Determinants of protein hyperthermostability: Purification and amino acid sequence of rubredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus and secondary structure of the zinc adduct by NMR

Journal Article · Tue Nov 12 04:00:00 EST 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00109a012· OSTI ID:5487379

Blake, P R; Summers, M F ^[1]; Park, J B; Bryant, F O; Aono, Shigetoshi; Adams, M W.W. ^[2]; Magnuson, J K; Eccleston, E; Howard, J B ^[3]

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1. Univ. of Maryland, Baltimore (United States)
2. Univ. of Georgia, Athens (United States)
3. Univ. of Minnesota, Minneapolis, MN (United States)

The purification, amino acid sequence, and two-dimensional {sup 1}H NMR results are reported for the rubredoxin (Rd) from the hyperthermophilic archaebacterium Pyrococcus furiosus, an organism that grows optimally at 100C. The molecular mass (5397 Da), iron content UV-vis spectrophotometric properties, and amino acid sequence are found to be typical of this class of redox protein. However, P. furiosus Rd is remarkably thermostable, being unaffected after incubation for 24 h at 95C. One- and two-dimensional {sup 1}H nuclear magnetic resonance spectra of the oxidized (Fe(III)Rd) and reduced (Fe(II)Rd) forms of P. furiosus Rd exhibited substantial paramagnetic line broadening, and this precluded detailed 3D structural studies. The apoprotein was not readily amenable to NMR studies due to apparent protein oxidation involving the free cysteine sulfhydryls. Secondary structural elements were determined from qualitative analysis of 2D Overhauser effect spectra. These structural elements are similar to those observed by X-ray crystallography for native Rd from the mesophile C. pasteurianum. From analysis of the secondary structure, potentially stabilizing electrostatic interactions involving the charged groups of residues Ala(1), Glu(14), and Glu(52) are proposed. These interactions, which are not present in rubredoxins from mesophilic organisms, may prevent the {beta}-sheet from unzipping' at elevated temperatures.

• DOE Contract Number: FG09-88ER13901
• OSTI ID: 5487379
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 30:45; 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
BACTERIA
BARYONS
BIOCHEMISTRY
CHEMICAL SHIFT
CHEMISTRY
ELEMENTARY PARTICLES
FERMIONS
HADRONS
MAGNETIC RESONANCE
METALLOPROTEINS
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEAR MAGNETIC RESONANCE
NUCLEONS
ORGANIC COMPOUNDS
PROTEINS
PROTONS
RESONANCE
RUBREDOXIN
THERMOPHILIC CONDITIONS