New quaternary structures

To test these possibilities, random segments of four to seven residues were inserted into the middle of the EcCM HI helix [95]. The individual libraries (designated L4, L5, L6 and L7) have a maximum theoretical diversity of 160,000 (204), 3.2 x 106 (205), 6.4 x 107 (206), and 1.28 x 109 (207) distinct members, respectively. In each case, transformation of chorismate mutase-deficient bacteria yielded roughly 107 clones, giving fully diverse and redundant coverage of the L4 and L5 libraries, 10 % sequence coverage of the L6 library, and 1 % coverage of the L7 library. 

No functional clones were found in the L6 library under selection conditions, but 0.05 %, 0.002 %, and 0.5 % of the L4, L5, and L7 libraries, respectively, complemented the genetic defect. Several active enzymes from the libraries were characterized biochemically (Fig. 3.14). Insertion of four or seven amino acid segments into the middle of the HI helix yielded monomeric enzymes with near wild-type activity, but these proteins were unstable and tended to aggregate. In contrast, insertion of a five amino acid segment (Cys-Phe-Pro-Trp-Asp) yielded a well-behaved hexameric chorismate mutase (as judged by analytical ultracentrifugation) that is about 200-fold less active than the wild-type protein. 

The properties of the hexameric species, when compared with those of the monomers, suggest that protein stability is an important driving force in the evolution of oligomeric proteins [95]. However, additional work is needed to elucidate how the subunits are organized and how quaternary structure influences function. 

On a more general note, loop length appears to play a more important structural role than might have been anticipated from other experiments [83]. The low percentage of 

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Packman, L.C., and Perham, R.N. (1982) Quaternary structure of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus studied by a new reversible cross-linking procedure with bis(imidoesters). Biochemistry 21, 5171-5175. 

Figure 4.3 Myoglobin and hemoglobin quaternary structures. (Reprinted from Figure 4.2 of Cowan, J. A. Inorganic Biochemistry, An Introduction, 2nd ed., Wiley-VCH, New York, 1997. Copyright 1997, Wiley-VCH.)...

The new quaternary pavine base (-)-caryachine A-metho salt was isolated from Cryptocarya chinensis (29), previously shown to yield both the levorotato-ry and racemic forms of caryachine (7) (30,31). The structure 16 was derived... 

The structure proof of the second new quaternary base, (—)-eschscholtzidine Al-metho salt (17), isolated as its chloride from the fruits of Thalictrum revo-lutum, was provided by spectral analyses and direct comparison of the compound with the N-metho salt prepared from authentic (—)-eschscholtzidine (8), also present in the plant (32). 

Spectral studies indicated that the new quaternary base dendro-wardine (C19H3204N-C1 mp 168-172° [ct] 5 —28°) isolated from this plant has structure 85a. When treated with lithium hydride in DMF there was generated 85b which on hydrogenolysis (Pt02) gave dihydronobilinone (85c) (53a). 

In this review, we shall focus on new approaches with particular emphasis on the subunit assembly in the cell after a brief summary of the functional aspects of quaternary structure, because the comprehensive review of the quaternary structure written by Klotz, Darnall and Langerman3) in 1975 is essentially valid. 

As demonstrated by the examples above, recent studies aimed at understanding the relationship between the quaternary structure and the physiological activity of oligomeric proteins have produced a considerable amount of information which explains many aspects of this phenomenon. In the process, a number of experimental methods have been used. The following sections provide an overview of new approaches which are generally applicable for elucidating the structure-function relationship of oligomeric proteins. 

During the period of time when the nature of the 19-nortestosterone acetate-dependent photoinactivation was under investigation, a new bacterial steroid isomerase was obtained from extracts of Pseudomonas putida (Biotype B) in nearly homogeneous form and some of its physical and enzymatic properties were characterized (64, 65, 66). The putida isomerase is similar in its molecular weight and quaternary structure to the testosteroni isomerase. Chemically, the most striking difference between the two isomerases is the presence of four residues of cysteine per polypeptide chain of the putida isomerase whereas no cysteine or cystine is present in the testosteroni isomerase. N-Terminal sequence analysis of the putida isomerase demonstrated substantial sequence homology between the two enzymes. 

Terihanine (8) was originally isolated as its 6-oxoderivative named oxyterihanine from Zcmthoxylum nitidum DC. (synonym Xanthoxylum nitidum (Roxb.) DC. Japanese name, teriha-zan o) [53], Later Ih-Sheng Chen isolated terihanine from Taiwan Z nitidum as a new quaternary alkaloid (private communication from Professor Hisashi Ishii, Chiba University, Japan). The structures of terihanine and oxyterihanine have been established by syntheris [54,55]. 

Alkaloid isolations and structural elucidations are summarized in Table 3. Reduction of a new quaternary alkaloid isolated from Berberis oblonga gave 6-hydroxy-7-methoxy-2-methyl-l,2,3,4-tetrahydroisoquinoline and the alkaloid was deduced to... 

An illustration of the probable nonadaptive origin of allostery is furnished by the wrapping of hemoglobin across orthologs in species with vastly different population size. Thus, this protein becomes richer in dehydrons and more prone to oligomerization in species with smaller population, with the majority of the new dehydrons located at the interface that promotes the quaternary structure of the homomer (Fig. 6.1c). 

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