Cys-tRNACys formation and cysteine biosynthesis in methanogenic archaea: two faces of the same problem?

Aminoacyl-tRNA (transfer RNA) synthetases are essential components of the cellular translation machinery as they provide the ribosome with aminoacyl-tRNAs. Aminoacyl-tRNA synthesis is generally well understood. However, the mechanism of Cys-tRNACys formation in three methanogenic archaea ( Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus and Methanopyrus kandleri) is still unknown, since no recognizable gene for a canonical cysteinyl-tRNA synthetase could be identified in the genome sequences of these organisms. Here we review the different routes recently proposed for Cys-tRNACys formation and discuss its possible link with cysteine biosynthesis in these methanogenic archaea.

Nucleotide-dependent conformational changes in a protease-associated ATPase HsIU.

BACKGROUND: The bacterial heat shock locus ATPase HslU is an AAA(+) protein that has structures known in many nucleotide-free and -bound states. Nucleotide is required for the formation of the biologically active HslU hexameric assembly. The hexameric HslU ATPase binds the dodecameric HslV peptidase and forms an ATP-dependent HslVU protease. RESULTS: We have characterized four distinct HslU conformational states, going sequentially from open to closed: the empty, SO(4), ATP, and ADP states. The nucleotide binds at a cleft formed by an alpha/beta domain and an alpha-helical domain in HslU. The four HslU states differ by a rotation of the alpha-helical domain. This classification leads to a correction of nucleotide identity in one structure and reveals the ATP hydrolysis-dependent structural changes in the HslVU complex, including a ring rotation and a conformational change of the HslU C terminus. This leads to an amended protein unfolding-coupled translocation mechanism. CONCLUSIONS: The observed nucleotide-dependent conformational changes in HslU and their governing principles provide a framework for the mechanistic understanding of other AAA(+) proteins.

Crystal structures of the HslVU peptidase-ATPase complex reveal an ATP-dependent proteolysis mechanism.

BACKGROUND: The bacterial heat shock locus HslU ATPase and HslV peptidase together form an ATP-dependent HslVU protease. Bacterial HslVU is a homolog of the eukaryotic 26S proteasome. Crystallographic studies of HslVU should provide an understanding of ATP-dependent protein unfolding, translocation, and proteolysis by this and other ATP-dependent proteases. RESULTS: We present a 3.0 A resolution crystal structure of HslVU with an HslU hexamer bound at one end of an HslV dodecamer. The structure shows that the central pores of the ATPase and peptidase are next to each other and aligned. The central pore of HslU consists of a GYVG motif, which is conserved among protease-associated ATPases. The binding of one HslU hexamer to one end of an HslV dodecamer in the 3.0 A resolution structure opens both HslV central pores and induces asymmetric changes in HslV. CONCLUSIONS: Analysis of nucleotide binding induced conformational changes in the current and previous HslU structures suggests a protein unfolding-coupled translocation mechanism. In this mechanism, unfolded polypeptides are threaded through the aligned pores of the ATPase and peptidase and translocated into the peptidase central chamber.

De novo heme proteins from designed combinatorial libraries.

We previously reported the design of a library of de novo amino acid sequences targeted to fold into four-helix bundles. The design of these sequences was based on a "binary code" strategy, in which the patterning of polar and nonpolar amino acids is specified explicitly, but the exact identities of the side chains is varied extensively (Kamtekar S, Schiffer JM, Xiong H, Babik JM, Hecht MH, 1993, Science 262:1680-1685). Because of this variability, the resulting collection of amino acid sequences may include de novo proteins capable of binding biologically important cofactors. To probe for such binding, the de novo sequences were screened for their ability to bind the heme cofactor. Among an initial collection of 30 binary code sequences, 15 are shown to bind heme and form bright red complexes. Characterization of several of these de novo heme proteins demonstrated that their absorption spectra and resonance Raman spectra resemble those of natural cytochromes. Because the design of these sequences is based on global features of polar/ nonpolar patterning, the finding that half of them bind heme highlights the power of the binary code strategy, and demonstrates that isolating de novo heme proteins does not require explicit design of the cofactor binding site. Because bound heme plays a key role in the functions of many natural proteins, these results suggest that binary code sequences may serve as initial prototypes for the development of large collections of functionally active de novo proteins.

A biotinylated undecylthiophene copolymer bioconjugate for surface immobilization: creating an alkaline phosphatase chemiluminescence-based biosensor.

Methodology is described for the creation of a molecular assembly consisting of the enzyme alkaline phosphatase immobilized onto a glass surface using a biotinylated conjugated copolymer, poly(3-undecylthiophene-co-3-thiophenecarboxaldehyde) 6-biotinamidohexanohydrazone. The biotinylated polymer is attached to the inside walls of a silanized glass capillary via hydrophobic interactions, and a streptavidin-conjugated alkaline phosphatase is interfaced with the polymer through the classical biotin-streptavidin interaction. Utilizing a simple optical setup, we can detect the activity of as little as approximately 0.1 fmol of alkaline phosphatase with this molecular assembly. The assembly is mechanically robust and retains the majority of bound enzyme activity for up to 30 days. We have utilized this molecular assembly for the detection of organophosphorus-based pesticides. Both paraoxon and methyl parathion inhibit the enzyme-mediated generation of chemiluminescence signal. We are able to detect paraoxon and methyl parathion concentrations down to 500-700 ppb.

Trace Analysis of Zn(II), Be(II), and Bi(III) by Enzyme-Catalyzed Chemiluminescence.

A novel technique for the trace analysis of metal ions Zn(II), Be(II), and Bi(III) in bulk solutions is discussed. This technique involves the generation of a chemiluminescence signal from alkaline phosphatase catalyzed hydrolysis of a phosphate derivative of 1,2-dioxetane. Zn(II) can be determined by two methods, reactivation of the alkaline phosphatase apoenzyme and inhibition of the native enzyme. Be(II) and Bi(III) can be determined quantitatively by inhibition of the native enzyme. Subppb to ppm level detection of Zn(II), Be(II), and Bi(III) has been achieved. Initial studies with mixed metals are also reported. The technique described is rapid and sensitive and can be readily applied to the microassay of heavy metal ions.

Chemiluminescence-based inhibition kinetics of alkaline phosphatase in the development of a pesticide biosensor.

The use and application of the enzyme alkaline phosphatase in a chemiluminescence assay are discussed. The enzyme catalyzes the hydrolysis of a macrocyclic phosphate compound generating a chemiluminescence signal. On the basis of inhibition of this signal, a methodology for the detection and quantitation of organophosphorus-based pesticides has been developed. The methodology is studied with alkaline phosphatase in the bulk aqueous phase, and detection of the signal is accomplished by a simple optical setup. Parts per billion level detection of paraoxon and methyl parathion in bulk solutions is achieved. The technique is rapid and sensitive and is applicable to the detection of most organophosphorus-based pesticides. The results from kinetic studies indicate a mixed type of inhibition of the enzyme by paraoxon and methyl parathion. The detection methodology forms an integral part of a biosensor under development and is adaptable to incorporating optical fibers for remote detection of pesticides.

Protein Motifs. 7. The four-helix bundle: what determines a fold?

The four-helix bundle motif occurs in many structural contexts and in proteins that are functionally diverse. The motif can be classified into individual folds on the basis of topological and geometric properties. It has been thoroughly investigated structurally by both nuclear magnetic resonance and x-ray crystallography. Many mutants of four-helix bundles have been generated, and the motif has also been the target of de novo design studies. Taken together, these studies provide an opportunity to examine many of the forces governing protein folding. In this article we consider the relative importance of the burial of hydrophobic residues, loss of conformational entropy, packing interactions, interhelical turn composition, and helical dipole interactions all within the context of a single folding motif. We conclude by examining why de novo designed four-helix bundle proteins possess flexible interiors, and possible mechanisms by which natural proteins may lock their cores into rigid structures.

Molecular assembly of proteins and conjugated polymers: Toward development of biosensors.

A molecular assembly in which a conjugated polymer is interfaced with a photodynamic protein is described. The conjugated polymer, functionalized with biotion, is designed such that it can be physisorbed on or chemically grown off a glass surface. The streptavidin-derivatized protein is immobilized on the biotinylated polymer matrix through the strong biotin-streptavidin interactions. The assembly, built on the surface of an optical fiber or on the inside walls of a glass capillary, forms an integral part of a biosensor for the detection of environmental pollutants such as organophosphorus-based insecticides. The Protein in the system can be replaced by any biological macromolecule of interest. We study one specific case, the enzyme alkaline phosphatase. The enzyme catalyzes a reaction producing an intermediate compound that chemiluminesces, and the chemiluminescence singnal is monitored to detect and quantify insecticides such as paraoxon and methyl parathion. Preliminary results indicate ppb level detection with response time less than 1 minute. (c) 1995 John Wiley & Sons, Inc.

Protein design by binary patterning of polar and nonpolar amino acids.

A general strategy is described for the de novo design of proteins. In this strategy the sequence locations of hydrophobic and hydrophilic residues were specified explicitly, but the precise identities of the side chains were not constrained and varied extensively. This strategy was tested by constructing a large collection of synthetic genes whose protein products were designed to fold into four-helix bundle proteins. Each gene encoded a different amino acid sequence, but all sequences shared the same pattern of polar and nonpolar residues. Characterization of the expressed proteins indicated that most of the designed sequences folded into compact alpha-helical structures. Thus, a simple binary code of polar and nonpolar residues arranged in the appropriate order can drive polypeptide chains to collapse into globular alpha-helical folds.