SNase mutants

Fluorescence Resonance Energy Transfer (FRET), Fig. 4 (a) Positions of cysteine residues used for donor/ acceptor labeling on mutants of SNase. (b) Microfluidic chip used for mixing and spERET detection, (c) FRET histogram for refolding of two of the SNase mutants... 

Figure 29. Hydration correlation functions c(t) probed by tryptophan W140 for wild-type SNase and four mutants, K110C, K133A, E129A, and K110A. The inset shows the correlation functions in the short time range. Note the similarity of the time scales and the difference in amplitudes.

Strikingly, the solvation dynamics for all mutants are nearly the same. All correlation functions can be best described by a double exponential decay with time constants of 0.67 ps with 68% of the total amplitude and 13.2 ps (32%) for D60, 0.47 ps (67%) and 12.7 (33%) for D60G, and 0.53 ps (69%) and 10.8 ps (31%) for D60N. Relative to SNase above, the solvation dynamics are fast, which reflects the neighboring hydrophobic environment. We also measured the anisotropy dynamics and, as shown in the inset of Fig. 33, the local structure is very rigid in the time window of 800 ps. This observation is consistent with the inflexible turn (-T30W31-) in the transition from the second /1-sheet and the second x-helix (Fig. 31). Thus, the three mutants, with a charged, polar, or hydrophobic reside around the probe (Fig. 34) but with the similar time scales of... 

Fig. 9.2. Ribbon diagram of Snase, PDB 1EYO [13]. The single tryptophan is shown in dark gray and one of the residues for which a number of site specific mutants has been studied, valine 66, is shown in black...

Staphylococcal nuclease (SNase) is one of the most powerful enzymes known in terms of its rate acceleration, with a catalytic rate that exceeds that of the non-enzymatic reaction by as much as 1016.211 This enzyme is a phosphodiesterase, and utilizes a Ca2+ ion for catalysis to hydrolyze the linkages in DNA and RNA. In addition to the metal ion, the active site has two Arg residues in a position to interact with the phosphoryl group, and a glutamate. X-ray structures212 215 of SNase have been solved for the wild-type enzyme and mutants, but the exact roles of active-site residues are still uncertain. SNase cleaves the 5 O-P nucleotide bond to yield a free 5 -hydroxyl group (Fig. 30). 

The putative general base catalyst, Glu-43, and electrophilic catalysts, Arg-35 and Arg-87, have been specifically mutated to Asp (9/, 92) and Lys (93) residues, respectively, in the author s laboratory to assess the roles of these amino acid residues in catalysis. Other amino acids were also introduced at these positions, but the present discussion will briefly outline the results obtained with the conservative substitutions. As mentioned previously, the rate acceleration characteristic of the SNase-catalyzed hydrolysis of DNA is approximately 10. The Asp substitution for Glu-43 (E43D) decreased the catalytic efficiency approximately 10, and the Lys substitutions for Arg-35 (R35K) and Arg-87 (R87K) decreased the catalytic efficiency approximately 10 and 10, respectively. While such decreases in catalytic efficiency have been used to describe quantitatively the roles of various active site residues in catalysis, such interpretation is clearly unwarranted in the case of these active site mutants of SNase. The melting temperatures of all three of these mutant enzymes differ significantly... 

Figure 4.3 Toward single amino acid resolution, (a) Some residue-resolved HX-MS results for stabilized double mutant (PI 17C/H124L) of SNase [51] compared with HX-NMR results (dashed curves) or with the calculated rate for an unprotected amide not measured by NMR (dotted curve), (b) Comparison of HX-NMR and HX-MS data for SNase plotted in terms of HX protection factor (Pf= measured HX rate/expected unprotected rate). Filled symbols indicate directly determined HX-MS D-occupancy. Open symbols, switchable sites due to incomplete MS peptide overlap, are paired with their apparent NMR identities (this does not alter the lit quality in b and c). Dotted lines show deviations of 3-fold and 10-fold from the identity line, (c) Population distribution of site-resolved protection factors computed from HX-MS data versus measured by NMR. Reproduced with permission from Ref [48], PNAS...

Figure 1 In a typical example of the perturbation approach, the strength of two selected Tyr-Glu hydrogen bonds in the structure of SNase was systematically studied by creating mutant enzymes and determining their folding energies.

The reason for this unpredictable sensitivity to amino acid changes is that the conservative or nonconservative substitutions seemingly entail minor but significant nonlocal structural changes affecting the active conformation (24). All of the Glu-43 mutant enzymes just mentioned show higher thermal stabilities than the wild-type SNase. 

Various mutant enzymes have also been produced at high yields. For example, SNase-H124L, which is threefold less active but more thermostable than the wild-type enzyme (53), has been cloned and overexpressed under the control of phage T7 promoter in a T7 RNA polymerase expression vector pET3A (54). 

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