Two-dimensional NMR studies of staphylococcal nuclease. 1. Sequence-specific assignments of hydrogen-1 signals and solution structure of the nuclease H124L-thymidine 3 prime ,5 prime -bisphosphate-Ca sup 2+ ternary complex

Journal Article · Tue Jan 09 04:00:00 EST 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00453a011· OSTI ID:7193189

Wang, J; Markley, J L ^[1]; LeMaster, D M ^[2]

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1. Univ. of Wisconsin, Madison (USA)
2. Northwestern Univ., Evanston, IL (USA)

Staphylococcal nuclease H124L is a recombinant protein produced in Escherichia coli whose sequence is identical with that of the nuclease produced by the V8 variant of Staphylococcus aureus. The enzyme-metal ion activator-nucleotide inhibitor ternary complex, nuclease H124L-thymidine 3{prime},5{prime}-bisphosphate-Ca{sup 2+}, was investigated by two-dimensional (SD) NMR techniques. Efficient overproduction of the enzyme facilitated the production of random fractionally deuterated protein, which proved essential for detailed NMR analysis. {sup 1}H NMR spin systems were analyzed by conventional 2D {sup 1}H({sup 1}H) methods: COSY, relayed COSY, HOHAHA, and NOESY. Assignments obtained by {sup 1}H NMR experiments were confirmed and extended by {sup 1}H-{sup 13}C and {sup 1}H-{sup 15}N heteronuclear NMR experiments. Spectra of the ternary complexes prepared with protein at natural abundance and at 50% random fractional deuteration provided the information needed for sequence-specific assignments of 121 of the 149 amino acid residues. Short- and intermediate-range NOE connectivities allowed the determination of secondary structural features of the ternary complex. The solution structure of this ternary complex shows a close correspondence to the crystal structure of the nuclease wt-thymidine 3{prime},5{prime}-bisphosphate-Ca{sup 2+} ternary complex.

• OSTI ID: 7193189
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:1; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
AMINO ACID SEQUENCE
AQUEOUS SOLUTIONS
BACTERIA
BARYONS
CARBON 13
CARBON ISOTOPES
CELL CONSTITUENTS
DEUTERIUM COMPOUNDS
DISPERSIONS
ELEMENTARY PARTICLES
ENZYME ACTIVITY
ENZYMES
ESTERASES
EVEN-ODD NUCLEI
FERMIONS
HADRONS
HYDROGEN COMPOUNDS
HYDROLASES
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
MIXTURES
MOLECULAR STRUCTURE
NITROGEN 15
NITROGEN ISOTOPES
NUCLEAR MAGNETIC RESONANCE
NUCLEASES
NUCLEI
NUCLEONS
ODD-EVEN NUCLEI
PHOSPHODIESTERASES
PLASMIDS
PROTONS
RESONANCE
SOLUTIONS
STABLE ISOTOPES
STAPHYLOCOCCUS
STRUCTURE-ACTIVITY RELATIONSHIPS