Nuclease solution structure

The solution structures of DNA duplexes containing the mutagenic lesions of benzo [a] pyrene-dtrihydroxybenz[a] anthracene, aminopyrene-dG, aminofluorene-dG and malondialdehyde-dG derivatives have been reported. In each case the lesion was shown to intercalate into the duplex causing only minimal disruption to the duplex structure. These structures have been used to study the nucleotide excision repair (NER) by the UvrABC nuclease system from E. coli of the bulky purine lesions. ... 

Wang JF, LeMaster DM, Markley JL (1990) Two-dimensional NMR studies of staphylococcal nuclease. 1. Sequence-specific assignments of hydrogen-1 signals and solution structure of the nuclease H124L-thymidine 3, 5 -bisphosphate-Ca2-i- ternary complex. Biochemistry 29(1) 88-101... 

Multidimensional NMR spectroscopy provides an alternative method for 3-D structure determination. In fact, this NMR technique is unique in the sense that protein structures can be determined in a solution and noncrystalline state (69, 70). Although still limited in capability, 4-D NMR techniques, in which a 3-D measurement based on N spectra is extended into a fourth dimension by spectra, have been successfully applied to the 153-aa interleukin lj8 (71), providing further insights into the dynamics and folding of the protein molecule. Probing crystalline enzymes by NMR spectroscopy seems to support the claim that crystal structures are representative of the solution structures for most functional purposes. Nevertheless, some differences have been noted when detailed microenvironments are studied. For instance, the pK of His-57 in the catalytic triad of a-lytic protease in crystal is 7.9, nearly one unit higher than that in solution (72). Certain regional sequences may be more flexible and disordered in solution than in the crystal, as has been shown by staphylococcal nuclease (73). 

The structure of the condensed chromatin fiber is still under discussion [1,23,54], with two competing models the original solenoid model of Finch and Klug [16], and the straight-linker model [12,14,55]. Assessing the structure in vivo or in situ has proven impossible thus far, due to technical limitations. Chromatin fibers released from nuclei into solution by nuclease treatment have been widely used as models for fiber structure such fibers are extended at low ionic strength and condensed at ionic strengths believed to be close to those found in vivo ( 150 mM Na" " or 0.35 mM Mg " "). The salt-induced fiber compaction has been extensively studied in the past but is still poorly understood in terms not only of the details of the structure but also in terms of the molecular mechanisms of the compaction process. 

Nuclease behaves like a typical globular protein in aqueous solution when examined by classic hydrodynamic methods (40) or by measurements of rotational relaxation times for the dimethylaminonaphth-alene sulfonyl derivative (48)- Its intrinsic viscosity, approximately 0.025 dl/g is also consistent with such a conformation. Measurements of its optical rotatory properties, either by estimation of the Moffitt parameter b , or the mean residue rotation at 233 nin, indicate that approximately 15-18% of the polypeptide backbone is in the -helical conformation (47, 48). A similar value is calculated from circular dichroism measurements (48). These estimations agree very closely with the amount of helix actually observed in the electron density map of nuclease, which is discussed in Chapter 7 by Cotton and Hazen, this volume, and Arnone et al. (49). One can state with some assurance, therefore, that the structure of the average molecule of nuclease in neutral, aqueous solution is at least grossly similar to that in the crystalline state. As will be discussed below, this similarity extends to the unique sensitivity to tryptic digestion of a region of the sequence in the presence of ligands (47, 48), which can easily be seen in the solid state as a rather anomalous protrusion from the body of the molecule (19, 49). 

In native proteins of known three-dimensional structure about 7% of all prolyl peptide bonds are cis (Stewart et al., 1990 MacArthur and Thornton, 1991). Usually, the conformational state of each peptide bond is clearly defined. It is either cis or trans in every molecule, depending on the structural framework imposed by the folded protein chain. There are a few exceptions to this rule. In the native states of staphylococcal nuclease (Evans et al., 1987), insulin (Higgins et al., 1988), and calbindin (Chazin et al., 1989) cis-trans equilibria at particular Xaa-Pro bonds have been detected in solution by NMR. In staphylococcal nuclease, the cis conformer of the Lys 116-Pro 117 bond can be selectively stabilized by bind-... 

DNase I stock solutions are stored at -2O C (1 mg/ml) in 5- xl aliquots (each aliquot is used once). Variation in the activity of DNase I preparations is often observed and the exact amount needed to introduce the desired number of nicks should be determined for each enzyme batch. Sometimes template switches occur which will result in snap-back structures (zero-binding nucleic acid), which remain S, nuclease-resistant upon denaturation. Rigby et al. (1977) suggested that this effect was due to a differential loss of 5 - 3 exonuclease activity upon storage leading to a displacement of the nicked strand and a template switch from the complementary... 

The metallobiochemistry of staphylococcal nuclease has been extensively investigated. It was shown early that the tripositive lanthanide ions, Ln, are potent competitive inhibitors of the enzyme, binding to it with K s of about 9/uM and acting with inhibitory constants of l-2 iM. The binding of Ln + ions enhances the binding of pdTp. Ln " ions, but not Ca, stabilize the enzyme toward tryptic proteolysis. The paramagnetism of Gd " was exploited in H and P resonance relaxation studies on pdTp bound in the ternary complex to determine that the structure in solution was consistent with the observed x-ray structure. Some differences were observed but these were of uncertain significance. 

In crystal structures of folded proteins the prolyl peptide bonds are generally either cis or trans in every molecule. There is, however, an increasing number of exceptions to this rule, and cis/trans equilibria have been found, in particular by 2D-NMR spectroscopy in solution. Examples include staphylococcal nuclease (Evans et al, 1987), insulin (Higgins etal., 1988), calbindin (Chazin et al., 1989 Kordel et al., 1990), scorpion venom Lqh-8/6 (Adjadj et al., 1997), human interleukin-3 (Feng et al., 1997), and the TB6 domain of human fibrillin-1 (Yuan etal., 1997 Yuan et al., 1998). 

With all the library members in the same solution at such a miniature scale, the choice of analytical methods to characterize the library is critical. In the first library, Liu and co-workers used MALDI-TOF to analyze the products from each step respectively. An increasingly complex MALDI spectra indicates the diversification of the library as each step takes place. However, the sensitivity and resolution of MALDI-TOF do not meet the needs to analyze large libraries. Instead, SI nuclease digestion was able to remove the DNA template, so that the small molecule portion can be directly analyzed with nanospray LC-MS (Figure 8.11). Remarkably, for the sampled analysis of one sub-library, the correct masses of 94% of all 1728 possible structures have been observed with clean background, proving the quality of the library synthesis and providing confidence for the subsequent in vitro selection. 

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