Repetitive amino acid sequences

Proteins are usually separated into two distinct functional classes passive structural materials, which are built up from long fibers, and active components of cellular machinery in which the protein chains are arranged in small compact domains, as we have discussed in earlier chapters. In spite of their differences in structure and function, both these classes of proteins contain a helices and/or p sheets separated by regions of irregular structure. In most cases the fibrous proteins contain specific repetitive amino acid sequences that are necessary for their specific three-dimensional structure. 

Fibrous proteins are long-chain polymers that are used as structural materials. Most contain specific repetitive amino acid sequences and fall into one of three groups coiled-coil a helices as in keratin and myosin triple helices as in collagen and p sheets as in silk and amyloid fibrils. 

Silk is produced from the spun threads from silkworms (the larvae of the moth Bombyx mori and related species). The main protein in silk, fibroin, consists of antiparallel pleated sheet structures arranged one on top of the other in numerous layers (1). Since the amino acid side chains in pleated sheets point either straight up or straight down (see p. 68), only compact side chains fit between the layers. In fact, more than 80% of fibroin consists of glycine, alanine, and serine, the three amino acids with the shortest side chains. A typical repetitive amino acid sequence is (Gly-Ala-Gly-Ala-Gly-Ser). The individual pleated sheet layers in fibroin are found to lie alternately 0.35 nm and 0.57 nm apart. In the first case, only glycine residues (R = H) are opposed to one another. The slightly greater distance of 0.57 nm results from repulsion forces between the side chains of alanine and serine residues (2). 

The majority of this matrix is composed of a protein called spectrin, a heterodimeric protein containing a 220-kDa a subunit and a similar but slightly larger /3 subunit. The highly repetitive amino acid sequences of both the a and /3 subunits give them a filamentous three-dimensional structure. As the a and /3 subunits of spectrin associate with one another, they form the flexible monomeric units that are used to create the membrane skeleton. 

The common feature of these protein polymers is the presence of repetitive sequence motifs which form defined secondary structures. These repetitive amino acid sequences offer the possibihty to construct artificial genes by mul-timerization of small synthetic oligonucleotide sequences and thus the build up of high molecular weight proteins. The constructed artificial genes can be incorporated into an expression plasmid, which can subsequently be transferred to a bacterial host for production of the desired polypeptide (Fig. 19). The most commonly used host is E. coli. 

The cDNA of the sialic-acid-specific lectin of S. sanguis codes for a polypeptide of 1435 residues (calculated mw of 158.4 kDa) with three unique domains, two of which consist of repetitive amino acid sequences [66]. The third, which resides near the earboxy terminus, contains 48% proline. The lectin bound to a single salivary glycoprotein of mw 400 kDa [66]. Binding was inhibited by sialic acid and was abolished by desialylation of the glycoprotein the best inhibitor was A -acetylneuraminyllactose. 

Figures 4 and 5 illustrate the use of these shape descriptors. As a first example, we have considered two proteins with a similar number of amino acid residues but radically different folding patterns. Figure 4 contrasts the backbone of ribonudease inhibitor ( = 456) and yeast hexokinase ( = 457). These structures are found in the Brookhaven Protein Data Bank (PDB) with the codes IBNH and 2YHX, respectively. Ribonudease inhibitor is a very unusual horseshoe-shaped protein, the first known 3D structure of a protein with a highly repetitive amino acid sequence. Table 1 gives their size and entanglement characterization in terms of Rq, A, N, andN. Protem IBNH is less compact and less entangled than 2YHX (note the smaller N and N values).

TABLE 2.1 Repetitive Amino Acid Sequences Found in Silkworm and Spider Fibroins... 

Coiled-coil a helices contain a repetitive heptad amino acid sequence pattern... 

Coacervation occurs in tropoelastin solutions and is a precursor event in the assembly of elastin nanofibrils [42]. This phenomenon is thought to be mainly due to the interaction between hydro-phobic domains of tropoelastin. In scanning electron microscopy (SEM) picmres, nanofibril stmc-tures are visible in coacervate solutions of elastin-based peptides [37,43]. Indeed, Wright et al. [44] describe the self-association characteristics of multidomain proteins containing near-identical peptide repeat motifs. They suggest that this form of self-assembly occurs via specific intermolecular association, based on the repetition of identical or near-identical amino acid sequences. This specificity is consistent with the principle that ordered molecular assembhes are usually more stable than disordered ones, and with the idea that native-like interactions may be generally more favorable than nonnative ones in protein aggregates. 

In view of the repetitive 3D structure of /3-solenoids, one might expect their amino acid sequences also to be repetitive. Indeed, some /8-solenoids... 

In contrast to polysaccharides, protein antigens do have tertiary structure in solution this leaves only a limited number of amino acid sequences exposed at the surface of the molecule. Of these determinants, few, if any, will be repetitive. It would therefore be ex-... 

To some extent, the properties of the protein are mainly determined by its primary structure (i.e., the amino acid sequence). The two kinds of structural protein, fibroin and spidroin have a distinct and highly repetitive primary structure, which results in specific secondary and tertiary... 

Isolation and Characterization of cDNA Clones Encoding the Poly-phenolic Protein. Characterization of the primary amino acid sequence of the mussel adhesive protein has been hindered by the large size of the protein and the repetitiveness of the amino acids. In such cases, the practical (and perhaps only) approach for determining the complete amino acid sequence is to clone DNA sequences encoding the protein and to deduce the amino acid sequence from the genetic code carried by that DNA. To accomplish this, we obtained mRNA from mussels and synthesized cDNA in vitro. 

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