This study shows, for the first time, the advantages of combining two transition-metal complexes as selective proteolytic reagents. In this procedure, cis-[Pt(en)(H(2)O)(2)](2+) is followed by [Pd(H(2)O)(4)](2+). In the peptide AcAla-Lys-Tyr-Gly-Gly-Met-Ala-Ala-Arg-Ala, the Pt(II) reagent cleaves the Met6-Ala7 peptide bond, whereas the Pd(II) reagent cleaves the Gly4-Gly5 bond. In the peptide AcVal-Lys-Gly-Gly-His-Ala-Lys-Tyr-Gly-Gly-Met-Ala-Ala-Arg-Ala, the Pt(II) reagent cleaves the Met11-Ala12 peptide bond, whereas the Pd(II) reagent cleaves the Gly3-Gly4 bond. All cleavage reactions are regioselective and complete at pH 2.0 and 60 degrees C. Each metal ion binds to an anchoring side chain and then, as a Lewis acid, activates a proximal peptide bond toward hydrolysis by the solvent water. The selectivity in cleavage is a consequence of the selectivity in this initial anchoring. Both Pt(II) and Pd(II) reagents bind to the methionine side chain, whereas only the Pd(II) reagent binds to the histidine side chain under the reaction conditions. Consequently, only methionine residues direct the cleavage by the Pt(II) reagent, whereas both methionine and histidine residues direct the cleavage by the Pd(II) reagent. The Pt(II) reagent cleaves the first bond downstream from the anchor, i.e., the Met-Z bond. The Pd(II) reagent cleaves the second bond upstream from the anchor, i.e., the X-Y bond in the X-Y-Met-Z and in the X-Y-His-Z segments. The diethylenetriamine complex [Pt(dien)(H(2)O)](2+) cannot promote cleavage. Its prior binding to the Met11 residue in the second peptide prevents the Pd(II) reagents from binding to Met11 and cleaving the Gly9-Gly10 bond and directs the cleavage by the Pd(II) reagent exclusively at the Gly3-Gly4 bond. Our new method was tested on equine myoglobin, which contains 2 methionine residues and 11 histidine residues. The complete methionine-directed cleavage of the Met55-Lys56 and Met131-Thr132 bonds by the Pt(II) reagent produced three fragments, suitable for various biochemical applications because they are relatively long and contain amino and carboxylic terminal groups. The deliberately incomplete histidine-directed cleavage of the long fragments 1.55 and 56.131 at many sites by the Pd(II) reagent produced numerous short fragments, suitable for protein identification by mass spectrometry. The ability of combined Pt(II) and Pd(II) complexes to cleave proteins with explicable and adjustable selectivity and with good yields bodes well for their greater use in biochemical and bioanalytical practice.

Complex cis-[Pt(en)(H(2)O)(2)](2+) promotes selective hydrolytic cleavage of two proteins, horse cytochrome c and bovine beta-casein. The cleavage is completed in 24 h under relatively mild conditions, at about pH 2.5, and a temperature as low as 40 degrees C. The results of HPLC and TSDS PAGE separations, MALDI mass spectrometry, and Edman sequencing showed that cleavage occurred exclusively at the peptide bond involving the C-terminus of each methionine residue, both such residues in cytochrome c and all six such residues in beta-casein. While having the same selectivity as cyanogen bromide (CNBr), the most common chemical protease, cis-[Pt(en)(H(2)O)(2)](2+) has several advantages. It is nonvolatile, easy to handle, and recyclable. Its cleavage is residue-selective, the rest of the polypeptide backbone remains intact, and the other side chains remain unmodified. It is applied in approximately equimolar amounts with respect to methionine residues, creates free amino and carboxylic groups, and cleaves even the Met-Pro bond, which is resistant to CNBr and most proteolytic enzymes. Finally the complex also works in the presence of the denaturing reagent sodium dodecyl sulfate. Experiments with the synthetic peptides, AcAla-Lys-Tyr-Gly-Gly-Met-Ala-Ala-Arg-Ala (termed Met-peptide) and AcVal-Lys-Gly-Gly-His-Ala-Lys-Tyr-Gly-Gly-Met-Ala-Ala-Arg-Ala (termed HisMet-peptide) as substrates, revealed structural and mechanistic features of the proteolytic reactions. We explain why two similar complexes with similar metal ions, cis-[Pt(en)(H(2)O)(2)](2+) and cis-[Pd(en)(H(2)O)(2)](2+), differ in selectivity as proteolytic reagents. The selectivity of cleavage is governed by the selectivity of the cis-[Pt(en)(H(2)O)(2)](2+) binding to the methionine side chain. The proteolytic activity is governed by the modes of coordination, which control the approach of the anchored Pt(II) ion to the scissile peptide bond. The cleavage occurs with a small, but significant, catalytic turnover of more than 18 after 7 days. The ability of cis-[Pt(en)(H(2)O)(2)](2+) to cleave proteins at relatively few sites, with explicable selectivity and catalytic turnover, bodes well for its use in biochemical practice.

Ribonucleoprotein particles (RNPs) are important components of all living systems, and the assembly of these particles is an intricate, often multistep, process. The 30 S ribosomal subunit is composed of one large RNA (16 S rRNA) and 21 ribosomal proteins (r-proteins). In vitro studies have revealed that assembly of the 30 S subunit is a temperature-dependent process involving sequential binding of r-proteins and conformational changes of 16 S rRNA. Additionally, a temperature-dependent conformational rearrangement was reported for a complex of primary r-protein S4 and 16 S rRNA. Given these observations, a systematic study of the temperature-dependence of 16 S rRNA architecture in individual complexes with the other five primary binding proteins (S7, S8, S15, S17, and S20) was performed. While all primary binding r-proteins bind 16 S rRNA at low temperature, not all r-proteins/16 S rRNA complexes undergo temperature-dependent conformational rearrangements. Some RNPs achieve the same conformation regardless of temperature, others show minor adjustments in 16 S rRNA conformation upon heating and, finally, others undergo significant temperature-dependent changes. Some of the architectures achieved in these rearrangements are consistent with subsequent downstream assembly events such as assembly of the secondary and tertiary binding r-proteins. The differential interaction of 16 S rRNA with r-proteins illustrates a means for controlling the sequential assembly pathway for complex RNPs and may offer insights into aspects of RNP assembly in general.

Coordination chemistry Z 0150 Palladium -[993 refs.]. -(KOSTIC, N. M.; DUTCA, L.-M.; Compr. Coord. Chem. II 6 (2004) 555-672; Dep. Chem., Iowa State Univ., Ames, IA 50011, USA; Eng.) -Schramke 43-239