The alphabet of intrinsic disorder: II. Various roles of glutamic acid in ordered and intrinsically disordered proteins

Uversky, Vladimir N

Poly-γ-glutamate (PGA) is a natural homopolymer synthesized by several bacteria, one archaea (Natrialba aegyptiaca) and one eukaryote (Cnidaria). 135 One of the most known sources of PGA is the Japanese specialty natto, a fermentation product made by Bacillus subtilis grown on soybean. 135 PGA is a highly soluble polyanionic polymer that sequesters water molecules and can be found in surface-bound and released forms. In structural studies, polyglutamic acid is traditionally used as a biopolymer with a well-characterized secondary structure response to changes in the environmental pH, where PGA is in a random coil-like conformation at neutral pH, but gains monomeric α-helical structure at acidic pH and is transformed into a β-sheet structure at alkaline pH. [136] [137] [138] Curiously, the addition of polylysine to an aqueous solution of polyglutamic acid homopolypeptide at EBD is not a structurally stable entity in the conventional sense, since for this protein region there are no folded states that exist for any appreciable amount of time. Instead, the EBD represents a time-average 3D region of a protein derived from the thermally driven motion of certain polypeptide chains, including those that are part of an otherwise stable folded protein. 163 Therefore, the EBD which is defined by the time-averaged occupancy of space by a polypeptide chain, can exclude lager molecules while allowing small molecules and water to move freely through it. It was proposed that since functions of EBD depend on the intrinsically rapid thermal motion of the polypeptide, and the free energy changes that result when that motion is confined, this domain can be used to control binding events, confer mechanical properties, and sterically control molecular interactions. 163 Obviously, to be able to serve as an EBD, a given fragment of a protein has to possess specific amino acid composition that would preclude it from folding. Therefore, EBDs are expected to possess low hydropathy and high net charge; i.e., in the CH-plot, they can be found well above the boundary separating compact and extended disordered proteins. One of the illustrative examples of biologically active EBDs (which are not tightly folded, but expected to have a very extended conformation) is given by side-arms of neurofilament (NF) proteins. 164 The side-arms of the NF heavy polypeptide, NF-H (which are ~600 amino acids long), were shown by rotary shadow electron microscopy to be ~85 nm long. Since there was not enough mass to form a stiff folded structure to occupy such a volume, it was proposed that the side-arms were not folded but were in constant thermal motion. 164 Analysis of the amino acid sequence of the porcine NF medium polypeptide (NF-M, which has an apparent molecular mass of 160 kDa and is one of the two high molecular mass components of mammalian neurofilaments) revealed that this protein has several peculiar features. 165 The N-terminal 436 residues contain a non-α-helical arginine-rich headpiece (residues 1-98) with multiple β-turns followed by a highly α-helical rod domain that forms double-stranded coiled-coils (residues 99-412), followed by a C-terminal tailpiece extension (approximately 500 residues) that represents an autonomous domain of unique amino acid composition, being characterized by a high content of lysines and particularly glutamic acids. 165 In human NF-M, there are 185 glutamic acids (20.2%), most of which are concentrated within the C-terminal tail, where glu