PubMed · 6797013
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G Hocman. 1981. Human thyroxine binding globulin (TBG).. https://doi.org/10.1007/3-540-10961-7_2
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Buckling transitions in superhelical DNA are sudden changes in shape that accompany a smooth variation in a key parameter, such as superhelical density. Here we explore the dependence of these transitions on the elastic constants for bending and twisting. A and C, important characteristics of DNA's bending and twisting persistence lengths. The large range we explore extends to other elastic materials with self-contact interactions, modeled here by a Debye-Hückel electrostatic potential. Our collective description of DNA shapes and energies over a wide range of p = A/C reveals a dramatic dependence of DNA shape and associated configurational transitions on p: transitions are sharp for large p but masked for small p. In particular, at small p, a nonplanar circular family emerges, in agreement with Jülicher's recent analytical predictions: a continuum of forms (and associated writhing numbers) is also observed. The relevance of these buckling transitions to DNA in solution is examined through studies of size dependence and thermal effects. Buckling transitions smooth considerably as size increases, and this can be explained in part by the lower curvature in larger plasmids. This trend suggests that buckling transitions should not be detectable for isolated (i.e., unbound) DNA plasmids of biological interest, except possibly for very large p. Buckling phenomena would nonetheless be relevant for small DNA loops, particularly for higher values of p, and might have a role in regulatory mechanisms: a small change in superhelical stress could lead to a large configurational change. Writhe distributions as a function of p, generated by Langevin dynamics simulations, reveal the importance of thermal fluctuations. Each distribution range (and multipeaked shape) can be interpreted by our buckling profiles. Significantly, the distributions for moderate to high superhelical densities are most sensitive to p, isolating different distribution patterns. If this effect could be captured experimentally for small plasmids by currently available imaging techniques, such results suggest a slightly different experimental procedure for estimating the torsional stiffness of supercoiled DNA than considered to date.
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Semisynthesis is a chemical technique of protein engineering that provides a valuable complement to directed mutagenesis. It is the method of choice when the structural modification requires, for example, a noncoded amino acid. The process involves specific and limited protein fragmentation, structural manipulation of the target sequence, and subsequent religation of fragments to give the mutant holoprotein. We suggested and demonstrated that mutagenesis and semisynthesis could be used synergistically to achieve protein engineering goals otherwise unobtainable, if mutagenesis was used to shuffle methionine residues in the yeast cytochrome c sequence (Wallace, C. J. A., Guillemette, J. G., Hibiya, Y., and Smith, M. (1991) J. Biol. Chem. 266, 21355-21357). These residues can not only be sites of specific cleavage by CNBr but also of spontaneous peptide bond synthesis between fragments in noncovalent complexes, which greatly facilitates the semisynthetic process. We have now used an informed "methionine scan" of the protein sequence to discover other useful sites and to characterize the factors that promote this extraordinary and convenient autocatalytic religation. Of eight sites canvassed, in a wide range of settings, five efficiently provoked peptide bond synthesis. The principal factor determining efficiency seems to be the hydropathy of the religation site. The mutants created have also provided some new insights on structure-function relationships in the cytochrome.