Sequence specificity, synthetic proteins

Figure 8.15 Sequence-specific protein-DNA interactions provide a general recognition signal for operator regions in 434 bacteriophage, (a) In this complex between 434 repressor fragment and a synthetic DNA there are two glutamine residues (28 and 29) at the beginning of the recognition helix in the helix-turn-helix motif that provide such interactions with the first three base pairs of the operator region.

Sequence-specific heteropolymers, as a class of synthetic molecules, are unique in that they must be made by chemical steps that add one monomer unit at a time. Moreover, to create truly protein-like structures, which typically have chain lengths of at least 100 monomers and a diverse set of 20 side chains (or more), extremely efficient and rapid coupHngs under general reaction conditions are necessary. For these reasons, soHd-phase synthesis is typically used, so that excess reagents can be used to drive reactions to completion, and subsequent reaction work-ups are quite rapid. 

Elastin-mimetic protein polymers have been fabricated into elastic networks primarily via y-radiation-induced, radical crosslinking of the material in the coacervate state [10]. Although effective, this method cannot produce polymers gels of defined molecular architecture, i.e., specific crosslink position and density, due to the lack of chemoselectivity in radical reactions. In addition, the ionizing radiation employed in this technique can cause material damage, and the reproducibility of specimen preparations may vary between different batches of material. In contrast, the e-amino groups of the lysine residues in polymers based on Lys-25 can be chemically crosslinked under controllable conditions into synthetic protein networks (vide infra). Elastic networks based on Lys-25 should contain crosslinks at well-defined position and density, determined by the sequence of the repeat, in the limit of complete substitution of the amino groups. 

A variation on the theme has been to map out protease specificity.28 A library of fusion proteins was constructed in a modular manner. The synthetic protein had an N-terminal domain that binds very tightly to an affinity column. This domain was connected to the C-terminal domain of M13 gene III by a randomized peptide sequence. The phages were then bound to the affinity support and treated with a protease. Phages that had a protease-susceptible site were cleaved from the support and eluted. This procedure was subsequently used to map out the specificity of furin,29 which is described in the next chapter. 

Ribonucleases are a widely distributed family of en-zymes that hydrolyze RNA by cutting the P—O ester bond attached to a ribose 5 carbon (fig. 8.12). A good representative of the family is the pancreatic enzyme ribonuclease A (RNase A), which is specific for a pyrimidine base (uracil or cytosine) on the 3 side of the phosphate bond that is cleaved. When the amino acid sequence of bovine RNase A was determined in 1960 by Stanford Moore and William Stein, it was the first enzyme and only the second protein to be sequenced. RNase A thus played an important role in the development of ideas about enzymatic catalysis. It was one of the first enzymes to have its three-dimensional structure elucidated by x-ray diffraction and was also the first to be synthesized completely from its amino acids. The synthetic protein proved to be enzymatically indistinguishable from the native enzyme. 

Recognition of a specific amino acid sequence from a protein or a specific tag peptide sequence is expected to lead to variety of medical and materials applications. Whereas the recognition of a single amino acid residue by synthetic hosts has been reported previously, the artificial recognition of a sequence of two or more amino acid residues has been quite limited. There are only a few reports on the... 

Based on these mechanisms a new frontier in the application of enzymes to biotechnology, including the development of synthetic enzymes (synzymes)82,83 will be exploited. Research on structure-function relationships between ribozymes and abzymes will lead to the development of a number of sequence specific catalysts, which will control expression of a specific gene or its products, and eventually to application as pathogen controls in agriculture and to clinical use. Sequence specific abzymes may also facilitate research on the primary structure determination of protein. 

The future importance of peptide vaccines lies in the fact that one could replace inactivated or attenuated microbial pathogens or toxins, which are high-molecular and therefore difficult to characterize and standardize, by highly specific synthetic peptides. Emini et al.157 have synthesized oligopeptides that prime the rabbit immune system and are effective against poliovirus. The amino acid sequence of the peptide vaccines 63 and 64 originate in the poliovirus VPt protein. 

We have already discussed primary structure in terms of the general character of amino acids and some specific examples of amino acid sequences in certain proteins will be discussed later. Our attention now is focused on secondary structure, or conformation as we called it when we discussed synthetic polymers. There are a number of factors that afreet the conformation of a polypeptide chain and a lot can be learned initially by just focusing on two of these steric restrictions on bond rotations and the strong driving force for amide groups to hydrogen bond to one another. 

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