structure refers to the linear sequence of the amino acids connected by the peptide bonds. The secondary structure consists of local packing of polypeptide chain into α-helices and β-sheets due to hydrogen bonds between peptide bond – central carbon backbone. Tertiary (3D) structure is a shape resulting from folding of secondary structures determined by interactions between side chains of amino acids. Quaternary structure describes the arrangement of the polypeptide chains in the multi subunit arrangement. Following translation, translocation or insertion into ER membrane, proteins are modified to assume their final structure and therefore function. Post-translational modifications change the chemical nature of the polypeptide chain through alterations to amino acid residues. Post-translational modifications take place in the ER and include folding, glycosylation, multimeric protein assembly and proteolytic cleavage leading to protein maturation and activation. They take place as soon as the growing peptide emerges in the ER and is exposed to modifying enzymes. Only properly folded, properly modified proteins are transferred to Golgi and secreted through the process of exocytosis; out of the cell for secretory proteins or merging with the plasma membrane for membrane proteins. Misfolded or unfolded proteins accumulate in the ER and cause upregulation of molecular chaperones through the ER overload response. If chaperones cannot fix the problem, defective protein would be transported back to cytosol, ubiquitinated and destroyed in proteasomes. Post-translational modifications take place also in cytosol. Some of them, for example phosphorylation can happen repeatedly throughout the life of the protein and are the frequent way of regulating protein activity (for example turning the enzyme on and off). In doing so, each protein becomes prepared for its specific and unique function in the cell, taking into the account local environmental conditions at the final destination place. Maturation of secretory and membrane proteins in the ER and GolgiFollowing translation all proteins go through the maturation process before they are ready to assume their cellular functions. The finishing touches are added to the protein in the form of disulfide bonds, attachment of sugars (glycosylation) or proteolytic cleavage, just to name a few. These modifications change the chemical nature of the amino acid’s side chain but they don’t affect primary sequence of amino acids assembled during translation. However, the modifying enzymes begin to work on the amino acids as soon as they emerge from the ribosomal tunnel. In reality, post-translational modifications take place co-translationally as the peptide elongates or grows and portrudes into the ER or cytosol. Let’s look in detail at a few posttranslational modifications that have the biggest influence on protein shape and hence their function. FoldingFolding is a post-translational modification that happens to every protein, whether its final habitation site is cytosolic, mitochondrial, intrinsic membrane or extracellular. Folding is based on the interactions between side chains of the amino acid residues in the protein chain. But achieving proper interactions might be more difficult than you think – after all there is an interference from other molecules, mostly water, surrounding them them. That’s where chaperones come into play. As in real life, they temporarily protect the maturing polypeptide and shelter it from making undesirable connections. Molecular chaperones or a story of heat shock proteins You have already heard about molecular chaperones in conjunction with translocation of mitochondrial proteins. Cytosolic chaperones are responsible for keeping newly synthesized matrix proteins unfolded until they made it to the translocation channel (Tom). Mitochondrial matrix chaperones sequester the peptide upon its arrival in the matrix and keep it unfolded until the entire length of the polypeptide chain makes it to the other side. Similarly, there is a pool of chaperones in the ER that aids the passage of nascent proteins during translocation into the ER until all parts of the polypeptide emerge and can be folded correctly. Disulfide bond formation Disulfide bonds bring distant parts of the protein together and create major folds. (Figure protein structure-disulfide. jpg wrapped in a text) They also link separate parts of protein together (see insulin molecule and its three disulfide bonds). However, they are also the “weakest” links and can be easily reduced or broken, as a means of inactivating an enzyme to terminate the reaction or stopping a hormone action when it is no longer needed. It all comes together in the ER, formation of multimeric proteinsER is also the place where the subunits come together to create multimeric proteins. Joining of separate polypeptide chains follows the same physical laws as protein folding, an attraction of the hydrophobic surfaces and reactivity of sulfhydryl groups in cysteines. Most often subunits are products of separate genes, and the ER serves as a waiting room for short peptides awaiting longer ones that take a longer time to assemble. Proteins have to be fully assembled before they are allowed to exit the ER and continue to Golgi. GlycosylationA unique glycosylation, attachment of mannose-6-phosphate, has a double duty for lysosomal proteins. It works as targeting signal for lysosomal enzymes and then protects them from being digested. Carbohydrate chains are added to proteins in a reaction called glycosylation, which occurs in the ER or Golgi. An N-linked glycosylated protein acquires its carbohydrate chain as a preformed oligosaccharide in the ER, and an O-linked glycosylated carbohydrate chain is added in the Golgi, one sugar at a time. N-linked glycosylation starts as soon as glycosylated residues, always asparagine or arginine, make it to the lumen of the ER as a nascent peptide, which is still unfolded. A preformed oligosaccharide is then transferred from an ER membrane-bound lipid carrier, dolichol, to the polypeptide chain. O-linked glycosylation that occurs in Golgi during later stages of protein maturation is catalyzed by sugar-specific glycosyl transferases. Sugars are transferred from the nucleotide precursors. This process is important in the formation of extracellular matrix proteins and will be discussed in later chapters. The most well-known O-linked glycosylated proteins and lipids are the ABO blood types and are the result of genetically coded presence or absence of two glycosyl transferases. All people have enzymes necessary for O blood group, and make O antigen. People with A blood group have an additional GalNAc transferase that adds the extra N-acetylgalactosamine, while people with B type have Gal transferase that adds the extra galactose. People with AB group have both of these enzymes and produce three antigens, O, A and B. Targeting to lysosomesGlycosylation is also used to deliver proteins to their proper destination. Delivery of resident lysosomal enzymes, most of which are hydrolases, is based on the attachment of special kind of sugar, mannose-6-phosphate to the precursor proteins in the Golgi. Future lysosomes proteins are first synthesized and targeted to the ER the same way as are future secreted proteins, by the N-terminal ER-targeting sequence. Once inside the ER, they are post-translationally modified and send to cis Golgi in the vesicles. Once in Golgi, they are glycosylated with mannose-6-P group. Mannose-6-phosphate binds to a receptor at trans-Golgi and in the process of the vesicular progression, the vesicle is delivered to the endosome, that later matures into lysosome. The newly delivered protein (lysosomal enzyme) remains in the lysosome and the attached mannose sugars protect the protein from being digested by other hydrolases. Proteolytic cleavageProteolytic cleavage is a common method of activation of proteins from the precursors . It happens late in protein maturation, usually just prior to exocytosis or even after secretion, in the blood or destination place. The examples of proteolytic cleavage by prohormone convertases are numerous and well known; prothrombin to thrombin, proinsulin to insulin or even multistep reactions like activation of vasoconstricting angiotensin II from angiotensinogen in 2 step reaction, with partially active angiotensin I being an intermediate step. Proteolytic cleavage is irreversible; once you sever a protein you can’t seal it back. Remember, that all of the hormones (secretory proteins) have already undergone their first proteolytic removal of ER targeting sequence, a pre- part of full coding sequence of pre-pro-hormone). Activation of protein by deletion of its part does not seem like the most efficient way of managing cellular resources, but it is the most straightforward way of activating fast-acting molecules by keeping the inactive forms of s in circulation. After all, it takes much less time to remove a “blocking” fragment then synthesize a new protein on demand. Reversible modifications turn on/off cellular processesPost-translational modifications are not reserved to early steps in maturation or confined to ER and Golgi. Especially in cytosol, they are reversible, and used to regulate protein activity. One of the most frequent methods of turning on/off catalytic activity of an enzyme is phosphorylation. Addition of high energy phosphate group that is very hydrophilic to side chains of hydrophobic amino acids, serine, threonine or tyrosine alters the interactions with substrates, creates binding sites or opens catalytic pockets. Phosphorylation is not always activating process; sometimes it interferes with substrate binding and stops the reaction. During fight or flight response, discussed in the chapter about stress, phosphorylation by protein kinase A has dual/divergent action; it activates one enzyme, glycogen phosphorylase and inhibit an enzyme with the opposing activity, glycogen synthase. Enzymes that catalyse the transfer and attachment of phosphate group are called kinases, enzymes responsible for hydrolysis of the bond are called phosphatases. Especially in recent years phosphatases have come to the prominence as their action, or lack thereof were implicated in prolonged activation of cellular signaling leading to serious consequences such as cancer. A special form of glycosylation is the transient attachment of glycosylphosphatidylinositol (GPI anchor) to the C-terminus a protein. Addition of GPI temporarily affixes the protein to the cell membrane where it participates in signal transduction. Other posttranslational modifications involved in intercellular signaling are described in No cell is an Island chapter. Which of the following is a postThese modifications include phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation and proteolysis and influence almost all aspects of normal cell biology and pathogenesis.
What are the postPosttranslational modifications (PTMs) are covalent processing events that change the properties of a protein by proteolytic cleavage and adding a modifying group, such as acetyl, phosphoryl, glycosyl and methyl, to one or more amino acids (1).
Which of the following are postWhat are the six major forms of PTM? phosphorylation, glycosylation, ubiquitination, sumoylation, protein cleavage, and addition of vitamin/mineral cofactors.
What is the most common postProtein phosphorylation (Figure 2) is the most commonly studied post-translational modification. It has been estimated that one-third of mammalian proteins may be phosphorylated, and this modification often plays a key role in modulating protein function.
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