Secondary structure engineering

Table 1 contains the metal-to-metal engineering property requirements for Boeing Material Specification (BMS) 5-101, a structural film adhesive for metal to metal and honeycomb sandwich use in areas with normal temperature exposure. The requirements are dominated by shear strength tests. Shear strength is the most critical engineering property for structural adhesives, at least for the simplistic joint analysis that is commonly used for metal-to-metal secondary structure on commercial aircraft. Adhesive Joints are purposefully loaded primarily in shear as opposed to tension or peel modes as adhesives are typically stronger in shear than in Mode I (load normal to the plane of the bond) loading. 

Altogether, the identification of the coordinating residues in the endogenous hDAT Zn2+ binding site followed by the engineering artificial sites have defined an important series of structural constraints in this transporter. This includes not only a series of proximity relationships in the tertiary structure, but also secondary structure relationships. The data also provided information about the orientation of TM7 relative to TM8. A model of the TM7/8 microdomain that incorporates all these structural constraints is shown in Fig. 4 (36). The model is an important example of how structural inferences derived from a series of Zn2+ binding sites can provide sufficient information for at least an initial structural mapping of a selected protein domain. 

In addition, Zn2+ was shown to inhibit dopamine uptake in a mutant containing an engineered tridentate zinc site, in which the i-4 site from His3757.60, Met3717.56, was replaced with histidine, whereas the introduction of histidines at the i-2, i-3, and i-5 position did not increase Zn2+ affinity (29). In contrast, histidines at positions i+2, i+3, and i+4 all resulted in potent inhibition of dopamine uptake by Zn2+. The incorporation of these data in a model of secondary structure provides evidence for an a-helical configuration of the extracellular portion of TM7, as well as the absence of well-defined secondary structure between positions 3757.6o and 37 97.64 (Fig. IB), thereby suggesting an approximate boundary between the C-terminal end of the helix and the beginning of EL4 (29). 

The pattern of accessibility of the engineered cysteines that react with the sulfhydryl-speciiic reagents along the primary amino acid sequence reflects the secondary structure of the region. If one side of an a hehx... 

Fig. 3. The hepatitis delta virus ribozyme. A Secondary structure of the genomic HDV ribo-zyme RNA used for the determination of the crystal structure [37]. The color code is reflected In the three dimensional structure B of this ribozyme. PI to P4 indicate the base-paired regions. Nucleotides in small letters indicate the U1 A binding site that was engineered into the ribozyme without affecting the overall tertiary structure. The yellow region indicates close contacts between the RNA and the U1 A protein...

Independently, Ruan etal. (1990) demonstrated that unnatural metal-ligating residues may likewise be utilized toward the stabilization of short a helices by transition metal ions (including Zn " ")—these investigators reported that an 11-mer is converted from the random coil conformation to about 80% a helix by the addition of Cd at 4°C. These results suggest that the engineering of zinc-binding sites in small peptides or large proteins may be a powerful approach toward the stabilization of protein secondary structure. 

The fastest-folding small proteins generally fold on much slower time scales than the time scale of formation of secondary structure. The speed record is currently held by lambda(6-85), a truncated, monomeric form of the N-terminal domain of lambda repressor, which refolds with a half-life of approximately 140 fjis. A thermostable lambda(6-85) variant with alanine substituted for glycine residues 46 and 48 in the third helix folds faster in dilute solutions of de-naturant, with an extrapolated half-life of less than 10 /us in water.13 Cold-shock protein CspB from Bacillus subtilis folds in about 1 ms.61 Engineered mutants of the P22 Arc repressor62 and CI263 fold in a fraction of a millisecond. 

RD Chen, A Greer, JM Hurley, AM Dean. Engineering secondary structure to invert coenzyme specificity in isopropylmalate dehydrogenase. In DR Marshak, ed. Techniques in Protein Chemistry VIII. New York Academic Press, 1997, pp 809-816. 

Ramesh, V, Amitabha De, and Nagaraja, V. (1994) Engineering hyperexpression of bacteriophage Mu protein C by removal of secondary structure at the translation initiation region. Prot. Engineer. 7,1053-1057. 

Enzymes and Recombinant DNA Technology. Recombinant DNA technology for whatever purpose depends absolutely upon enzymes. Keys to the rapid advancement of biochemical engineering have been 1) understanding of the primary and secondary structures of... 

Smolke CD, Keasling JD (2002) Effect of gene location, mRNA secondary structures, and RNase sites on expression of two genes in an engineered operon. Biotechnol Bioeng... 

Rational protein engineering aims to exchange and fuse secondary structure elements and domains at precise locations, based on predictions through rational means, to form a functional hybrid structure. Despite the great complexity involved in rational protein engineering, two outstanding experimental successes were recently reported for /1-barrel and f>/a-barrel structures. 

---