Deletion of the Ω-Loop in the Active Site of Staphylococcal Nuclease. 2. Effects on Protein Structure and Dynamics

Donna M. Baldisseri, Dennis A. Torchia, Leslie B. Poole, John A. Gerlt

Research output: Contribution to journalArticle

Abstract

It has been shown (Poole et al., 1991) that deletion of residues 44–49 from the sequence of staphylococcal nuclease (E43 SNase) results in an enzyme (E43 ΔSNase) that is significantly more active than D43 SNase, an enzyme that differs from the wild-type enzyme by deletion of a single methylene group. In addition, both E43 ΔSNase and D43 ΔSNase are significantly more stable than their respective parent enzymes. Herein we use high-resolution 2D and 3D NMR spectroscopy to characterize the solution conformations of the four enzymes in order to better understand their differences in stability and activity. The backbone assignments of E43 SNase were extended to the three mutant proteins (uniformly 15N-enriched) by using 2D HSQC, 3D HOHAHA-HMQC, and 3D NOESY-HMQC spectra. The NOE patterns observed for E43 and D43 SNase in solution are consistent with the crystal structures of these proteins. The NOESY data further show that the intact and deleted proteins have essentially the same structures except that (a) the disordered Ω-loops in the intact proteins are replaced by tight type II′ turns, formed by residues 43–50-51-52, in the deleted proteins and (b) the orientation of the D43 side chain in crystalline D43 SNase differs from that found for D43 ΔSNase in solution. Except for regions neighboring the Ω-loops, the intact and deleted proteins show nearly identical amide 15N and 1H chemical shifts. In contrast, there are widespread, small and similar, chemical shift differences (a) between E43 SNase and D43 SNase and (b) between E43 ΔSNase and D43 ΔSNase. This observation indicates that deletion of the E43 γ-methylene group causes smalt, widespread, and similar changes in the structures of E43 SNase and E43 ΔSNase.

Original languageEnglish (US)
Pages (from-to)3628-3633
Number of pages6
JournalBiochemistry
Volume30
Issue number15
DOIs
StatePublished - Apr 1 1991

ASJC Scopus subject areas

  • Biochemistry

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