Staphylococcal nuclease folding

Jacobs, M. D. Fox, R. O. (1994). Staphylococcal nuclease folding intermediate charaterized by hydrogen exchange and NMR spectroscopy. Proc. Natl. Acad. Sci. USA 91,449-453. 

Nishimura, C., Riley, R., Eastman, P. and Fink, A. L, 2000, Fluorescence energy transfer indicates similar transient and equilibrium intermediates in staphylococcal nuclease folding. Journal of Molecular Biology 299, 1133-1146. 

J. Woenckhaus, R. Khling, T. Thiyagarajan, K. C. Littrell, S. Seifert, C. A. Royer, R. Winter, Pressure-jump small-angle X-ray scattering detected kinetics of staphylococcal nuclease folding, Biophys. J. 80 (2001) 1518-1523. 

En me Mechanism. Staphylococcal nuclease (SNase) accelerates the hydrolysis of phosphodiester bonds in nucleic acids (qv) some 10 -fold over the uncatalyzed rate (r93 and references therein). Mutagenesis studies in which Glu43 has been replaced by Asp or Gin have shown Glu to be important for high catalytic activity. The enzyme mechanism is thought to involve base catalysis in which Glu43 acts as a general base and activates a water molecule that attacks the phosphodiester backbone of DNA. To study this mechanistic possibiUty further, Glu was replaced by two unnatural amino acids. 

Zhang O., Kay L. E., Shortle D. and For-man-Kay J. D. Comprehensive NOE characterization of a partially folded large fragment of staphylococcal nuclease Deltal 31 Delta, using NMR methods with improved resolution. J. Mol. Biol. (1997) 272(1) 9-20. 

Studies of proteolytic fragments of staphylococcal nuclease (Tan-iuchi and Anfinsen, 1969) and RNase A (Taniuchi, 1970) seemed to support this view. Taniuchi (1970), in summary remarks, said Thus, the minimum information of the specific folding of a protein requiring almost the entire amino acid sequence is observed with both staph-yloccocal nuclease and bovine pancreatic ribonuclease. ... 

Uversky, V.N., A.S. Kamoup, D.J. Segel, S. Seshadri, S. Doniach, and A.L. Fink. 1998. Anion-induced folding of Staphylococcal nuclease characterization of multiple equilibrium partially folded intermediates. J Mol Biol 278 879-894. 

The hydrolysis of dinucleotides by the staphylococcal nuclease has been recently studied in Laskowski s laboratory (12). In 12 dinucleotides of the type d-N pN p the susceptibility to hydrolysis varied about 100-fold and appeared to depend primarily on the type of base in the p position. In addition, the corresponding d-NpN dinucleosidemonophos-phates were more resistant to hydrolysis, and the pNpN 5 -phosphodinu-cleotides were most resistant of all. This work would appear to be consistent with our view of the pdTp binding site of the nuclease at high resolution. 

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-... 

Hilser, V. J. Freire, E. (1996a). Predicting the Equilibrium Protein Folding Pathway Structure-Based Analysis of Staphylococcal Nuclease. Proteins In Press. 

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). 

Figure 6.S. Fluorescence emission specUa of native (N) (pH 7.5) and acid-unfolded (Ua) (pH 2.0) staphylococcal nuclease K46C labeled with lAEDANS. Excitation of tryptophan residue ( e = 295 nm) meteases the emission ai 485 nm of lAEDANS as the result of Trp I.AEDANS energy transfer. This increase k less import art in the nnfolded slate than in llie folded one Source Nishhnura. C. Ril. R., Eastman. P. and Fkik. A. L. 2000, Journal of Molecular Biokigy. 299,1133 1146.

Zhi Z, Liu P, Wang P, Huang Y, Zhao XS (2011) Domain-specific folding kinetics of staphylococcal nuclease observed through single-molecule FRET in a microfluidic mixer. Chem Phys Chem 12(18) 3515-3518... 

The experiments of Epstein et al. (1971), already referred to, on the reversible folding of staphylococcal nuclease on change of pH from 3.2 (unfolding conditions) to 6.7, provided a good example of a record with two time constants. They reported that the faster process (12 s" at 25 °C) was not significantly affected by temperature (13 s at 38 °C) while the slower... 

Epstein, H. F., Schechter, A. N., Chen, R. F. Anfinsen, C. B. (1971). Folding of staphylococcal nuclease kinetic studies of two processes in acid renaturation. Joitrnal of Molecular Biology, 60,49 508. 

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). 

Staphylococcal nuclease has been extensively used as a model protein for the study of enzyme mechanisms, stability, and the kinetics of (re)folding. In recombinant DNA technology, SNase is primarily used as a nonspecific endonuclease, as is DNase I which generates 5 -phosphonucleotides (Section I, this chapter). Staphylococcal nuclease has also served as the prototype model for a new generation of nucleases called hybrid nucleases, which can cleave DNA and RNA with tailored site specificities. 

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