2D ¹H and 3D ¹H‐¹⁵N NMR of zinc‐rubredoxins: Contributions of the β‐sheet to thermostability

Abstract

Based on 2D 'H-IH and 2D and 3D 'H-"N NMR spectroscopies, complete ' H NMR assignments are reported for zinc-containing Clostridium pasteurianum rubredoxin (Cp ZnRd). Complete 'H NMR assignments are also reported for a mutated Cp ZnRd, in which residues near the N-terminus, namely, Met 1 , Lys 2, and Pro 15, have been changed to their counterparts, (-), Ala and Glu, respectively, in rubredoxin from the hyperthermophilic archaeon, Pyrococcusfuriosus (Pf Rd). The secondary structure of both wild-type and mutated Cp ZnRds, as determined by NMR methods, is essentially the same. However, the NMR data indicate an extension of the threestranded &sheet in the mutated Cp ZnRd to include the N-terminal Ala residue and Glu 15, as occurs in Pf Rd. The mutated Cp Rd also shows more intense NOE cross peaks, indicating stronger interactions between the strands of the 0-sheet and, in fact, throughout the mutated Rd. However, these stronger interactions do not lead to any significant increase in thermostability, and both the mutated and wild-type Cp Rds are much less thermostable than Pf Rd. These correlations strongly suggest that, contrary to a previous proposal [Blake PR et al., 1992, Protein Sci I : 1508-15211, the thermostabilization mechanism of Pf Rd is not dominated by a unique set of hydrogen bonds or electrostatic interactions involving the N-terminal strand of the 0-sheet. The NMR results also suggest that an overall tighter protein structure does not necessarily lead to increased thermostability.Keywords: Clostridium pasteaurianum; 2D NMR; 3D NMR; mutated rubredoxin; protein secondary structure; protein thermostability; rubredoxin; zinc-containing rubredoxin The group of bacterial and archaeal iron-sulfur proteins known as rubredoxins appears to be ideal for delineating molecular determinants of non-heme metalloprotein structure, thermostability, and redox properties. Rds have in common the following favorable characteristics for such investigations: small size (typ- ically 52-54 residues), a very simple redox active Fe(SCys), site (whose Cys residues are underlined in Scheme I), a large and continually expanding database of amino acid sequences (Zeng et al., 1995), at least five high-resolution X-ray crystal structures (Sieker et al., 1994), a characteristic structural motif featuring a three-stranded @-sheet, and well-characterized proteins from several mesophiles and from the hyperthermophilic archaeon Pyrococcus furiosus. Pf Rd is known to be remarkably thermostable compared to Rds from mesophilic organisms (Blake et al., 1991;Day et al., 1992). However, there are no obvious distinguishing structural or spectroscopic features of the FeS, site in Pf Rd compared to Rds from mesophiles. Instead, based on the NMR solution structure of the zinc-substituted Pf Rd (Blake et al., 1992) and the X-ray crystal structure of Pf FeRd (Day et al., 1992), a unique set of hydrogen bonds and electrostatic interactions was proposed to inhibit "unzipping" of the threestranded @-sheet at high temperatures. This inhibition cou...

Miniaturized metalloproteins: Application to iron–sulfur proteins

“…Material). Finally, the pattern of the amide and ␣CH proton chemical shifts is similar to that observed for Zn(II)-Rd (25,26). We are presently refining a starting model by restrained molecular dynamic simulations.…”

“…Several investigations are being carried out on both natural Rds (17)(18)(19)(20)(21)(22)(23) and their mutants (24)(25)(26)(27)(28)(29)(30)(31)(32) and on model systems (33)(34)(35)(36)(37)(38)(39) to identify the factors tuning the properties of the active site.…”

“Iron priming” guides folding of denatured aporubredoxins

“…Factors such as the nature of the intracellular iron donor, intracellular metal complexing agents and macromolecular crowding could conceivably affect these time scales. The fact that holoCpRds and holoPfRds can be recombinantly expressed in high yields in Escherichia coli [17,30], which contains no native Rd, indicates that a Rd-specific metallochaperone is not required (although a mixture of ZnRds and FeRds is typically obtained in this heterologous expression system [18]). …”

“…More than 20 X-ray crystal structures of Rds have been deposited in the Protein Data Bank [15], and NMR-derived solution structures of holoRds are also available [16]. Since they contain no inorganic sulfide or other cofactors, Rds may not require a specialized machinery for iron incorporation in vivo [17,18], and, in fact, no Rd metallochaperones have been reported. Rds, therefore, constitute a seemingly ideal prototype for investigating the relationships between non-heme iron binding and folding into the native protein structure.…”

Rubredoxin-related Maturation Factor Guarantees Metal Cofactor Integrity during Aerobic Biosynthesis of Membrane-bound [NiFe] Hydrogenase

“…These observations are in marked contrast to common rubredoxins that have a red color, bind iron at high affinity, and are usually rather stable (47,48). When provided in excess, nickel, zinc, or cadmium can substitute for the iron in rubredoxins (48).…”