Nuclease structure

These observations on the complementation of fragments of nuclease are consistent with the concept that almost the entire amino acid sequence of nuclease is required for the minimum information necessary to determine a stable, functional nuclease structure. Furthermore, the native conformation of nuclease cannot be formed during assembly from the NH2-terminus until the polypeptide chain has been extended beyond residue 126 (80). Finally, it appears that stable and functional structures can be formed in several ways when the minimum requirement for information is fulfilled (81). 

At the time we solved the crystal structure of Serratia nuclease, comparative sequence data, mutagenesis data and chemical modification data were not available. After we completed the building and refinemoit of the Serratia nuclease structure, visual inspection did not reveal the active site location. Since there were no other nuclease structures to use to help locate the active site, we analyzed the structure in terms of its electrostatic surface potential. Inspection of electrostatic maps calculated in GRASP (32) revealed that one side of the nuclease monomer displayed a broad area of positive electrostatic potential while the opposite side displayed negative electrostatic... 

Tucker, P.W., Hazen, E.E., Colton, F.A. Staphylococcal nuclease reviewed a prototypic study in contemporary enzymology. III. Correlation of fhe three-dimensional structure with the mechanisms of enzymatic action. Mol. Cell. Biochem. 

Figure 36-2. Model for the structure of the nucleosome, in which DNA is wrapped around the surface of a flat protein cylinder consisting of two each of histones H2A, H2B, H3, and H4 that form the histone octamer. The 146 base pairs of DNA, consisting of 1.75 superhelical turns, are in contact with the histone octamer. This protects the DNA from digestion by a nuclease. The position of histone HI, when it is present, is indicated by the dashed outline at the bottom of the figure.

Messenger RNAs exist in the cytoplasm as ribonu-cleoprotein particles (RNPs). Some of these proteins protect the mRNA from digestion by nucleases, while others may under certain conditions promote nuclease attack. It is thought that mRNAs are stabihzed or destabilized by the interaction of proteins with these various structures or sequences. Certain effectors, such as hormones, may regulate mRNA stability by increasing or decreasing the amount of these proteins. 

Shortle has focused on the unfolded state for more than a decade, leading up to his recent demonstration using residual dipolar couplings that staphylococcal nuclease retains global structure in 8 M urea. His chapter on The Expanded Denatured State sets the stage. Dunker etal. then explore the complementary world of disordered regions within... 

In this review, NMR analysis of the denatured state of staphylococcal nuclease is briefly reviewed in nontechnical language. Most of the work has come from the author s laboratory over the past eight years. The initial experiments, which only measure local structural parameters, reported small amounts of persisting helical structure, two turns, and indirect evidence for perhaps a three-strand beta meander. When applied to the denatured state in 6 M urea, the same experiments indicated that most of these features are lost. 

II. Nuclease A131A Local Structure A. The Protein System... 

Fig. 1. Schematic diagram of nuclease A131A in the folded conformation. The alpha helices and beta strands are labeled. NMR analysis suggests the two turns and one helix in black are modestly populated in the denatured state, whereas the shaded helix is slightly populated. Strands / l-/ 2-/ 3 form an extended structure about which littie is known. Reproduced from Barron, L. D., Hecht, L., Blanch, E. W., and Bell, A. F. (2000). Prog. Biophys. Mol Chem. 73, 1-49. 2000, with permission from Elsevier Science.

The changes in structure of denatured nuclease as a function of urea concentration (Fig. 3) suggest that, as hydrophobic interactions are weakened and the backbone becomes more highly solvated, the chain expands gradually. The data presented by Millet et al. in this volume suggest that this expansion does not continue asymptotically as predicted by simple polymer physical chemistry. This is the behavior expected for a polypeptide chain trapped in a small region of conformation space. Most, perhaps all, of the conformations accessible in the expanded denatured state may have a native-like topology. 

---