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

Abstract

The purification, amino acid sequence, and two-dimensional 1H NMR results are reported for the rubredoxin (Rd) from the hyperthermophilic archaebacterium Pyrococcus furiosus, an organism that grows optimally at 100 degrees C. The molecular mass (5397 Da), iron content (1.2 +/- 0.2 g-atom of Fe/mol), UV-vis spectrophotometric properties, and amino acid sequence (60% sequence identity with Clostridium pasteurianum Rd) 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 95 degrees C. One- and two-dimensional 1H 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. However, high-quality NMR spectra were obtained for the Zn-substituted protein, Zn(Rd), enabling detailed NMR signal assignment for all backbone amide and alpha and most side-chain protons. Secondary structural elements were determined from qualitative analysis of 2D Overhauser effect spectra. Residues A1-K6, Y10-E14, and F48-E51 form a three-strand antiparallel beta-sheet, which comprises ca. 30% of the primary sequence. Residues C5-Y10 and C38-A43 form types I and II amide-sulfur tight turns common to iron-sulfur proteins. These structural elements are similar to those observed by X-ray crystallography for native Rd from the mesophile C. pasteurianum. However, the beta-sheet domain in P. furiosus Rd is larger than that in C. pasteurianum Rd and appears to begin at the N-terminal residue. 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.

X‐ray crystal structures of the oxidized and reduced forms of the rubredoxin from the marine hyperthermophilic archaebacterium pyrococcus furiosus

“…The hydrogen-bonding pattern between the first and second @-strands is interrupted by a G1 type j3 bulge (Richardson et al, 1978) that occurs between residues Gly 9 and Tyr 10. The overall hydrogen-bonding pattern (see Table 2) is in close agreement with the pattern reported in the 'H-NMR secondary structure analysis (Blake et al, 1991). The hydrophobic core contains six aromatic residues -Trp 3, Tyr 10, Tyr 12, Phe 29, Trp 36, and Phe 48 -as well as the hydrophobic aliphatic residue Leu 32 (see Fig.…”

“…As expected from the growth conditions for P. furiosus, the rubredoxin from this organism (RdPf) exhibits significant thermostability, as the UV-visible and EPR properties of the protein are unaffected after a 24-h incubation at 95 "C (Blake et al, 1991). In contrast, rubredoxins from mesophilic bacteria are less thermostable; RdCp is rapidly denatured at 80 "C (Lovenberg & Sobel, 1965), whereas RdDg loses 50% of its visible absorption after -2 h at 80°C (Papavassilou & Hatchikian, 1985).…”

“…Sequence alignment for the rubredoxins from anaerobic bacteria (Blake et al, 1991). The top five sequences are for the rubredoxins whose X-ray structures have been solved.…”

“…Residue numbers correspond to the RdPf sequence, which has one less residue at the amino-terminus than the other rubredoxins. The abbreviations and references are: RdPf, Pyrococcus furiosus (Blake et al, 1991); RdDg, Desulfovibrio gigas (Bruschi, 1976a);…”

“…furiosus and the isolation of rubredoxin (RdPf) were as previously described (Bryant & Adams, 1989;Blake et al, 1991). Crystals of the oxidized form of rubredoxin were grown using the hanging drop method by equilibrating a 4-pL drop against a solution containing 30% 2-methyl-2,4-pentanediol, 2.2 M (NH4)2S04, and 0.1 M Tris-HCI, pH 8.…”